cell models of and therapies for eye diseases

专利摘要:
Cellular models for eye disease, methods and compositions for treating eye disease are provided.
公开号:BR112020001940A2
申请号:R112020001940-2
申请日:2018-07-31
公开日:2020-08-18
发明作者:Richard R. Yang；Stephen H. TSANG
申请人:Reflection Biotechnologies Limited；
IPC主号:

专利说明:

[1] [1] This application claims priority under 35 USC § 119 (e) for US Application No. 62 / 539,473 entitled “CELLULAR MODELS OF AND THERAPIES FOR OCULAR DISEASES” filed on July 31, 2017. The contents in its all of the above are hereby incorporated by reference.
[2] [2] Bietti's Crystalline Dystrophy (BCD, tcc Bietti's Corneoretinal Crystalline Dystrophy, Bietti's Crystalline Retinopathy, Bietti's Retinal Dystrophy (OMIM 210370)) is a rare, blunt, autosomal retinal dystrophy, characterized by several fine yellow-white crystalline deposits of low gloss on the posterior pole of the retina, associated with atrophy of the retinal pigment epithelium (RPE), lumps of pigments and choroidal sclerosis. It was first identified by Dr. G. B. Bietti in 1937. Background photographs and SD-OCT images of patients with BCD showed that deposits in the lens were located mainly on the retinal side of the retinal pigment epithelium (RPE). (H. Kojima, A. Otani, K. Ogino and others. “Outer circular retinal structures in patients with Bietti crystalline retinopathy.” British Journal of Ophthalmology, vol. 96, pp. 390–393, 2012.) . Crystalline deposits in the corneal limbus have been estimated to occur in a quarter to a third of people with BCD (Kaiser-Kupfer et al. Clinical biochemistry and pathologic correlations in Bietti's crystalline dystrophy, Am J Ophthalmol., 1994, 118: 569 –82). In some cases, crystal deposits on the lenses are also observed (Chung et al., J Ophthalmol. 57: 447-450, 2013). In an advanced stage, patients with BCD have advanced choroidal sclerosis, decrease or absence of crystalline deposits
[3] [3] Clinically, BCD is progressive and associated with dystrophy and degeneration of RPE. Accentuated asymmetry between the eyes of the same patient is common. The age of onset and progression of the disease varies among patients with BCD, even within the same family. Most patients develop night blindness, restricted visual field, poor color vision, macular degeneration and reduced visual acuity between the 2nd and 4th decades of life and progress to legal blindness between the 3rd and 6th decades of life.
[4] [4] Located between the vessels of the choriocapillaris and external segments sensitive to the light of the photoreceptors, the RPE is a monolayer of pigmented cells that interacts closely with photoreceptors (cones and rods) in maintaining visual function. A key function of RPE is to nourish and remove waste products from the photoreceptors that are on the sensorineural retina. Other RPE functions include, but are not limited to: light absorption, epithelial transport, spatial ion buffering, visual cycle, phagocytosis, secretion and immune modulation (Strauss, 2005, The retinal pigment epithelium in visual function. Physiol Rev 85: 845-81 ). In this way, dysfunction and degeneration of RPE cause photoreceptor dysfunction and degeneration that result in loss of vision. Given BCD is associated with progressive dystrophy and degeneration of RPE, RPE is critical for the purposes of both study and treatment of BCD.
[5] [5] BCD is a rare disease. One source estimated the BCD incidence rate to be 1: 67,000 (ghr.nlm.nih.gov/condition/bietti-crystalline-dystrophy # statistics on the World Wide Web). Another source estimated that the prevalence of BCD is 2.5% of all patients with PR (3 BCD index patients out of 121
[6] [6] Currently, there is no approved treatment for BCD, and patients eventually go blind. There is a strong unmet medical need to develop life-changing treatment options for patients suffering from this rare disease. CYP4V2
[7] [7] CYP4V2 (Cytochrome P450, Family 4, Subfamily V, Polypeptide 2, (OMIM 608614), synonym: CYP4AH1) is one of the proteins in the cytochrome P450 superfamily and a member of the cytochrome P450 heme-thiolate subfamily 4 (CYP4). Cytochromes P450s (CYPs) are important heme-containing proteins, known for their monooxygenase reaction. They are involved in the metabolism of xenobiotic and endogenous compounds, such as steroids and fatty acids. Human CYPs are mainly membrane-associated proteins located either on the inner membrane of the mitochondria or on the reticulum.
[8] [8] The human CYP4 family consists of 12 genes and 10 pseudo-dogenes. The human CYP4V2 gene (HGNC: 23198) is located at 4q35 and has 11 exons. Mutations in the CYP4V2 gene cause BCD (Li et al., Am J Hum Genet. 74: 817-826, 2004). Although CYP4V2 is expressed in almost all tissues, it is expressed at high levels in the retina and RPE and at slightly lower levels in the cornea, tissues that show the main clinical findings of BCD (Li et al., Am J Hum Genet. 74: 817-826, 2004; Nakano M, Kelly EJ, Rettie AE: Expression and Characterization of CYP4V2 as a Fatty Acid omega- Hydroxylase. Drug Metab Dispos 2009; Nakano M, Kelly EJ, Wiek C, Hanenberg H, Rettie AE: CYP4V2 in Bietti's crystalline dystrophy: ocular localization, metabolism of omega-3-polyunsaturated fatty acids, and functional deficit of the p.H331P variant. Mol Pharmacol 2012; 82: 679-686).
[9] [9] Since CYP4V2 is a relatively new member of the P450 family and BCD is a rare disease, the function of CYP4V2 has not been studied extensively. Previous studies have shown that CYP4V2 protein is predominantly active in fatty acid metabolism. Abnormalities in fatty acids and their metabolism have been demonstrated in serum, lymphocytes and skin fibroblasts of patients with BCD (Lee J, Jiao X, Hejtmancik JF and others: The metabolism of fatty acids in human Bietti crystalline dystrophy. Invest Ophthalmol Vis Sci 2001; 42: 1707-1714; Lai T, Chu KO, Chan KP and others: Alterations in serum fatty acid concentrations and desaturase activities in Bietti crystalline dystrophy unaffected by CYP4V2 genotypes. Invest Ophthalmol Vis Sci 2010; 51: 1092 -1097). Another study showed that CYP4V2 is an omega-3-polyunsaturated fatty acid (PUFA) hydroxylase and a P450 highly expressed in the transformed human RPE cell line ARPE-19 (Nakano M, Kelly EJ, Wiek C, Hanenberg H, Rettie AE: CYP4V2 in Bietti's crystalline dystrophy: ocular localization, metabolism of omega-3-polyunsaturated fatty acids, and functional deficit of the p.H331P variant. Molecular pharmacology 2012; 82: 679-686).
[10] [10] Several mutations have been identified in the CYP4V2 gene and causing BCD, with at least one mutation in each of the gene's 11 exons. The most common CYP4V2 mutation among BCD patients is c.802-8_810del17insGC (referring to a deletion at base 17 with two bases (GC) inserted into the site beginning 8 bases from the intron 6 end of the CYP4V2 gene, also referred to as IVS6-8 del / insGC See SEQ ID NO: 46 showing human CYP4V2 genomic DNA region sequence comprising the c.802-8_810del17insGC mutation and SEQ ID NO: 47 showing the corresponding wild type sequence. The c.802- 8_810del17insGC mutation is illustrated in the following sequence that shows the junction of human CYP4V2 intron 6-exon 7. The sequence of intron 6 is shown in lower case and the exon sequence 7 in upper case. in 17 bps and the insertion of GC are in brackets): caa aca gaa gca tgt gat tat cat tca aa (tca tac agG TCA TCG CT) (GC) GAA CGG GCC AAT GAA ATG AAC GCC AAT GA (SEQ ID NO : 46)) resulting in the leap of exon 7 (Xiao et al., Biochem Bi-res Res Commun. 409: 181-6, 2011; Meng et al., 2014, Mol Vis., 20: 1806-14; Wada et al., Am J Ophthalmol. 139: 894-9, 2005; Jiao et al., European Journal of Human Genetics (2017) 25, 461-471). A recent study estimated that the age of the c.802- 8_810del17insGC mutation would be 1,040-8,200 generations in the Chinese population and 300-1100 generations in the Japanese. See Jiao et al., European Journal of Human Genetics (2017) 25, 461-471.
[11] [11] Various types of CYP4V2 mutations have been found associated with BCD, including, but not limited to, erroneous, in duplicate, union site, structure change, deletion, insertion, indel, nonsense, polymorphisms ( for example, single nucleotide polymorphisms) and premature termination, as well as entire deletion of the CYP4V2 gene. A summary of selection CYP4V2 mutations among patients with human BCD is provided in Table 1 here and can be found in various publications and online databases, for example, LOVD (databases.lovd.nl/shared / genes / CYP4V2 on the World Wide Web), OMIM (omim.org/allelicVariant/608614 on the World Wide Web) and ClinVar (ncbi.nlm.nih.gov/clinvar term=608614 XNXXMIM) on the World Wide Web). Table 1. CYP4V2 mutations for selection among patients with
[12] [12] This is a selection list only and may not contain all pathological CYP4V2 mutations / variants among BCD patients identified and reported so far. The mutations are related to reference sequences (NM_207352.3) and (NP_997235.3). New pathological CYP4V2 mutations among BCD patients are being continuously identified. All pathological CYP4V2 mutations / variants identified and identified in the future associated with BCD are hereby incorporated by reference. Inherited Retinal Degenerations (IRDs)
[13] [13] Inherited Retinal Degenerations (IRDs) is a major cause of blindness. Currently, more than 200 genes are known to be involved in IRDs and related disorders. Retinitis pigmentosa (RP) is the main form of IRDs in humans. There are three general modes of inheritance for RP (autosomal dominant, autosomal recessive and X-linked). The worldwide incidence rate of PR was estimated to be one in 4000, with autosomal recessive PR being responsible for 50% -60% PR (Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. Lancet. 2006; 368: 1795–809). A study in Europe estimated that the prevalence of BCD is 2.5% of all patients with PR and approximately 10% of people with autosomal recessive non-syndromic PR (Mataftsi A, Zografos L, Millá E, Secrétan M, Munier FL Bietti's crystalline corneoretinal dystrophy: a cross-sectional study.
[14] [14] Li A, Jiao X, Munier FL, Schorderet DF, Yao W, and others (2004) Bietti crystalline corneoretinal dystrophy is caused by mutations in the novel CYP4V2 gene. Am J Hum Genet 74: 817-826.
[15] [15] Xiao X, Mai G, Li S, Guo X, Zhang Q (2011) Identification of CYP4V2 mutation in 21 families and overview of mutation spectrum in Bietti crystalline corneoretinal dystrophy. Biochem Biophys Res Comm. 409: 181-186.
[16] [16] Shan M, Dong B, Zhao X, Wang J, Li G, and others (2005) Novel mutations in the CYP4V2 gene associated with Bietti crystalline corneoretinal dystrophy. Mol Vis 11: 738-743.
[17] [17] Rossi S, Testa F, Li A, Yaylacioglu F, Gesualdo C, and others (2013) Clinical and genetic features in Italian Bietti crystalline dystrophy patients. Br J Ophthalmol 97: 174-179.
[18] [18] Lin J, Nishiguchi KM, Nakamura M, Dryja TP, Berson EL, and others (2005) Recessive mutations in the CYP4V2 gene in East Asian and Middle Eastern patients with Bietti crystalline corneoretinal dystrophophy. J Med Genet 42: e38.
[19] [19] Manzouri B, Sergouniotis PI, Robson AG, Webster AR, Moore A (2012) Bietti crystalline retinopathy: report of retinal crystal deposition in male adolescent siblings. ARCH OPHTHALMOL 130: 1470-1473.
[20] [20] Lai TY, Ng TK, Tam PO, Yam GH, Ngai JW, and others (2007) Genotype phenotype analysis of Bietti's crystalline dystrophy in pa-
[21] [21] Parravano M, Sciamanna M, Giorno P, Boninfante A, Vara- no M (2012) Bietti crystalline dystrophy: a morpho-functional evalua- tion. Doc Ophthalmol 124: 73-77.
[22] [22] Wada Y, Itabashi T, Sato H, Kawamura M, Tada A, and others (2005) Screening for mutations in CYP4V2 gene in Japanese patients with Bietti's crystalline corneoretinal dystrophy. Am J Ophthalmol 139: 894-899.
[23] [23] Zenteno JC, Ayala-Ramirez R, Graue-Wiechers F (2008) Novel CYP4V2 gene mutation in a Mexican patient with Bietti's crystal-line corneoretinal dystrophy. Curr Eye Res 33: 313-318.
[24] [24] Lee KY, Koh AH, Aung T, Yong VH, Yeung K, and others (2005) Characterization of Bietti crystalline dystrophy patients with CYP4V2 mutations. Invest Ophthalmol Vis Sci 46: 3812-3816.
[25] [25] Yokoi Y, Sato K, Aoyagi H, Takahashi Y, Yamagami M, and others (2011) A Novel Compound Heterozygous Mutation in the CYP4V2 Gene in a Japanese Patient with Bietti's Crystalline Coronoretinal Dystrophy. Case Rep Ophthalmol 2: 296-301.
[26] [26] Haddad NM, Waked N, Bejjani R, Khoueir Z, Chouery E, et al. (2012) Clinical and molecular findings in three Lebanese families with Bietti crystalline dystrophy: report on a novel mutation. Mol Vis 18: 1182-1188.
[27] [27] Fu Q, Wang F, Wang H, Xu F, Zaneveld JE, et al. (2013) Next-generation sequencing-based molecular diagnosis of a Chinese patient cohort with autosomal recessive retinitis pigmentosa. Invest Ophthalmol Vis Sci 54: 4158-4166.
[28] [28] Song Y, Mo G, Yin G (2013) A novel mutation in the CYP4V2 gene in a Chinese patient with Bietti's crystalline dystrophy. Int Ophthalmol 33: 269-276.
[29] [29] Jin ZB, Ito S, Saito Y, Inoue Y, Yanagi Y, and others (2006) Clinical and molecular findings in three Japanese patients with crystal-line retinopathy. Jpn J Ophthalmol 50: 426-431.
[30] [30] Halford S, Liew G, Mackay DS, Sergouniotis PI, Holt R, Broadgate S, Volpi EV, Ocaka L, Robson AG, Holder GE, Moore AT, Michaelides M, Webster AR. Detailed phenotypic and genotypic char-acterization of bietti crystalline dystrophy. Ophthalmology. 2014; 121: 1174-84.
[31] [31] Houfa Yin, Chongfei Jin, Xiaoyun Fang, Qi Miao, Yingying Zhao, Zhiqing Chen, Zhaoan Su, Panpan Ye, Yao Wang and Jinfu Yin, Molecular Analysis and Phenotypic Study in 14 Chinese Families With Bietti Crystalline Dystrophy. PLoS One 9 (4), e94960. 2014 Apr 16.
[32] [32] Xiao Hong Meng, Hong Guo, Hai Wei Xu, Qi You Li, Xin Jin, Yun Bai, Shi Ying Li, Zheng Qin Yin, Identification of novel CYP4V2 gene mutations in 92 Chinese families with Bietti's coronal neoretinal dystrophy, Molecular Vision (2014); 20: 1806-1814.
[33] [33] Galuh DN Astuti, Vincent Sun, Miriam Bauwens, Ditta Zobor, Bart P Leroy, Amer Omar, Bernhard Jurklies, Irma Lopez, Huanan Ren, Volkan Yazar, Christian Hamel, Ulrich Kellner, Bernd Wissinger, Susanne Kohl, Elfride De Baere, Rob WJ Collin, and Robert K Koenekoop, Novel insights into the molecular pathogenesis of CYP4V2-associated Bietti's retinal dystrophy, Mol Genet Genomic Med. 2015 January; 3 (1): 14–29.
[34] [34] Xiaodong Jiao, Anren Li, Zi-Bing Jin, Xinjing Wang, Ales- sandro Iannaccone, Elias I Traboulsi, Michael B Gorin, Francesca Simonelli and J Fielding Hejtmancik, Identification and Population His- tory of CYP4V2 mutations in Patients with Bietti Crystalline Corneoretinal Dystrophy, European Journal of Human Genetics (2017) 25, 461-
[35] [35] In one aspect, a model of cell disease including a cell line is provided. Such a disease model includes (a) a stem cell provided by an individual or reprogrammed from a cell provided by an individual or (2) a cell derived from a stem cell provided by an individual or reprogrammed from a cell provided by an individual, comprising one or more mutations in a target gene.
[36] [36] In some embodiments, the stem cell is an induced pluripotent stem cell (iPS). In some modalities, the stem cell is an embryonic stem cell (ES), somatic (or adult) stem cell or mesenchymal stem cell (MSC). In some modalities, the cell provided from an individual is of any type of cell and / or any tissue of the individual. In some embodiments, the cell provided by an individual is a skin cell, a fibroblast or blood cell. In some embodiments, the cell provided from an individual is a skin fibroblast or a peripheral blood mononuclear cell (PBMC). In some modalities, the cell provided from an individual is a urinary cell, a renal epithelial cell, a hair follicle or a dermal papillary cell.
[37] [37] In some embodiments, the stem-derived cell is an eye cell. In some embodiments, the eye cell is a retinal pigment epithelium (RPE) cell, photoreceptor cell (PRC, including rod cell, cone cell and photoreceptor progenitor cell), retinal cell, corneal cell, epithelial cell of the cornea (CPB), optic nerve cell, lens cell, choroidal endothelial cell (CE), optic nerve cell or choroidal cell. In some modalities
[38] [38] In some modalities, the mutation is endogenous to the individual. In some modalities, the mutation is exogenous to the individual. In some embodiments, the mutation is introduced artificially through genetic editing or genetic manipulation. In some modalities, the cell line comprises a plurality of mutations that are endogenous and / or exogenous to the individual.
[39] [39] In some embodiments, the individual is a mammal. In some modalities, the individual is a human.
[40] [40] In some embodiments, the target gene comprises a gene shown in Table 4. In some embodiments, the target gene comprises a gene CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, 1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4F2, CYP4F2, CYP4F2 CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A gene or a CYP4V2 gene, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACH2, FH2, , MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND , FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or mutated or defective CYP46A that encodes a protein having defective or partial function or activity. In some embodiments, the target gene is CYP4V2.
[41] [41] In some embodiments, the cell line comprises an iPS cell. In some embodiments, the cell line comprises an iPS-RPE cell. In some embodiments, the cell line comprises an iPS-photoreceptor cell (iPS-PRC), an iPS-epithelial corneal cell (iPS-CEC), an iPS-choroidal iPS-endothelial (CE) cell, an iPS-corneal cell , an iPS-choroidal cell, an iPS-optic nerve cell, an iPS-ocular cell or an iPS-neuron cell. In some embodiments, the CYP4V2 mutation in the cell line is endogenous to the individual. In some modalities, the individual has a pathological mutation in the CYP4V2 gene or in an orthologist of the CYP4V2 gene.
[42] [42] In some modalities, the individual has at least one mutation shown in Table 1. In some modalities, the individual has inherited retinal degeneration (IRD) or retinitis pigmentosa (RP). In some modalities, the individual has Bietti's Crystalline Dystrophy (BCD, Bietti's Corneoretinal Crystalline Dystrophy, Bietti's Crystal Retinopathy, Bietti's Retinal Dystrophy) or is at risk of developing BCD.
[43] [43] In some embodiments, the cell line comprises a mutation in CYP4V2 that is exogenous to the individual and is artificially introduced through genetic editing or genetic manipulation.
[44] [44] In some embodiments, the cell line comprises an iPS cell, ES cell, MSC, or adult stem cell, or an RPE cell, photoreceptor cell, corneal epithelial cell, choroidal endothelial cell (CE) or choroidal cell derived from an iPS cell, ES cell, MSC or adult stem cell. In some embodiments, the iPS cell or other type of stem cell is characterized by one or more of the following: a. the unique morphology of iPS, ES or MSC; B. one or more pluripotency markers, such as Oct-4, Sox-2, SSEA4,
[45] [45] In some embodiments, the iPS-RPE cell or the RPE cell derived from other types of stem cells is characterized by: a. morphology: pigment and hexagonal shape and / or b. one or more of the following biomarkers, retinaldehyde-binding protein 1 (RLBP1, pseudonym: CRALBP), RPE65, BESTROPHIN-1, MITF, LRAT, RDH5, PAX6, MERTK, TYR, ZO-1 and / or VINCULIN.
[46] [46] In another aspect, a human cell model of BCD or a cell model of CYP4V2 function is provided. Such a model includes an iPS cell or iPS cell line or an iPS-RPE cell or iPS-RPE cell line derived from a cell or cell line of a patient with BCD or derived from a cell or cell line with mutations in CYP4V2 artificially created.
[47] [47] In some embodiments, the cell line has an abnormal biochemical profile in one or more compounds in the following groups of compounds: (i) fatty acids, (ii) ceramides, (iii) sphingomyelins, (iv) sphingosine, (v) sphinganine or (vi) hydroxy fatty acids, compared to a corresponding cell line for healthy control. In some embodiments, the cell line has an abnormal biochemical profile in one or more compounds shown in Table 2 compared to the corresponding cell line for a healthy control. Cell Disease Model Manufacturing Method:
[48] [48] In another aspect, a method of fabricating an iPS-derived BCD disease model is provided. Such a method includes obtaining cells from an individual having endogenous mutations in the CYP4V2 gene or cells without any endogenous mutations in the CYP4V2 gene, but exogenous CYP4V2 mutation is artificially introduced through genetic editing or genetic manipulation at this stage or any one of the stages that follow; induction of pluripotency in cells or reprogramming of cells to produce iPSCs; culture of iPSCs under conditions that result in differentiation of iPSCs in desired eye cells, thereby producing an iPS-derived ocular cell line.
[49] [49] In some embodiments, the cells obtained from individuals are somatic cells. In some modalities, cells obtained from individuals are skin cells, fibroblasts, blood cells, peripheral blood mononuclear cells (PBMC) or eye cells. In some embodiments, cells obtained from individuals are urinary cells, renal epithelial cells, a hair follicle or dermal papillary cells. In some embodiments, eye cells are retinal pigment epithelial cells (RPE), corneal epithelial cells (ECCs), photoreceptor cells (PRCs), coronal endothelial cells (EC), optic nerve cells, retinal cells, retinal cells of the cornea or choroidal cells. In some embodiments, pluripotency is induced or cells are reprogrammed using one or more of the OCT4, SOX2, KLF4 and c-MYC transcription factors
[50] [50] In some embodiments, the mutation is pathological. In some modalities, the cell line comprises one or more mutations among the mutations shown in Table 1. In some modalities, the cell line is heterozygous for the mutation. In some modalities, the cell line is homozygous for the mutation.
[51] [51] In some embodiments, the cell disease model exhibits abnormal levels in one or more compounds from the following groups of compounds compared to those in a relevant cell line for healthy control: (i) fatty acids, ( ii) ceramides, (iii) sphingomyelin, (iv) sphingosine, (v) sphinganine or (vi) acidic
[52] [52] In one aspect, a method of detecting abnormalities or phenotype in a cell model of disease is provided. Such a method typically includes assessing and comparing the levels of one or more compounds between a patient's cell line (or a genetically edited or manipulated cell line comprising an exogenous mutation in the gene causing such a disease) and healthy control, where the one or more compounds are selected from the groups that follow: (i) fatty acids, (ii) ceramides, (iii) sphingomyelin, (iv) sphinxosine, (v) sphinganine and / or (vi) hydroxy fatty acids .
[53] [53] In some embodiments, one or more of the evaluated compounds are shown in Table 2. In some embodiments, the identification and / or assessment of compound levels is performed using LC-MS, LC-MS / MS, GC-MS, GC-MS / MS and / or FIA-MS / MS. In some embodiments, the disease cell model comprises a mutated or defective gene shown in Table 4. In some embodiments, the disease cell model comprises a mutated or defective gene among the CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAXIT, PDG, PX, TEK, TXNRD2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4, CPC CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A.
[54] [54] In another aspect, a method of evaluating a test agent for therapeutic efficacy against BCD is provided. Such a method typically includes contacting the cells of an iPS-RPE cell line derived from a patient with BCD or an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene as a result of artificial genetic manipulation or editing with a test agent. ; and assessment of cells for normalization to levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid sequence in cells; an increase in the amount of CYP4V2 polypeptides in cells; and / or improved cell structure, morphology or function, compared to before contact by such a test agent; in which normalization at levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid in the cells; an increase in the amount of CYP4V2 polypeptides in cells; and / or improved cell structure, morphology or function, compared to before treatment by such a test agent, is indicative of a test agent that exhibits therapeutic efficacy against BCD.
[55] [55] In some embodiments, test agents are selected from the group consisting of nucleic acids or analogues thereof, vectors containing nucleic acid sequence or coding polypeptides, polypeptides or analogues thereof, antibodies, agents chemicals, small molecules and / or any combination thereof. In some modalities, cells are evaluated using PCR techniques, immunoassays, sequencing, biochemical assay, function assay, microscopy or combination thereof.
[56] [56] In another aspect, a method of evaluating the effectiveness or efficiency of a formulation, vector or construct comprising a test agent for BCD is provided. Such a method typically includes contacting multiple cell samples from an iPS-RPE cell line derived from a patient with BCD or an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene as a result of artificial genetic manipulation or editing with a test agent formulated or packaged in various formulations, vectors or constructs; and evaluation of cell samples for normalization at levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid in the cells; an increase in the amount of CYP4V2 polypeptides in cells; improved cell structure, morphology or function; and / or tolerance or cell death, compared to before treatment by such a test agent and / or cell samples treated by the same test agent, but formulated or packaged in a different formulation, vector or construct, to determine and compare the efficiency or effectiveness of such a formulation, vector or construct; in which cells are evaluated using PCR techniques, immunoassays, sequencing, biochemical assay, cell viability assay, microscopy or combination thereof.
[57] [57] In one aspect, an effective and safe dosing range assessment method for a BCD test agent is provided. Such a method typically includes contacting multiple cell samples from an iPS-RPE cell line derived from a patient with BCD or an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene as a result of artificial genetic editing or manipulation with a test agent at a different dose for each cell sample; evaluation of cell samples for normalization at levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid sequence
[58] [58] In another aspect, a method of assessing the effectiveness or efficiency of a device or method of administration for delivering a therapeutic agent to the retina or retinal cells is provided. Such a method typically includes (i) contacting a cell sample from an iPS-RPE cell line derived from a patient with BCD or an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene as a result of editing or manipulation artificial genetics with a test agent without using the device or method of administration; (ii) contacting another cell sample from an iPS-RPE cell line derived from a patient with BCD or an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene as a result of artificial genetic editing or manipulation with the agent testing the same dosage as in (i) using the device or method of administration; (iii) evaluation and comparison of cell samples from (i) and (ii) for normalization to levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid sequence in cells; an increase in the amount of CYP4V2 polypeptides in cells; improved cell structure, morphology or function; tolerance or cell death; and / or the levels of the test agent in the cells, compared to before treatment by such test agent and / or treatment by the same test agent of the same dose, but without using the device or method of administration, to determine the effectiveness or efficiency of such management device or technique; in which cells are evaluated using PCR techniques, immunoassays, sequencing, biochemical assay, function assay, microscopy or combination thereof.
[59] [59] In some embodiments, the retinal cells are RPE cells. CRISPR Gene Editing Therapy
[60] [60] In one aspect, a composition is provided that includes: (a) a CRISPR guide RNA targeting a nucleic acid sequence (the “target sequence”) of or within 100 bp for the CYP4V2 gene and (b) a functional CRISPR (Cas) associated protein. In some embodiments, such a composition may further include (c) a donor nucleic acid sequence comprising all or a portion of a wild-type sequence or a functional sequence of the CYP4V2 gene for correction, disruption or replacement of the CYP4V2 gene or a portion of it.
[61] [61] In some embodiments, one or more components of the same are provided in the form of a DNA molecule encoding such a component, an mRNA molecule encoding such a component, an RNA molecule, a polypeptide and / or a ribonucleoprotein (RNP) or protein-RNA complex. In some embodiments, two or more components of the same are in a separate molecule or combined into a molecule or complex, are in separate vectors or combined into a vector, are in one or more nucleic acid complexes, are in one or more most complex of RNP. In some embodiments, the donor nucleic acid sequence is provided in a single-stranded donor oligonucleotide (ssODN) or vector. In some embodiments, the vector is a plasmid, a recombinant AAV vector, a recombinant lentivirus vector and / or a combination thereof.
[62] [62] In some respects, a composition including a cell with a pathological CYP4V2 mutation that contains any of the compositions described here is provided. In some embodiments, (a) the CRISPR guide RNA comprising (i) a CRISP RNA (crRNA) that comprises a protospacer element sequence that is complementary to the target sequence of or within 100 bps for a target gene ( the “target gene”) and a sequence that corresponds to a complementary region of the transactivating crRNA (tracrRNA) and (ii) a tracrRNA that comprises a region that is complementary to the corresponding region of the crRNA and a sequence that interacts with a CRISPR 9-associated protein (Cas9) and (b) the functional CRISPR-associated protein comprises Cas9.
[63] [63] In some embodiments, the protospacer element is about 20 bases, about 19 bases, about 21 bases, about 19-21 bases, about 18-22 bases, or about 16-24 bases. In some modalities, crRNA and tracrRNA are in separate molecules. In some modalities, crRNA and tracrRNA are combined into a single guide RNA (sgRNA). In some embodiments, sgRNA is about 88-150 bp.
[64] [64] In some embodiments, Cas9 comprises a mutant Cas9 or Cas9 orthologist selected from: Streptococcus pyogenes (SpCas9), SpCas9 nickase (Cas9n D10A), SpCas9 (D1135E), eS- pCas9, SpCas9-HF1, SpCas9 VRER, SpCas9 VQR, SpCas9EQR, Staphylococcus aureus (SaCas9), Neisseria Meningitidis, Streptococcus thermophilus, Streptococcus pneumnoniae, Campylobacter coli, Campylobacter jejuni, Streptococcus mutans, Pasteurella multocida, bifidobi
[65] [65] In some embodiments, (a) the CRISPR guide RNA comprises a crRNA comprising a sequence of protospacer element that is complementary to the target sequence of or within 100 bps for a target gene (b) the associated protein the functional CRISPR comprises Cpf1. In some embodiments, the protective element is about 20 bases, about 21 bases, about 22 bases, about 23 bases, about 24 bases, about 19-25 bases, about 18-26 bases or about 16-28 bases.
[66] [66] In some embodiments, the proto-spacer sequence is selected from the group consisting of SEQ ID NOs: 48 to 52 or shares at least 85% sequence identity with one of SEQ ID NOs: 48 to 52 for use with a Cas protein that has NGG as a motif-adjacent motif (PAM) to target the c.802-8_810del17insGC mutation of the CYP4V2 gene. In some embodiments, the donor nucleic acid sequence is selected from SEQ ID NOs: 56 and 57 (ie, the two donor model sequences) or shares at least 90% sequence identity with one of SEQ ID NOs: 56 and 57, or a sequence that is complementary to it, for use to correct, disrupt or replace the c.802-8_810del17insGC mutation in the CYP4V2 gene. Claims of
[67] [67] CRISPR gene therapy method In another aspect, a method of treating or preventing BCD in an individual or a cell with a mutated CYP4V2 gene is provided. Such a method includes (i) identifying the pathological mutation in the individual or cell through sequencing; (ii) find PAM sites related to Cas within the region extending from about 100 bps upstream of the first nucleotide involved in the mutation to about 100 bps downstream of the last nucleotide involved in the mutation; (iii) to identify several protospace element sequences directing the relevant CYP4V2 sequence for each MAP site identified in (ii); (iv) evaluate the activity level of each CRISPR guide RNA comprising a sequence of protospacer element identified in (iii) and an out-of-target editing profile based on the sequence of protospacer element and MAP; (v) select one or more CRISPR guide RNA projects based on (iv); (vi) designing one or more donor nucleic acid sequences based on homology-based repair (HDR) for correction, disruption or replacement of the targeted CYP4V2 mutation; (vii) constructing CRISPR, Cas guide RNA and donor nucleic acid sequence as provided in composition claims 1 to 18; (viii) optionally validate and further select the components of (vii) in an isolated cell of the individual; either an iPS cell derived from the individual or a cell differentiated from a stem cell derived from the individual or the genomic DNA isolated from the individual or a cell isolated or derived from it to assess the level of activity and / or editing profile outside the target; and (ix) administering the components in (viii) to the individual or the cell through an administration system selected from the group consisting of a ri- bonucleoprotein or protein-RNA complex, a vector, a protein, a molecule nucleic acid, a nanoparticle, a liposome, a micelle, a virosome, a nucleic acid complex, and / or a combination thereof, in which administration is carried out by electroporation or by lipid-mediated transfection , or nucleofection, or viral transduction or injection, or a combination thereof; (x) where for treatment in cells in vitro, a selection marker including, without limitation, GFP, EGFP or resistance to puromycin is optionally added or incorporated into the components in (viii).
[68] [68] In one aspect, a genetic editing composition is provided to correct or replace the c.802- 8_810del17insGC mutation in a CYP4V2 gene in an individual in vivo or in a cell in vitro. Such a composition typically includes: (i) a CRISPR guide RNA comprising a proto-spaencing element sequence selected from one of SEQ ID NOs: 48 to 52 or sharing at least 80% sequence identity with one of the following quenches in SEQ ID NOs: 48 to 52; (ii) a donor nucleic acid sequence selected from one of SEQ ID NOs: 56 and 57 or shares at least 90% sequence identity with one of SEQ ID NOs: 56 and 57 or a sequence that is complementary the same; and (iii) a Cas9 protein (exemplary sequence shown in SEQ ID NO: 58), optionally containing 1, 2, 3 or more NLS, and / or a selection marker including, without limitation, GFP or EGFP.
[69] [69] In some embodiments, an optional G nucleotide is added before the sequence of the protospace element. In some embodiments, the CRISPR guide RNA includes a crRNA (exemplary sequence (excluding the 5 'protospacer element sequence) shown in SEQ ID NO: 53) and a tracrRNA (exemplary sequence shown in SEQ ID NO: 54); and the sequence of the protospace element is contained in the crRNA. In some embodiments, the CRISPR guide RNA includes a single guide RNA (sgRNA) comprising the sequence of the protospacer element (exemplary sgRNA sequence (excluding the 5 'protospacer element sequence) shown in SEQ ID NO: 55) .
[70] [70] In some embodiments, one or more components of (i), (ii) and (iii) are provided in the form of a DNA molecule encoding such a component, an mRNA molecule encoding such a component, a nucleic acid molecule, a vector, an RNA molecule, a polypeptide, a ribonucleoprotein (RNP) or protein-RNA complex and / or a combination thereof. Cell Therapy Claims for BCD, Autologous Cell Therapy for Eye Disease and Combination Treatment Cell Therapy for BCD Allogeneic Cell Therapy or Autologous Cell Therapy without genetic repair for BCD
[71] [71] In some respects, a method of treating or preventing an eye disease in an individual is provided, in which the disease is associated with a pathological genetic or epigenetic change in the CYP4V2 gene. Such a method typically includes administering a cell composition to the individual, wherein the cell composition includes: retinal pigment epithelial cells (RPE), photoreceptor or photoreceptor progenitor cells (PRCs), corneal epithelial cells (SCCs), choroidal endothelial cells (CE) and / or other eye cells derived from a stem cell.
[72] [72] In some embodiments, the stem cell is an embryonic stem cell (ES), an iPC cell, an MSC, an adult stem cell, or a tissue-specific stem cell. In some modalities, the stem cell is from or derived from one or more individuals without BCD or without a pathological CYP4V3 gene. In some embodiments, the stem cell is from or derived from one or more individuals with pathological mutations in the CYP4V2 gene. In some ways, the individual is a human individual. Genetically repaired autologous cell therapy for BCD
[73] [73] In another aspect, a cell composition is provided that includes (a) a stem cell reprogrammed from an isolated cell from or a stem cell isolated from an individual affected by
[74] [74] In some embodiments, the stem cell reprogrammed from an individual's isolated cell is an iPC cell. In some embodiments, the iPS cell is reprogrammed from any cell in any tissue of the individual. In some embodiments, the iPS cell is reprogrammed from a skin cell, a blood cell, a urinary cell, a hair cell, a fibroblast, a peripheral blood mononuclear cell (PBMC), a renal epithelial cell, a hair follicle or a dermal papillary cell. In some embodiments, the individual's isolated stem cell is an MSC, an adult stem cell, or a tissue-specific stem cell. In some embodiments, the cell differentiated from a stem cell is an ocular cell. In some embodiments, the differentiated cell of a stem cell is an RPE cell, a PRC, a rectal cell, a corneal cell, a choroidal cell, an ECC or an EC cell. In some embodiments, the differentiated cell of a stem cell is an iPS-RPE, iPS-PRC, iPS-CEC or iPS-CE cell
[75] [75] In some embodiments, (i) the cell isolated from an individual affected by BCD or having pathological mutations in the CYP4V2 gene for use to reprogram in an iPSC, (ii) the stem cell isolated from an individual or iPS cell reprogrammed from an isolated cell of an individual affected by BCD or having pathological mutations in the CYP4V2 gene or (iii) the cell differentiated from an individual's isolated stem cell or an iPS cell reprogrammed from an isolated cell from an individual affected by BCD or having pathological mutations in the CYP4V2 gene, is genetically repaired to improve the effect of the mutated CYP4V2 gene. In some modalities,
[76] [76] In another aspect, a method of treating or preventing an eye disease in an individual affected by BCD or having pathological genetic or epigenetic changes in the CYP4V2 gene is provided. Such a method typically includes administration of any of the autologous CYP4V2 cell compositions described herein to the subject, wherein the cell composition includes: retinal pigment epithelium (RPE) cells, photoreceptors or photoreceptor progenitor cells (PRCs), epithelial cells of the cornea (SCCs), choroidal endothelial cells (EC) and / or other eye cells derived from an individual's stem cell.
[77] [77] In some embodiments, the stem cell is an iPC cell, an MSC, an adult stem cell, or a tissue-specific stem cell. In some embodiments, the iPS cell is reprogrammed using one or more of the OCT4, SOX2, KLF4 and c-MYC transcription factors. In some embodiments, the genetically repaired cells demonstrate one or more of the following: normalization to levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid sequence in cells; an increase in the amount of functional CYP4V2 polypeptides; and / or improved cell structure, morphology or function, compared to before the genetic repair was performed.
[78] [78] In some embodiments, the number of cells administered is about 1,000 to about 100 million cells in a single administration. In some modalities, administration is by injection. In some modalities, administration is through sub-retinal injection. In some modalities, administration is through intravitreal injection. In some modalities, administration is through direct retinal injection. In some modalities, administration is by injection into the cornea. In some modalities, administration is through any other method of administration that effectively delivers cells to the sub-retinal site, the posterior segment or the cornea of the individual's eye. In some embodiments, cells are administered by injecting a cell suspension. In some embodiments, cells are administered as part of a sheet, matrix, base or tissue.
[79] [79] In some embodiments, RPE cells are administered using natural and / or synthetic bases to generate a functional RPE monolayer. In some embodiments, the individual is a human individual. Genetically repaired autologous cell therapy for eye diseases
[80] [80] In another aspect, a cell composition is provided that includes (a) a stem cell reprogrammed from a cell isolated from or a stem cell isolated from an individual affected by a disease caused by a mutated or defective gene or a gene encoding a protein having defective or par-
[81] [81] In some embodiments, the stem cell reprogrammed from an individual's isolated cell is an iPS cell. In some embodiments, the iPS cell is reprogrammed from any cell in any tissue of the individual. In some embodiments, the iPS cell is reprogrammed from a skin cell, a blood cell, a urinary cell, a hair cell, a fibroblast, a peripheral blood mononuclear cell (PBMC), a renal epithelial cell, a hair follicle or a dermal papillary cell. In some embodiments, the individual's isolated stem cell is an MSC, an adult stem cell, or a tissue-specific stem cell.
[82] [82] In some modalities, the gene is involved in ocular development or function and / or mutation which causes or is a risk factor for causing an eye disease. In some modalities, the gene is involved in neuronal development or function and / or mutation which causes or is a risk factor for causing a neurodegenerative disease. In some embodiments, the gene is a cytochrome P450 gene. In some modalities, the gene is shown in Table 4.
[83] [83] In some embodiments, the gene includes a CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXC2, FOXC2, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47, MT47 GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA,
[84] [84] In some embodiments, the cell differentiated from a stem cell is any cell type. In some embodiments, the cell differentiated from a stem cell is an eye cell. In some embodiments, the cell differentiated from a stem cell is an RPE cell, a PRC, a retinal cell, a corneal cell, a choroidal cell, an ECC, a CE cell or an optic nerve cell . In some embodiments, the differentiated cell of a stem cell is an iPS-RPE, iPS-PRC, iPS-CEC or iPS-CE cell. In some modalities, the differentiated cell of a stem cell is a neuron.
[85] [85] In some embodiments, (i) the isolated cell of an individual affected by a disease caused by a mutated or defective gene or a gene encoding a protein having defective or partial function or activity for use to reprogram in a iPSC, (ii) the stem cell isolated from an individual or iPS cell reprogrammed from an isolated cell from an individual affected by a disease caused by a mutated or defective gene or a gene encoding a protein having defective or partial function or activity or (iii) the cell differentiated from a stem cell isolated from an individual or an iPS cell reprogrammed from a cell isolated from an individual affected by a disease caused by a mutated or defective gene or a gene encoding a protein having defective or partial function or activity is genetically repaired to improve the effect of the mutated or defective gene.
[86] [86] In some modalities, genetic repair is performed before reprogramming in an iPS cell. In some modalities, genetic repair is performed after reprogramming to an iPS cell. In some modalities, genetic repair is performed before the differentiation of the stem cell or iPS cell. In some modalities, genetic repair is performed after differentiation of the stem cell or iPS cell. In some modalities, genetic repair is through gene transfer therapy. In some modalities, genetic repair is through gene transfer therapy using any composition or method of any of the claims related to gene therapy. In some modalities, genetic repair is through gene editing therapy. In some modalities, genetic repair is through gene editing therapy using any composition or method of any of the claims related to CRISPR gene therapy.
[87] [87] In another aspect, a method of treating or preventing a disease in an individual affected by a disease caused by a defective or mutated gene or a gene encoding a protein having a defective or partial function or activity shown in Table 4 is provided. Such a method typically includes administration of an autologous cell composition as described herein to the subject, wherein the cell composition includes: retinal pigment epithelium (RPE) cells, photoreceptors or photoreceptor progenitors (PRCs), cells,
[88] [88] In yet another aspect, a method is provided for autologically treating an individual. Such a method typically includes (i) providing cells from an individual having an eye disease; (ii) inducing pluripotency in the individual's cells to produce iPSCs; (iii) genetically repairing one or more mutations in a mutated or defective gene shown in Table 4 in the individual's derived iPSCs; (iv) differentiate iPSCs in eye cells; (v) alternative to step (iii), genetically repairing the iPS eye cells through gene transfer therapy; (vi) introducing the iPS-ocular cells into the individual, thereby autologously treating the individual with eye disease.
[89] [89] In some embodiments, the stem cell is an iPS cell, an MSC, an adult stem cell, or a tissue-specific stem cell. In some embodiments, the iPS cell is reprogrammed using one or more transcription factors OCT4, SOX2, KLF4 and c-MYC. In some embodiments, the genetically repaired cells demonstrate one or more of the following: an increase in non-defective target gene nucleic acid sequence in the cells; an increase in the amount of functional polypeptides encoded by the target gene in cells; improved cell structure, morphology or function and / or improved or normalized biochemical functions in cells, compared to before genetic repair is performed. In some modalities, the number of cells administered is about 1,000 to about 100 million cells in a single administration.
[90] [90] In some modalities, administration is by injection. In some modalities, administration is through injections
[91] [91] In some modalities, the disease is associated with a genetic or epigenetic change or risk factor in the individual. In some embodiments, the disease is photoreceptor degeneration, degeneration of the retinal pigment epithelium cell, retinal degeneration, corneal degeneration and / or choroidal disorders. In some modalities, the disease is an inherited retinal degeneration (IRD). In some modalities, the disease is retinitis pigmentosa (RP). In some modalities, the disease is Bietti's Crystalline Dystrophy (also known as Bietti's Corneoretinal Crystalline Dystrophy; BCD). In some modalities, the disease is related to neurological degeneration. In some embodiments, the disease is corneal dystrophy. In some modalities, the individual has BCD or is at risk of developing BCD.
[92] [92] In some embodiments, the cells are fibroblasts, blood cells or eye cells. In some modalities, cells are obtained from urine or hair or hair follicles. In some modalities, eye cells are cells of the pigment epithelium
[93] [93] In some modalities, the genetic or epigenetic alteration is selected from the group consisting of mutation, an insertion, a single nucleotide polymorphism, improper methylation, improper demethylation and combinations thereof. In some embodiments, the genetic or epigenetic alteration is a mutation. In some modes, the genetic or epigenetic change in the individual's iPS eye cells has been genetically repaired using genetic editing. In some modalities, the genetic editing method uses a zinc-finger nuclear, TALEN technology or CRISPR technology. In some embodiments, the genetic or epigenetic change in the individual's iPSC-ocular cells has been genetically repaired using gene transfer. In some embodiments, the gene transfer method uses a recombinant AAV vector or another viral or non-viral vector to deliver a healthy copy of the target gene (for example, cDNA) to the cells to be transplanted.
[94] [94] In some modalities, the administration stage occurs before the onset of symptoms of the disease or after the onset of symptoms of the disease. In some embodiments, administration is to the eye or another organ or tissue comprising neurons. In some modalities, administration is by injection. In some modalities, administration is through sub-retinal or intravitreal injection. In some modalities, administration is through direct retinal injection. In some modalities, administration is by injection into the cornea. In some modalities, the administration is through any other method that efficiently delivers the cells to the sub-retinal site, the posterior segment or the cornea of the individual's eye.
[95] [95] In some modalities, the method also includes, before administration or transplantation, genotypic analysis of cells to identify the presence or absence of the genetic or epigenetic alteration in one or more genes shown in Table 4. In some but modalities, the genetic or epigenetic alteration is a mutation. In some embodiments, the mutation is in the CYP4V2 nucleic acid molecule. In some embodiments, the method also includes, prior to administration, an assessment of the individual's eye to identify the area (s) and extent of damaged or retained photoreceptor cells, retinal cells or corneal cells.
[96] [96] In some modalities, the method also includes, following administration, monitoring of the individual. In some modalities, monitoring includes performing a non-invasive retinal image, corneal tests, perimetry, ERG, OCT, visual acuity tests and / or functional studies. In some modalities, monitoring includes assessing the individual for an immune response. In some modalities, the method also includes, following administration, evaluation of the individual's eye to identify the area (s) and extent of damaged, retained or retained cells, retinal cells or corneal cells. Cell Therapy Claims - RNP RNP Claims
[97] [97] In another aspect, a composition is provided that includes: (a) a CRISPR guide RNA targeting a nucleic acid sequence (the “target sequence”) from or within 100 bps to a target gene (the “target gene”) and (b) a protein associated with functional CRISPR, in a ribonucleoprotein (RNP) or protein-RNA complex.
[98] [98] In some embodiments, the composition further includes (c) a donor nucleic acid sequence including all or a portion of a wild-type sequence or a functional sequence of the target gene for correction or replacement of such target gene or a portion of it. In some modalities, the target gene is involved in eye development or function and / or mutation, which causes or is a risk factor for causing an eye disease. In some modalities, the target gene is involved in neuronal development or function and / or mutation which causes or is a risk factor for causing a neurodegenerative disease.
[99] [99] In some embodiments, the target gene is a cytochrome P450 gene. In some embodiments, the target gene includes a gene shown in Table 4 that is mutated or defective or encodes a protein having a defective or partial function or activity. In some embodiments, the donor nucleic acid sequence is provided in a single-stranded donor oligonucleotide (ssODN) or a vector.
[100] [100] In some embodiments, (a) the CRISPR guide RNA including (i) a CRISPR RNA (crRNA) that includes a protospace element sequence that is complementary to the target sequence from or within 100 bps for a target gene and a sequence that corresponds to a complementary region of the transactivating crRNA (tracrRNA) and (ii) a tracrRNA that includes a region that is complementary to the corresponding region of the crRNA and a sequence that interacts with a protein associated with CRISPR 9 (Cas9) and (b) the protein associated with functional CRISPR comprises Cas9.
[101] [101] In some embodiments, the protospace element is about 20 bases, about 19 bases, about 21 bases, about 19-21 bases, about 18-22 bases, or about 16-24 bases. In some embodiments, the crRNA and tracrRNA are in different nucleic acid molecules. In some embodiments, crRNA and tracrRNA are combined into a single guide RNA (sgRNA). In some embodiments, the sgRNA is about 88-150 bp.
[102] [102] In some embodiments, Cas9 comprises a Cas9 orthologist or a mutant Cas9 selected from: Streptococcus pyogenes (SpCas9), SpCas9 nickase (Cas9n D10A), SpCas9 (D1135E), eS- pCas9, SpCas9-HF1, SpCas9 VRER, SpCas9 VQR, SpCas9EQR, Staphylococcus aureus (SaCas9), Neisseria Meningitidis, Streptococcus thermophilus, Streptococcus pneumnoniae, Campylobacter coli, Campylobacter jejuni, Streptococcus mutans, bacteriform, bacteriellae enterococcus faecalis.
[103] [103] In some embodiments, (a) the CRISPR guide RNA comprises a crRNA that comprises a sequence of protospacer element that is complementary to the target sequence of or within 100 bp for a target gene and (b) the protein associated with functional CRISPR comprises Cpf1. In some embodiments, the proto-spacer element is about 20 bases, about 21 bases, about 22 bases, about 23 bases, about 24 bases, about 19-25 bases, about 18-26 bases or about 16-28 bases. In some modalities, the CRISPR-associated protein, Cas9, or Cpf1, further comprises one, two, three or more nuclear localization sequences (NLS) at the N-terminus and / or C-terminus and / or a selection marker, in - including, without limitation, GFP or EGFP.
[104] [104] In some embodiments, the protospacer element is 100% complementary to the target sequence or contains 1, 2, 3, 4 or 5 nucleotide mismatches corresponding to the target sequence. In some embodiments, the crRNA sequence further comprises a G nucleotide optionally added to the crRNA sequence immediately before the protospace element. In some ways, the CRISPR guide RNA, crRNA and / or tracrRNA, or sgRNA, is chemically modified.
[105] [105] In some embodiments, the donor nucleic acid sequence is no more than about 1kb, 800bp, 600bp, 500bp, 400bp, 300bp, 280bp, 260bp, 240bp, 220bp or 200bp for a donor nucleic acid sequence provided in an ssODN and no more than about 30kb, 25kb, 20kb, 15kb, 10kb, 9kb, 8kb, 7kb, 6kb, 5kb, 4,5kb, 4kb, 3,5kb, 3kb, 2,5kb, 2kb, 1 , 5kb, 1kb, 0.5kb, 0.2kb or 0.1kb for a donor nucleic acid sequence provided in a vector. In some embodiments, the wild-type version of the target gene encodes an enzyme.
[106] [106] In some embodiments, the target gene includes a CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXC1, FOXC1, FOXC1, FOXC1 MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, MT-ND4, MT-ND4, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F11, CYP4F11, , CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, OFPA1, NRX, NDFPA , PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA , SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP4Zuta or CYP4 muta or CYP46 muta or defective that encodes a protein having defective or partial function or activity.
[107] [107] In some modalities, any one or more components of the same including the CRISPR guide RNA, protein associated with
[108] [108] In another aspect, a method of treating an individual's disease caused by a mutated or defective gene or a gene encoding a protein having defective or partial function or activity is provided. Such a method includes disruption, correction or replacement of such a gene by administering to the individual any of the compositions described herein.
[109] [109] In another aspect, a method of treating an eye disease or improving an individual's related risk factor caused by a mutated or defective gene or a gene encoding a protein having defective function or activity is provided. partial or partial. Such a method includes disruption, correction or replacement of such a gene by administering to the individual any of the compositions described here.
[110] [110] In another aspect, a method of treating a neurodegenerative disease or improving an individual's related risk factor caused by a mutated or defective gene or a gene encoding a protein having defective function or activity is provided. partial. Such a method includes disruption, correction or replacement of such a gene by administering to the individual any of the compositions described here.
[111] [111] In another aspect, a method of treating a disease or improving an individual's related risk factor caused by a mutated or defective cytochrome P450 gene or a cytochrome P450 gene encoding a protein is provided defective or partial function or activity. Such a method includes disruption, correction or substitution of such a gene by administering to the individual any of the compositions described here.
[112] [112] In some embodiments, the mutated or defective gene, or gene encoding a protein having defective or partial function, activity, disrupted, corrected or replaced is a mutated or defective version of a gene shown in Table 4 or a version of a gene shown in Table 4 that encodes a protein having defective or partial function or activity. In some modalities, the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is present in fibroblasts, blood cells, RPE, photoreceptors, retinal, corneal, choroidal, ocular, optic nerve , neuron or stem cells or any type of cells derived from a stem cell.
[113] [113] In some embodiments, the present composition is administered to fibroblasts, blood cells, RPE, photoreceptors, retinal, corneal, choroidal, ocular, optic nerve, neuron or stem cells or any type of cells derived from a stem cell. In some modalities, administration is carried out through electroporation or through lipid-mediated transfection or nucleofection or viral transduction or injection or a combination thereof. In some embodiments, any one or more components thereof including the CRISPR guide RNA, CRISPR-associated protein and / or the donor nucleic acid sequence are administered to the individual or cells via an administration system. selected from the group consisting of a ribonucleoprotein or protein-RNA complex, a nanoparticle, a liposome, a micelle, a virosome, a nucleic acid complex and / or a combination thereof.
[114] [114] In some modalities, treatment is performed for an individual in vivo. In some modalities, treatment is performed
[115] [115] In some embodiments, the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is replaced. In some embodiments, the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, has one or more mutations corrected or replaced. In some embodiments, the mutated or defective gene, or gene encoding a protein having a defective or partial function or activity, is disrupted.
[116] [116] In some embodiments, the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, has 1-20, 21-40, 41-60, 61-80, 81-100, 101-1000 , 1001- 10,000 base pairs of nucleotides or mutations that have been broken, corrected or replaced. In some embodiments, a region of the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is disrupted, corrected, or replaced. In some embodiments, a region of less than about 10, 8, 6, 4, 2 or 1 kb of the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is disrupted, corrected, or replaced.
[117] [117] In some embodiments, the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is disrupted, corrected or replaced by nucleotide insertion and / or deletion. In some embodiments, the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is disrupted, corrected, or replaced in an allele or both. In some embodiments, two or more CRISPR guide RNAs, CRISPR-associated proteins and / or different donor nucleic acid sequences are used to disrupt, correct or replace one or more mutations or defects in the mutated or defective gene, or gene encoding a protein having defective or partial function.
[118] [118] In some embodiments, the individual is a mammal. In some modalities, the individual is a human. In some modalities, the method improves ocular development or function or prevents ocular, retinal or corneal degeneration. In some modalities, the method improves neurological development or function or prevents neural degeneration. In some modalities, the method improves expression or function of a P450 enzyme.
[119] [119] In some embodiments, a repair directed to homology based on the donor nucleic acid sequence resulted in an intron and / or an exon of the target gene. In some embodiments, a repair directed to homology based on the donor nucleic acid sequence resulted in a target acceptor of the target gene. Such a method may further include (c) a donor nucleic acid sequence comprising all or a portion of a target gene shown in Table 4 with a mutation or alteration to generate a mutated or altered target gene or a portion thereof .
[120] [120] In some ways, a method of generating a cell disease model of a disease caused by a mutated or defective gene, or a gene encoding a protein having a defective or partial function or activity, by generating a mutation in such a gene it is provided. Such a method includes administering to the cells a healthy version of such a gene using any of the compositions described here. In some modalities, administration is performed through electroporation or through lipid-mediated transfection or nucleofection or viral transduction or microinjection or combination thereof. In some embodiments, the cells are fibroblasts, blood cells, RPE cells, photoreceptors, retinal, coronal, ocular, optic nerve, neuron or stem cells or any type of cells derived from a stem cell.
[121] [121] In yet another aspect, a composition is provided that includes a cell with a mutated or defective gene shown in Table 4.
[122] [122] In another aspect, a composition is provided that includes a cell with a CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOX, CISD2, FOX , FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RP, MER , MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F12, CYP4F12, CYP4F11, CYP4F8, mutated or defective comprising a composition of any of these claims.
[123] [123] In some embodiments, the vector is an AAV vector. In some embodiments, the protospace element sequence is selected from the group consisting of SEQ ID NOs: 48 to 52 or shares at least 80% sequence identity with one of SEQ ID NOs: 48 to 52 for use with a Cas protein that has NGG as an adjacent protospacer motif (PAM) to target the c.802-8_810del17insGC mutation of the CYP4V2 gene. In some modalities,
[124] [124] In one aspect, a nucleic acid molecule including the nucleic acid sequence of SEQ ID NO: 2 encoding a human CYP4V2 protein or a nucleic acid sequence sharing at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 2 is provided.
[125] [125] In another aspect, an expression cassette including a nucleic acid molecule as described herein and one or more regulatory sequence operably linked to the nucleic acid sequence is provided. In yet another aspect, a vector including a nucleic acid molecule as described here or an expression cassette as described here is provided.
[126] [126] In some embodiments, the vector is a viral vector. In some modalities, the viral vector is selected from the group consisting of a recombinant adenovirus vector, a recombinant lentivirus vector, a recombinant herpes simplex virus vector, a recombinant Sendai virus vector and a recombinant retrovirus vector. In some embodiments, the vector is a recombinant adenoassociated virus (rAAV) vector or a plasmid. In some embodiments, the vector is a plasmid or a non-viral vector. In some modalities, the non-viral vector is selected from the group consisting of naked nucleic acids, liposomes, dendrimers and nanoparticles.
[127] [127] In some embodiments, a host cell including any of the nucleic acid molecules described here and / or any of the compositions described here. In some modalities, the host cell is a bacterial cell, an E. coli cell, a plant cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a HEK293, HeLa, Vero, V27, A549, K562, B50, WI38, Hep G2 or BHK cell.
[128] [128] In another aspect, the use of any of the nucleic acid molecule described here, any of the expression cassettes described here, or any of the vectors described here, to express the product encoded by that molecule nucleic acid, in a bacterial cell, or an insect cell, a plant cell, a mammalian cell, an RPE cell, a photoreceptor or photoreceptor progenitor (PRC), a retinal cell, a corneal cell, a eye cell, neuron, neuronal cell, blood cell, epithelial cell, somatic cell, iPS cell, ES cell, MSC, adult stem cell, stem cell or any cell derived from a stem cell. EFS and / or SPA related claims
[129] [129] In another aspect, a self-complementing adeno-associated virus (scAAV) vector including a short 1α elongation factor (EFS) promoter and / or a small polyadenylation (polyA) signal (SPA) operably linked to a molecule of nucleic acid encoding a polypeptide, an interfering RNA molecule or an oligonucleotide is provided. In some embodiments, the EFS promoter consists of a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 35 and the SPA consists of a nucleic acid sequence having at least 80% SEQ sequence identity ID NO: 36.
[130] [130] In some embodiments, the scAAV vector is administered to a cell so that the product encoded by the nucleic acid molecule is expressed in the cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a retinal cell, a corneal cell, a choroidal cell, an eye cell, a brain cell, a neuron, a neuronal cell, an iPS cell, an ES cell, an MSC, a stem cell or any cell derived from a stem cell.
[131] [131] In one aspect, a method is provided to reduce immune responses to viral vectors and preserve transduction efficiency in gene therapy and / or maximize therapeutic effect for patients different from the same genetic disease. Such a method includes (a) establishing a group of more than one recombinant viral vector (eg, rA-AVs) with sufficient transduction efficiency in the type of target cell for gene therapy. The viral vector group can be expanded by creating variants with mutations in the antigenic region or other mutations or variants in the capsids of said viral vectors and such mutations or variants confirmed with sufficient transduction efficiency in the target cells relevant to the disease (for example, in iPS-RPE cell lines for gene therapy with CYP4V2 for BCD); (b) detecting pre-existing neutralizing antiviral vector antibodies (NAbs) against different viral vector serotypes and / or mutations or capsid variants in the individual in need of gene therapy, and / or testing and comparing viral vectors different in specific patient cells (for example, iPS-RPE cells) derived from such an individual; (c) select a viral vector from the group of viral vectors with sufficient transduction efficiency with the lowest cross-reactivity with the pre-existing NAbs in the individual and / or a viral vector with the best phenotype rescue result in specific cells of individual patient, such a viral vector group comprising different capsid-modified serotypes and viral vectors (for example, including, without limitation, mutant capsid AAVs and / or capsid protein variant AAVs); (d) use of the viral vector selected from (c) for administration to the individual; and (e) repetition of (b) to (d) (only the part related to pre-existing NAbs) before each time the individual requires an administration of genetic therapy, including, without limitation, an administration of follow-up to the same eye or an administration to the contralateral eye, or to another organ.
[132] [132] In another aspect, a composition is provided for treating or preventing a disease in an individual, including an effective amount of a vector and a pharmaceutically acceptable carrier. Typically, the vector includes a nucleic acid molecule or a non-pathogenic variant thereof encoding a non-mutant or functional CYP4V2 protein operably linked to a regulatory sequence.
[133] [133] In some modalities, the disease is Bietti's Crystalline Dystrophy (also known as Crystalline Cornororetinal Dystrophy; BCD). In some modalities, the disease is associated with a genetic or epigenetic change in the individual. In some modalities, the disease is photoreceptor degeneration, retinal pigment epithelium cell degeneration, retinal degeneration, corneal degeneration or choroidal degeneration. In some modalities, retinal degeneration is retinitis pigmentosa (RP). In some modalities, retinal degeneration is an inherited retinal degeneration (IRD). In some modalities, the disease is BCD. In some embodiments, the disease is corneal dystrophy. In some modalities, the individual has BCD or is at risk of developing BCD.
[134] [134] In one aspect, a vector including a nucleic acid molecule or a non-pathogenic variant thereof is provided encoding a non-mutant or functional CYP4V2 protein operably linked to a regulatory sequence.
[135] [135] In some embodiments, the vector is a viral vector. And bad-
[136] [136] In some embodiments, the AAV genome or AAV capsid protein is from either AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or one other naturally derived serotype or isolate or Glade from AAV or any derivative or hybrid thereof. In some embodiments, rAAV is a pseudo-typified AAV (for example, AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 4, AAV2 / 6, AAV2 / 7, AAV2 / 12, AAV2 / 10 and AAV2 / 9) . In some modalities, the rAAV is a hybrid AAV (for example, AAV-DJ, AAV-DJ / 8 or AAV-DJ / 9). In some modalities, rAAV is developed through directed evolution and / or rational design (for example, AAV 7m8 or AAV-PHP.B).
[137] [137] In some embodiments, rAAV comprises one or more mutations of the capsid (for example, YF, KR, TA, S-A and / or TV mutations (for example, AAV2 with one or more capsid mutations within Y444F , Y500F, Y730F, Y252F, Y272F, Y700F, Y704F and T491V, or the corresponding mutation for a different AAV serotype (for example, AAV2 / 8 (Y733F), AAV2 (Y444F + Y500F + Y730F) and AAV2 (quadY -F + TV))). In some modalities, the rAAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Anc80, rh10 and In some modalities, the rAAV vector is selected from the group consisting of AAV2 / 5, AAV2 / 8, AAV2 / 8 (Y733F), AAV2 (Y444F + Y500F + Y730F), AAV2 / 1, AAV2 / 4, AAV2 / 9 , AAV2 / 6, AAV2 / 7, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, Anc80, AAV 7m8, AAV-DJ, ShH10, AAV-PHP.B or a hybrid, a- rived or variant thereof.
[138] [138] In some embodiments, the rAAV vector is either a single-strand AAV vector or a self-supplementing AAV vector (scA-AV). In some embodiments, the vector is a plasmid or a non-viral vector (for example, naked nucleic acids, liposomes, dendrimers and nanoparticles).
[139] [139] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises: (i) the human CYP4V2 protein (SEQ ID NO: 4); (ii) a variant of (for example, amino acid change and / or coupling variant) human CYP4V2 protein or a functional CYP4V2 protein (for example, SEQ ID NO: 5); (iii) one or more fragments of a functional CYP4V2 protein (for example, SEQ ID NO: 6); (iv) all or part of sequences from one or more of the CYP4V2 orthologist from other species; (v) all or part of sequences of one or more other P450 proteins, including, but not limited to, other CYP4 and CYP46A1 proteins, (vi) a polypeptide that can improve, treat or stop one or more biochemical abnormalities in one or more of the genes listed in Table 4 in a patient cell (for example, a BCD patient's iPS-RPE cell) and / or (vii) a combination of the above.
[140] [140] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises all or part of the amino acid sequence shown in SEQ ID NO: 4, 5 or 6. In some embodiments, the non-mutant or CYP4V2 protein functional code encoded by the nucleic acid sequence comprises all
[141] [141] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises a polypeptide having at least 80% amino acid sequence identity (for example, at least 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) with any of the selected sequences from the group consisting of SEQ ID NOs: 4-29. In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises sequence elements from FxxGxxxCxG and ExxR (SEQ ID NOs: 30 and 31). In some embodiments, the non-mutant or functional CYP4V2 protein is a compound or agent that can improve, treat or stop one or more biochemical abnormalities in one or more of the genes listed in Table 4 in a patient's cell (for example, the iPS cell - RPE of a patient with BCD).
[142] [142] In some embodiments, the nucleic acid molecule encodes a non-mutant or functional CYP4V2 protein of any of claims 43-50. In some embodiments, the nucleic acid molecule encodes a non-mutant or functional CYP4V2 protein
[143] [143] In some embodiments, the regulatory sequence comprises a promoter. In some embodiments, the promoter is a RPE cell specific promoter, a rectal cell specific promoter, a corneal cell specific promoter, an ocular cell specific promoter or a constitutive promoter. In some embodiments, the promoter is a mammalian beta-actin promoter or a viral promoter.
[144] [144] In some embodiments, the promoter is selected from the group consisting of a CAG promoter (hybrid chicken early CMV / beta-actin enhancer promoter, also known as CAGGS promoter, CB promoter or CBA promoter), a beta promoter - chicken actin, a small CBA promoter (smCBA), a CBSB promoter, or a CBh promoter, another beta-actin promoter such as a human beta-actin promoter, a short elongation factor 1 alpha (EFS) promoter , a short promoter 1 elongation factor alpha
[145] [145] In some embodiments, the promoter is a CAG promoter (hybrid chicken early CMV / beta-actin enhancer promoter, also known as CAGGS promoter, CB promoter or CBA promoter), a short 1-elongation factor (EFS) promoter , a short elongation factor 1 alpha promoter (EF-1 alpha) or a CMV promoter or a derivative or hybrid thereof. In some embodiments, the regulatory sequence comprises an enhancer.
[146] [146] In some embodiments, the enhancer is a viral enhancer, including, without limitation, a WPRE enhancer, an HPRE enhancer, a CTE enhancer or a derivative or hybrid thereof. In some embodiments, the regulatory sequence comprises a polyadenylation signal (polyA). In some embodiments, the polyA signal is a bovine growth hormone polyadenylation signal (polyA bGH), a small polyA signal (SPA), a human growth hormone polyadenylation signal (polyA hGH), a SVA polyA signal , a late SV40 polyA signal or a derivative or hybrid thereof. In some embodiments, the regulatory sequence comprises a Kozak sequence (SEQ ID NO: 37 or 38).
[147] [147] In some embodiments, the composition is formulated with a carrier and additional components suitable for the specific route of administration.
[148] [148] In another aspect, a host cell including any of the vectors described here is provided.
[149] [149] In another aspect, a method of treating or preventing an eye disease in an individual is provided, the method including administering a vector to the individual, in which the vector comprises
[150] [150] In one aspect, a method of preventing, stopping or slowing the speed of progression of or improving the dysfunction, dystrophy, disorder, degeneration and / or death of an eye cell is provided, the method including administering a vector to the ocular cell, where the vector comprises a nucleic acid molecule or a non-pathogenic variant thereof encoding a human CYP4V2 protein or a functional CYP4V2 protein operably linked to a regulatory sequence.
[151] [151] In some embodiments, the disease is Bietti's Crystalline Dystrophy (also known as Bietti's Corneoretinal Crystalline Dystrophy; Bietti's Crystalline Retinopathy; Bietti's Retinal Dystrophy; BCD). In some modalities, the individual is affected by other clinically defined ophthalmic conditions (for example, inherited retinal degeneration (IRD), retinitis pigmentosa (RP) or corneal dystrophy) caused by mutations in the CYP4V2 gene. In some modalities, eye disease is photoreceptor degeneration, retinal pigment epithelium cell degeneration, retinal degeneration, corneal dystrophy or BCD.
[152] [152] In some embodiments, the vector is a viral vector. In some modalities, the viral vector is selected from the group consisting of a recombinant adeno-associated virus (rAAV) vector, a recombinant adenovirus vector, a recombinant lentivirus vector, a recombinant herpes simplex virus vector, a Sendai virus vector recombinant and a recombinant retrovirus vector. In some embodiments, the viral vector is an rAAV vector. In some modalities, the rAAV vector comprises a VP1, VP2 or VP3 capsid protein from any serotype of AAV1, AAV2, AAV3, AAV4, AAV5,
[153] [153] In some embodiments, the ITR AAV 5 'of the rAAV vector is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or another sero naturally derived type or isolate or Glade of AAV or mutations, chimeras, variants or fusions thereof. In some modalities, the ITR AAV 3 'of the rAAV vector is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or another serotype naturally derivative or isolate or Glade of AAV or mutations, chimeras, variants or fusions thereof. In some embodiments, the rAAV is a chimeric AAV, a scrambled AAV, or an AAV with modified capsid. In some embodiments, rAAV is a pseudo-typified AAV (for example, AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 4, AAV2 / 6, AAV2 / 7, AAV2 / 12, AAV2 / 10 and AAV2 / 9) . In some modalities, rAAV is a hybrid AAV (for example, AAV-DJ, AAV-DJ / 8 or AAV-DJ / 9). In some modalities, rAAV is developed through directed evolution and / or rational design (for example, AAV 7m8 or AAV-PHP.B).
[154] [154] In some embodiments, rAAV comprises one or more mutations of the capsid (for example, YF, KR, TA, S- A and / or TV mutations (for example, AAV2 with one or more capsid mutations within Y444F , Y500F, Y730F, Y252F, Y272F, Y700F, Y704F and T491V, or the corresponding mutation for a different AAV serotype (for example, AAV2 / 8 (Y733F), AAV2 (Y444F + Y500F + Y730F) eAAV2 (quadY- F + TV))). In some modalities, the rA-AV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Anc80, rh10 and ShH10. In some modalities, the rAAV vector is selected
[155] [155] In some embodiments, the rAAV vector is either a single-strand AAV vector or a self-complementing AAV vector (scA-AV). In some embodiments, the vector is either a plasmid vector or a non-viral vector. In some modalities, the non-viral vector is selected from the group consisting of naked nucleic acids, liposomes, dendrimers and nanoparticles.
[156] [156] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises: (i) the human CYP4V2 protein (SEQ ID NO: 4); (ii) a variant of (for example, amino acid change and / or coupling variant) of the human CYP4V2 protein or a functional CYP4V2 protein (for example, SEQ ID NO: 5); (iii) one or more fragments of a functional CVP4V2 protein (for example, SEQ ID NO: 6); (iv) all or part of sequences from one or more of the CYP4V2 orthologist from other species; (v) all or part of sequences of one or more other P450 proteins, including, but not limited to, other CYP4 and CYP46A1 proteins; (vi) a polypeptide that can improve, treat or stop one or more biochemical abnormalities in one or more genes listed in Table 4 in a patient cell (for example, a BCD patient's iPS-RPE cell) and / or ( vii) a combination of the above.
[157] [157] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises all or part of the amino acid sequence shown in SEQ ID NO: 4, 5 or 6. In some embodiments, the non-mutant or CYP4V2 protein functional code encoded by the nucleic acid sequence comprises all
[158] [158] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises all or part of the amino acid sequence selected from the group consisting of CYP4V2 (or CYP4V2 orthologists) from chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, frog, horse, rabbit and fruit fly (SEQ ID NOs: 19-29) and derivatives, hybrids, variants and / or fragments thereof. In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises a polypeptide having at least 80% amino acid sequence identity (for example, at least 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity - quency) with any of the selected sequences from the group consisting of SEQ ID NOs: 4-29.
[159] [159] In some embodiments, the non-mutant or functional CYP4V2 protein encoded by the nucleic acid sequence comprises sequence elements of FxxGxxxCxG and ExxR (SEQ ID NOs: 30 and 31). In some embodiments, the non-mutant or functional CYP4V2 protein is a compound or agent that can improve, treat, or stop one or more biochemical abnormalities in one or more of the genes listed in Table 4 in a patient cell (for example, the iPS cell -RPE of a patient with BCD). In some embodiments, the nucleic acid molecule encodes a non-mutant or functional CYP4V2 protein of any one of claims 91-97. In some embodiments, the nucleic acid molecule encodes a protein
[160] [160] In some embodiments, the regulatory sequence comprises a promoter. In some embodiments, the promoter is a RPE cell-specific promoter, a rectal cell-specific promoter, a corneal cell-specific promoter or an ocular cell-specific promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a mammalian beta-actin promoter or a viral promoter.
[161] [161] In some embodiments, the promoter is selected from the group consisting of a CAG promoter (hybrid chicken early CMV / beta-actin enhancer promoter, also known as CAGGS promoter, CB promoter or CBA promoter), a promoter chicken beta-actin, a small CBA promoter (smCBA), a CBSB promoter, or a CBh promoter, another beta-actin promoter such as the human beta-actin promoter, a short elongation factor 1 alpha (EFS) promoter, a short elongation factor 1 alpha promoter (EF-1 alpha), a CMV promoter, a PGK promoter, a UBC promoter, a GUSB promoter, a UCOE promoter, a VMD2 promoter (vitelliform macular dystrophy 2; also known as BEST1), an RPE65 promoter or a hybrid or derivative thereof. In some modalities, the promoter is a CAG promoter (hybrid chicken early CMV / beta-actin enhancer promoter, also known as
[162] [162] In some embodiments, the regulatory sequence comprises an enhancer. In some embodiments, the enhancer is a viral enhancer, including, without limitation, a WPRE enhancer, an HPRE enhancer, a CTE enhancer or a derivative or hybrid thereof. In some embodiments, the regulatory sequence comprises a polyadenylation signal (polyA). In some embodiments, the polyA signal is a bovine growth hormone polyadenylation signal (polyA bGH), a small polyA signal (SPA), a SV40 polyA signal, a human growth hormone polyadenylation signal (polyA hGH), a late SV40 polyA signal or a derivative or hybrid thereof. In some embodiments, the regulatory sequence comprises a Kozak sequence (SEQ ID NO: 37 or 38).
[163] [163] In some embodiments, for in vitro treatment, the target cell is infected at a dose (MOI) of about 1 x 10 ^ 3 GC to about 1 x 10 ^ 6 GC per cell (GC: genomic copies, measurement of genomes containing AAV particles (tcc / vector genome (vg) or genome particles (gp)). In some modalities, for in vivo administration to an individual's eye, a single administration may be of the order from about 1 x 10 ^ 6 to 2 x 10 ^ 13 GC (for example, a high dose range of about 1 x 10 ^ 11 GC to about 1 x 10 ^ 12 GC, a dose range average of about 1 x 10 ^ 10 GC to about 1 x 10 ^ 11 GC, a low dose range of about 1 x 10 ^ 9 GC to about 1 x 10 ^ 10 GC, a very low dose range from about 1 x 10 ^ 6 GC to about 1 x 10 ^ 9 GC and a very high dose range of about 1 x 10 ^ 12 GC to about 2 x 10 ^ 13 GC) or any dose within those ranges that is sufficient to provide the desired effect.
[164] [164] In some modalities, the administration stage occurs before the onset of symptoms of the disease or after the onset of symptoms of the disease. In some modalities, administration is to the eye. In some modalities, administration is through sub-retinal injection. In some modalities, administration is through intravitreal injection. In some modalities, administration is through direct retinal injection. In some embodiments, administration is by any other method of administration that effectively delivers the vectors to the sub-retinal site, to the posterior segment of the eye, to the corneal cells or RPE, to the photoreceptor cells or epithelial cells of the individual's cornea .
[165] [165] In some embodiments, administration is through administration to the cornea. In some embodiments, administration to the eye is achieved through administration into the bloodstream. In some modalities, administration is through eye drops. In some modalities, administration is through lens administration. In some modalities, administration is in the sub-retinal space, to the cornea, the lens or the vitreous. In some embodiments, eye cells are selected from the group consisting of retinal pigment epithelial cells (RPE), photoreceptor cells (PRCs), corneal epithelial cells (SCCs), choroidal endothelial cells (SC), retinal cells, corneal cells, lens cells, ganglion cells, optic nerve cells and / or choroidal cells, as well as said cell types derived from a stem cell (including, without limitation, an iPSC, an ES cell, an MSC, an adult stem cell and / or a tissue-specific stem cell).
[166] [166] In some modalities, the methods described here may also include identification of an individual having BCD or at risk of developing BCD. Use of EFS and / or SPA in an rAAV vector comprising a sequence
[167] [167] In one aspect, a composition including a recombinant adenoassociated virus (rAAV) vector comprising a short promoter and 1α elongation factor (EFS) and / or a small polyadenylation (polyA) signal (SPA) linked operably to a nucleic acid molecule encoding a CRISPR-associated protein (Cas) is provided.
[168] [168] In some embodiments, the EFS promoter consists of a nucleic acid sequence having at least 80% sequence identity of SEQ ID NO: 35 and the SPA consists of a nucleic acid sequence having at least 80% sequence identity sequence of SEQ ID NO: 36. In some embodiments, the Cas encoded by the nucleic acid sequence operably linked to the EFS promoter and / or the SPA is a Cas9 or Cpf1.
[169] [169] Host cells including an rAAV as described here are provided. In some embodiments, the host cell is a bacterial cell, an E. coli cell, a plant cell, an insect cell or a mammalian cell. In some embodiments, the cell is a somatic cell or a stem cell. In some modalities, the host cell is a retinal cell, a corneal cell, a choroidal cell, an eye cell, a brain cell, a neuron, a neuronal cell, an iPS cell, an ES cell, an MSC , an adult stem cell, a specific tissue cell, a stem cell, or any cell derived from a stem cell. In some embodiments, the rAAV vector is administered to a host cell so that the Cas encoded by the nucleic acid molecule is expressed in the cell. In some embodiments, the host cell comprising any cell of any one of claims 131 to 134.
[170] [170] Other features and advantages of the invention will be apparent from the Detailed Description, Description of the Drawings and Examples and also from the claims. All publications, patent applications, patents, sequences, database records and other references mentioned here are incorporated by reference in their entirety. DESCRIPTION OF DRAWINGS
[171] [171] The invention is further illustrated in the figures and drawings that follow, which do not limit the scope of the invention described in the claims. Cell Line Patent:
[172] [172] Figure 1: iPS cell lines derived from patients with BCD (a) iPS cells generated from fibroblasts from skin biopsy samples from patients with BCD: (i) iPS cells from patient 1 (P1) (ii) iPS cells from patient 2 (P2) (iii) characterization of P1 and P2 iPS cell lines by Oct-4, Sox-2 and SSEA-4 markers (iv) characterization of P1 and P2 iPS cell lines by Nanog and Tra-1- markers 60 (b) iPS cells generated from a patient with BCD and healthy control from peripheral blood mononuclear cells (PBMC) from blood samples: (i) phase contrast images from iPS cell lines (ii ) AP staining results from iPS cell lines (c) iPS cell karyotype images derived from a BCD patient showing apparently normal human karyotype.
[173] [173] Figure 2: iPS-RPE cell lines derived from patients with BCD:
[174] [174] Figure 3: QRT-PCR results of CYP4V2 expression in iPS-RPE samples. (WT (controls). WT AVE (average of controls). P1 (Patient 1 with BCD). P1-AAV8 (sample of P1 treated by AAV8.CYP4V2fv, MOI = 1.5x10e4 GC / cell).
[175] [175] Figure 4: QRT-PCR results of CYP4V2op expression in iPS-RPE samples. WT (controls) WT AVE (average of controls). P1 and P2 (Patient 1 and Patient 2 with BCD). P1-AAV2 (sample of P1 treated by AAV2.CYP4V2op in 2x10e4 GC / cell MOI). P2-AAV2 (sample of P2 treated by AAV2.CYP4V2op in 2x10e4 GC / cell MOI)). P2-scAAV1 (sample of P2 treated by scAAV1.CYP4V2op in 2x10e4 GC / cell MOI).
[176] [176] Figure 5: images of cell viability of iPS-RPE samples without exposure to blue light. WT (controls). P1 and P2 (Patient 1 and Patient 2 with BCD). Red (dead / diseased cells); Green (living / healthy cells). Figure 5 (a): Red only. Figure 5 (b): Red and green.
[177] [177] Figure 6: Cell viability images of iPS-RPE samples after 1 hour of exposure to blue light. WT (controls). P1 and P2 (patient 1 and patient 2 with BCD). Red (dead / diseased cells); Green (living / healthy cells). Figure 6 (a): Red only. Figure 6 (b): Red and green. Gene therapy:
[178] [178] Figure 7: Scheme and annotations for exemplary CYP4V2 expression cassettes and recombinant AAV vectors (rAAV) (a) CYP4V2 expression cassette (with an enhancer) packaged in single-strand AAV vectors (ssAAV) ) (b) CYP4V2 expression cassette (without an enhancer) packaged in single-strand AAV vectors (ssAAV) (c) CYP4V2 expression cassette packaged in self-complementary AAV vectors (scAAV) or ssAAV.
[179] [179] Notes: A CYP4V2 expression cassette (as shown flanked by AAV ITRs) can be packaged in an rAAV vector with capsid of any AAV serotype or a hybrid or variant thereof. ITRs: inverted terminal repetitions (can be AAV2 ITRs or ITRs from other AAV serotypes). Sequences of exemplary AAV2 ITRs shown in SEQ ID NOs: 42 and
[180] [180] Figure 8: Cell viability images of iPS-RPE samples derived from a patient with BCD after 1 hour of exposure to blue light (without treatment with AAV.CYP4V2 vs. treated by AAV2.CYP4V2op or scAAV1.CYP4V2op in MOI of 1x10e5 GC / cell). P1 and P2 (Patient 1 and Patient 2 with BCD). Red (dead / diseased cells); Green (living / healthy cells). Figure 8 (a): Red only. Figure 8 (b): Red and green.
[181] [181] Figure 9: Cell viability images of iPS-RPE samples derived from a patient with BCD after 1 hour of exposure to blue light (without treatment with AAV.CYP4V2 vs. treated with AAV5.CYP4V2op, AAV5.CYP4V2st or AAV8. CYP4V2fv in MOI 1x10e5 GC / cell). P1 (Patient 1 with BCD). Red (dead / diseased cells); Green (living / healthy cells). Figure 9 (a): Red only; Figure 9 (b): Red and green.
[182] [182] Figure 10: Cell viability images of iPS-RBE samples derived from a patient with BCD after 1 hour of exposure to blue light (without treatment with AAV.CYP4V2 vs. treated with AAV5.CYP4V2op, scAAV1.CYP4V2op or scAAV5. CYP4V2op in MOI of 1x10e4 GC / cell). P2 (Patient 2 with BCD). Red (dead / diseased cells); Green (living / healthy cells). Figure 10 (a): Only red. Figure 10 (b): Red and green.
[183] [183] Figure 11: cell viability images of iPS-RPE samples derived from a patient with BCD after 1 hour of exposure to blue light (without treatment with AAV.CYP4V2 vs. treated with scA- AV9.CYP4V3op in 1x10e5 GC MOI /cell). P1 (Patient 1 with BCD). Red (dead / diseased cells); Green (living / healthy cells). Figure 11 (a): Red only. Figure 11 (b): Red and green. Cell Therapy:
[184] [184] Figure 12 shows a region of the CYP4V2 sequence and the position of the guide RNAs (gRNAs) designed with respect to the c.802-8_810del17insGC mutation and primers (orange arrows) for assay of gRNA activity.
[185] [185] Figure 13 shows an in vitro surveying assay. Tracks 1: amplicon + Cas9; 2: amplicon + g1 + Cas9; 3: amplicon + g2 + Cas9; 4: amplicon + g3 + Cas9; 5: amplicon + g4 + Cas9: 6: amplicon + g5 + Cas9; 7: amplicon only; M: 1 kb DNA marker.
[186] [186] Figure 14 is a sequence comparison confirming the origin of the DNA used in the surveying assay. Upper: untreated amplicon; Medium: amplicon fragment treated with g2; Lower: CYP4V2 loci indicating mutation site.
[187] [187] Figure 15 is a vector construction illustration of gRNA.
[188] [188] Figure 16 is a vector map of gRNA (using g1 as an example), Cas9 coexpression plasmid and PuroR pX459-hSpCas9-2A-Puro.
[189] [189] Figure 17 shows the position of the gRNA (using g1 as an example) relative to the U6 promoter in plasmid pX459-hSpCas9-2A-Puro. The “G” nucleotide between the U6 promoter and the gRNA is to increase the transcription efficiency directed by the U6 promoter. It is optional and not necessary when a different promoter is used or when the gRNA starts with a "G" nucleotide. DEFINITIONS
[190] [190] It should be understood that as used in the descriptive report and claims, “one” or “one” can mean one or more, depending on the context in which it is used. In this way, for example, reference to “a cell” can mean “at least one cell” or “more than one cell”.
[191] [191] The term “about” or “approximately” or the symbol “~” refers to within a range of about 10% (inclusive) of a given value or state. Unless otherwise clear from the context, all numerical values provided here can be modified by the term about.
[192] [192] The term “AAV.CYP4V2” refers to a recombinant adeno-associated virus (AAV) vector comprising a polynucleotide encoding a functional CYP4V2 protein.
[193] [193] The term “CYP4V2 gene therapy” refers to the introduction of a functional CYP4V2 protein or a polynucleotide encoding a functional CYP4V2 protein in a cell and / or an individual. See detailed discussion in the description.
[194] [194] The term “effective amount” or “effective dosage” or “therapeutically effective dosage” refers to an amount of a compound (for example, a vector) and / or cells sufficient and / or suitable for perform treatment when administered to an individual in need of such treatment. The effective amount will vary depending on the specific activity of the therapeutic agent being used, the severity of the patient's disease state and age, physical condition, the existence of other disease states and the individual's nutritional status. Still, other medication and / or treatment that the patient may be receiving will affect the determination of the effective amount of the therapeutic agent to be administered. See description here for more detailed discussion.
[195] [195] The term "treatment" or "treating" refers to the administration of a composition as disclosed herein (for example, an AAV comprising a transgene and / or cells) to an individual for purposes including 1) preventing or protecting against the disease or condition, that is, preventing the clinical symptoms from developing; 2) inhibit the disease or condition, that is, stop, slow down, improve or suppress the development of clinical symptoms; 3) relieve the disease or condition, that is, cause the regression of clinical symptoms; and / or 4) replace and / or restore the loss of function of the diseased cells, tissue and / or organ. In some modalities, the term "treatment" or "treatment" refers to the alleviation of the disease or condition; that is, cause the regression of clinical symptoms. In some modalities, the term "treatment" or "treat" alternatively or additionally refers to the prophylactic treatment of an individual in need of it. Prophylactic treatment can be carried out by providing an appropriate dose of a therapeutic agent to an individual at risk of suffering a disease, thereby substantially preventing the onset of the disease. It will be understood by those skilled in the art that it is not always possible to distinguish between “prevent” and “suppress”, since final inductive events or events may be unknown or latent, or the patient may not be sure until well after the occurrence of the event or events. Thus, as used here, the term “prophylaxis” is intended as an element of “treatment” to understand both “prevent” and “suppress” as defined herein.
[196] [196] The term "individual" refers to an animal, such as a mammal, for example, a human. The methods described here can be useful in human therapeutic, preclinical and veterinary applications. In some modalities, the individual is a mammal and in some modalities, the individual is human.
[197] [197] A “variant” is a protein with sequence homology to a biologically active reference protein that retains at least a portion of the therapeutic and / or biological activity of the biologically active protein. For example, a variant protein can be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity compared to the reference biologically active protein. The term “biologically active protein” includes proteins deliberately modified, such as through site-directed mutagenesis, insertions or accidentally through mutations. A "variant" includes a "fragment", which is a truncated form of a native or non-native biologically active protein that retains at least a portion of the therapeutic and / or biological activity.
[198] [198] The term “nucleic acid” is used here to refer to all forms of nucleic acid, polynucleotides and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include DNA, cDNA and genomic RNA. Polynucleotides include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Polynucleotides can be single, double or triple, linear or circular, and can be of any length. A sequence or structure of a particular polynucleotide can be described here according to the sequence provision convention in the 5 'to 3' direction.
[199] [199] The term “sequence variant” means genes or polypeptides that have been modified compared to their native or original sequence by one or more nucleotide or amino acid insertions, deletions and / or substitutions. Insertions can be located at one or both ends of the gene or protein and / or they can be positioned
[200] [200] As used herein, the term "therapy" or "treatment" can be applied either in vivo to an individual or in vitro in a cell.
[201] [201] As used here, a plasmid is a type of vector.
[202] [202] As used herein, the term "genetically repaired" or "genetic repair" refers to a cell that originally carries a genetic defect (for example, a mutation or pathological change) in a gene, its genetic defect having been repaired either through gene correction or disruption in the cell's genomic DNA or mRNA (hereinafter referred to as “genetic editing”, “gene editing therapy” or “gene correction”) or through transfer or supplementation of gene from an exogenous nucleic acid molecule to the cell that expresses a functional protein that corresponds to the defective gene (hereinafter referred to as “gene transfer therapy” or “gene therapy”).
[203] [203] As used herein, the term “percent sequence identity“ or “sequence identity” should be determined and calculated as follows. In calculating the sequence identity (percentage), two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (that is, the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value (and rounded to the nearest next largest integer (for example, 65.01% should be rounded to 66% and considered 66% for the present purposes)). It will be understood that the length of the aligned region can be a portion of one or both of the sequences up to the entire net length size (without lacuna) of the shorter sequence.
[204] [204] The term "adeno-associated virus vector" refers to a nucleic acid derived from any AAV serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 serotype , AAV12, or any other virus or serotype that shares homologues in its capsid protein sequence with the capsid protein of an AAV serotype. The term “recombinant adeno-associated virus” or “rAAV” refers to an infectious, replicating defective virus composed of an AAV protein shell encapsulating a nucleic acid molecule of interest, which is flanked in one or both sides by AAV ITRs. As used herein, reference to a particular AAV serotype means an AAV having at least one capsid protein for this AAV serotype. For example, the term "AAV2" refers to an AAV having at least one AAV serotype 2 capsid protein.
[205] [205] The term “CYP4V2” refers to Cytochrome P450 4V2 or Cytochrome P450, family 4, subfamily V, polypeptide 2 (sometimes referred to as CYP4AH1) and its orthologists in other species. Mutations in CYP4V2 have been associated with BCD (see, for example, Li et al., Am J Hum Genet. 74: 817-826, 2004) and retinitis pigmentosa (see, for example, Wang et al., PLOS ONE 7 : e33673, 2012). The full-length genomic human CYP4V2 gene is about 22,053 bp in length and can be found at, for example, genecards.org/cgi-bin/carddisp.pl gene=CYP4V2&keywords=CYP4V2 on the World Wide Web. As used here , the term “hCYP4V2” refers to a human CYP4V2 gene or protein. It should be understood that hCYP4V2 and CYP4V2 can refer to a gene or protein that contains a genetic or epigenetic change or a gene or protein that does not contain a genetic or epigenetic change.
[206] [206] As used herein, the term "functional CYP4V2" refers to a protein or nucleotide molecule that, when expressed,
[207] [207] The term “eye cell” refers to any cell in, or associated with, eye function including, without limitation, a retinal cell, a bipolar retinal cell, a photoreceptor cell or a photoreceptor progenitor cell ( including rod and / or cone, together (“PRCs”), a ganglion cell, a retinal pigment epithelium cell (RPE), a choroidal epithelial cell (EC), a corneal epithelial cell (ECC) ), a choroidal cell or a corneal cell or an optic nerve cell.
[208] [208] The term “loss of function” or “dysfunction” refers to a decrease in, or loss of, cellular function (eg, photoreceptor function, photoreceptor cell function, pigment epithelium cell function of retina, lens function, choroid function or corneal function) compared to a non-diseased, normal, or comparable cell
[209] [209] The term "transgene" refers to a donor nucleic acid that is intended or has been introduced into a cell or organism. Transgenes include any gene, such as a gene or cDNA set shown in Table 4.
[210] [210] The terms “pharmaceutically acceptable formulation” and “physiologically acceptable formulation” and “pharmaceutically acceptable carrier” mean a biologically acceptable, fat, liquid or solid formulation, or mixtures thereof, which is suitable for a or more routes of administration, in vivo administration, in vitro administration or contact, and may include a formulation or carrier used in therapies for other diseases (for example, gene therapy or cell therapy for other eye diseases). A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, for example, the material can be administered to an individual without causing substantial undesirable biological effects. In this way, such a pharmaceutical composition can be used, for example, in administering a protein, a polynucleotide, a plasmid, a viral vector or a nanoparticle to a cell or an individual. Such compositions include, without limitation, solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (for example, oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension, coatings, isotonic agents and promotion or delay of absorption, compatible with pharmaceutical administration or contact or administration in vivo or in vitro. Aqueous and non-aqueous solvents, solutions and suspensions can include suspending agents, lubricating agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microspheres, powder, granules and crystals. Supplementary active compounds (for example, preservatives, antibacterial, antiviral and antifungal and immunosuppressive agents) can also be incorporated into the compositions. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or administration, as shown here or known to one skilled in the art. In this way, pharmaceutical compositions include carriers, diluents or excipients suitable for administration through various routes.
[211] [211] The term "crRNA" refers to CRISPR RNA, which contains both the protospacer element and additional nucleotides that are complementary to tracrRNA.
[212] [212] The term “tracrRNA” refers to the transactivation crRNA, which hybridizes to the crRNA and binds to a Cas9 protein activating the complex to create double strand breaks at specific sites within the genomic sequence.
[213] [213] The term “sgRNA” refers to a single guide RNA, which combines crRNA and tracrRNA, which are separate molecules in the native CRISPR / Cas9 system in S. pyogenes, in a single RNA constant .
[214] [214] The term “MAP” refers to an “adjacent protospace spacer” which is a short sequence in any strand of the genome known by CRISPR nucleases as a cutting site. PAM varies with the nuclease (eg Cas9, Cpfl, etc.). The sequence of the protospace element is generally directly upstream of the MAP site.
[215] [215] The term “protospace element” (also referred to as “guide RNA” or “CRISPR gRNA” or “gRNA” or g1, g2, g3, g4, g5, etc.) refers to the portion of the crRNA ( or sgRNA) that is complementary to the target genomic DNA sequence.
[216] [216] DHA: Docosahexaenoic acid, a polyunsaturated omega-3 fatty acid, also known as 22: 6 (ω-3) or C22: 6 n3.
[217] [217] AA: Araquidonic Acid, a polyunsaturated omega-6 fatty acid, also known as 20: 4 (ω-6) or C20: 4 n6 or ARA.
[218] [218] PBS (+): phosphate buffered saline (PBS) with Calcium and Magnesium.
[219] [219] PBS (-): phosphate buffered saline (PBS) without calcium or magnesium.
[220] [220] Developing an appropriate BCD disease model and determining the molecular level phenotype for BCD are critical for BCD-related research, drug development and testing, and treatment options for BCD. They are also important for the study of CYP4V2 function. As shown in the Background section here, the clinical phenotype of BCD was characterized, established and studied 80 years ago, the genetic mutations causing BCD have been identified for a decade. However, there is still a gap between the clinical phenotype (for example, crystal-like deposits on the retina of patients with BCD) and the underlying CYP4V2 mutations.
[221] [221] Previous studies on BCD have found abnormal fatty acid levels in patients with BCD, including fibroblasts, lymphocytes and serum. For example, in Lee et al., The Metabolism of Fatty Acids in Human Bietti Crystalline Dystrophy, Invest Ophthalmol Vis Sci. 2001 Jul; 42 (8): 1707-14, the researchers used a pulse method to study abnormalities in fibroblasts and lymphocytes from patients with BCD. Fibroblasts and lymphocytes from a patient with BCD and normal control were incubated with [(14) C] 18: 3n-3 or [(14) C] 18: 2n-6. Fibroblasts from patients with BCD showed less conversion of 18: 3n-3, but not 18: 2n-6, to polyunsaturated fatty acids (PU-FAs) than those of normal individuals. In another study (Lai et al., Alterations in Serum Fatty Acid Concentrations and Desaturase Activities in Bietti Crystalline Dystrophy Unaffected by CYP4V2 Genotypes, Invest Ophthalmol Vis Sci 2010; 51: 1092–7), the researchers used GC-MS to analyze serum fatty acid concentrations in serum samples from patients with BCD and control. The study found a higher concentration of octadecanoic acid (18: 0) in serum from patients with BCD than in control subjects, as well as a lower concentration of octadecadienoic acid (18: 1n-9) than in control subjects. In addition, the concentration of total monounsaturated fatty acid was significantly lower in BCD than in the control. In yet another study (Nakano et al., CYP4V2 in Bietti's Crystalline Dystrophy: Ocular Localization, Metabolism of omega-3- Polyunsaturated Fatty Acids, and Functional Deficit of the p.H331P Variant, Mol Pharmacol 82: 679–686, 2012 ) that did not use samples from patients with BCD as the object of study, the results suggested that the enzyme CYP4V2 has omega-hydroxylase activity in relation to omega-3-PUFAs.
[222] [222] It is important to confirm that the abnormal fatty acid levels in fibroblasts and serum from patients with BCD really exist in RPE cells from patients with BCD, which are the disease-causing cells for BCD. In this way, a model of BCD disease allowing direct investigation into RPE cells of patients with BCD is desired to gain more understanding of BCD disease pathology and CYP4V2 functions, as well as to evaluate the effectiveness of potential treatment options. However, given the location and rarity of BCD RPEs, it is not practical to obtain native RPE cells from patients with BCD.
[223] [223] The present description provides cell models and methods of BCD to generate cell models of BCD. Cellular models of BCD consist of BCD patient-specific stem cells (including, without limitation, induced pluripotent stem cells (iPSCs), embryonic stem cells (ES), somatic (or adult) stem cells, mesenchymal stem cells (MCS)) and ocular cells (including, without limitation, RPE cells, photoreceptor cells (rod or cone), photoreceptor progenitor cells, corneal epithelial cells, lens cells and / or choroid cells) derived from any or stem cell from a patient with BCD. In addition to patient-specific stem cells, a BCD cell model can also be generated by creating CYP4V2 mutations in cells of individuals without BCD and such cells can be ES cells, iPS cells or other stem, or any cells that can be reprogrammed into stem cells, or any eye cells (whether derived from a stem cell or not).
[224] [224] Induced pluripotent stem cell technology provides an alternative for disease modeling for animal models. However, not all diseases have been successfully modeled using iPSC. (Urbach, A., Bar-Nur, O., Daley, G. Q. & Benvenisty, N. Differential Modeling of Fragile X Syndrome by Human Embryonic Stem Cells and Induced Pluripotent Stem Cells. Cell Stem Cell 6, 407-411
[225] [225] Methods of producing induced pluripotent stem cells (iPSCs) are known in the art. Virtually all types of somatic cells can be used as the source cell for reprogramming iPSC. In short, iPSCs can be made by introducing a particular set of proteins (for example, nucleic acids encoding a particular set of proteins or by direct protein administration) into cells. It would be understood by the converse that an exemplary, non-limiting method is through the introduction of one or more transgenes encoding one or more of OCT4, SOX2, KLF4 and / or c-MYC (for example, the “Yamanaka Factors”). In some modalities, reprogramming uses all four transcription factors. In some embodiments, one, two or three transcription factors can be used. Li et al., Stem Cells, 2009; 27: 2992–
[226] [226] Various methods (for example, Sendai virus, episomal method, with or without small molecules) can be used to generate iPSCs, see Examples section, see also, for example, Hubbard et al., J Vis. Exp., 2014, 92: 52009. Still, production methods
[227] [227] Any type of stem cells can be used in the generation of the BCD cell model described here including, without limitation, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), stem cells embryonic (ES), mesenchymal stem cells, adult stem cells or tissue-specific stem cells. Stem cells for use in the methods described here can be pluripotent, multipotent or totipotent stem cells.
[228] [228] As used here, the term “pluripotent” refers to a cell capable of at least developing into one of ecto-dermal, endodermal and mesodermal cells. In one embodiment, the term “pluripotent” refers to cells that are totipotent and multipotent. As used herein, the term “totipotent” cell refers to a cell capable of growing in all cell lines. As used here, the term “multipotent” refers to a cell that is not terminally differentiated. The pluripotent cells of the present description can be any stem cells or produced from non-pluripotent cells, such as fibroblasts, using methods of induction, de-differentiation and nuclear transfer known in the art. The pluripotent cells described here, whether stem cells or produced from non-pluripotent cells, may be from an individual having BCD or mutations of CYP4V2 or from a healthy individual not having BCD for use as a control or for use to create artificial CYP4V2 mutations.
[229] [229] Virtually any cell type can be reprogrammed into iPS cells. See discussion in the subsection entitled “Cell Origination” here. B. Differentiation of iPSCs
[230] [230] BPS patient iPS cells were differentiated into iPS-RPE cells (or another type of eye cell (for example, iPS-CEC, iPS-CE or iPS-PRC cells). Methods for differentiating iPSC cells into RPE or another type of eye cell (for example, CEC and PRC) are known. See, for example, the section Examples, Hayashi et al., 2012, PLoS One, 7 (9): e45435; Songstad, et al., Investigative Ophthalmology & Visual Science December 2015, Vol.56, 8258-8267; and Lamba et al., PLoS One. 2010 Jan 20; 5 (1): e8763. For example, induced pluripotent stem cells (iPSCs) reprogrammed from cells can be produced to differentiate more in, for example, RPE cells (referred to here as “iPS-RPE”), corneal epithelial cells (referred to here as “iPS-CEC”), photoreceptor cells (or progenitor of photoreceptors ; referred to here as "iPS-PRC") or choroidal iPS-endothelial cells (CE) (referred to here as "iPS-CE").
[231] [231] Differentiated cells, for example, iPS-RPE cells, were tested for biological functions (as described here and in the Examples section) to assess their biochemical defects / abnormalities compared to iPS-RPE cells from healthy controls.
[232] [232] iPS-RPE cell lines produced as described here exhibit morphology (for example, pigmentation and hexagonal shape) and / or express one or more biomarkers that are indicative of RPE cells. Biomarkers for RPE cells (and iPS-RPE cells) are known and include, without limitation, one or more of RLBP1 (tcc CRALBP), RPE65, BESTROPHIN-1, MITF, VINCULIN, LRAT, RDH5, PAX6, MERTK, TYR and / or ZO-1, and can be used to determine or confirm that differentiation of RPE has occurred. Similarly, biomarkers for CECs (and iPS-CECs) and PRCs (and iPS-PRCs) are known and include, for example, cytokeratin 12 and cytokeratin 3 for corneal epithelial cells; and Crx for photoreceptors, recoverin for sticks and cones and Nrl for sticks.
[233] [233] Through iPS reprogramming and RPE differentiation methods as described in the Examples section, patient-specific iPS and iPS-RPE cells with BCD were successfully generated. C. Biochemical Assay for Identifying Biochemical Defects / Abnormalities and Cell Viability Assay to Assess PRE Atrophy in iPS-RPE Cells from Patients with BCD
[234] [234] A set of biochemical assays has been developed and used to evaluate and determine the phenotype in iPS-RPE cells specific to patients with BCD.
[235] [235] First, a more complete list of fatty acids was included in the applicant's biochemical assay. In a previous study that identified abnormal serum fatty acid levels in patients with BCD, the samples were tested for the following fatty acids, 16: 0, 16: 1, 18: 0, 18: 1n-9, 18 : 2n-6, 18: 3n-3, 20: 3n-6, 20: 4n-6, 22: 5n-3, 22: 6n-3, 24: 0 and 24: 1. The study found a higher concentration of octadecanoic acid (18: 0) in the serum of patients with BCD than in control subjects, as well as a lower concentration of octadecadienoic acid (18: 1n-9) than that in control subjects. To determine whether the same fatty acid abnormalities exist in BPS patient-specific iPS-RPE cells and whether there are more abnormalities in other fatty acids, a biochemical assay comprising more fatty acids was developed (see Table 2) using LC-MS .
[236] [236] In addition, to determine whether BPS patient-specific iPS-RPE cells carry other abnormalities in addition to fatty acids, other lipid species were included in the assay, including ceramides (Cer), sphingomyelin (SM) and sphingosine and sphinx - ganina (SOSA), to analyze the phenotype in a BCD disease model and determine the biochemical functions of the CYP4V2 protein. See Table 2 for a list of different species and compounds included in the biochemical assay used to test specific BPS patient-specific iPS-RPE cells.
[237] [237] Surprisingly, the test results (see Examples section) showed that specific BPS patient-specific iPS-RPE cells have a different fatty acid abnormality profile than those found in serum from BCD patients.
[238] [238] The eye is a light-sensitive organ of the human body. BCD begins with atrophy of RPE, which in turn causes death of photoreceptors and loss of vision. A key function of RPE is light absorption (Strauss, 2005, The retinal pigment epithelium in visual function. Physiol Rev 85: 845-81). Exposure to ambient light can affect the development and progression of human retinal degenerations such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). The use of light exposure in models of eye disease is an adequate model system for studying retinal degenerations. Exposure to light including exposure to blue light has been widely used in retinal research (Dual roles of polyunsaturated fatty acids in retinal physiology and pathophysiology associated with retinal degeneration, Masaki Tanito & Robert Anderson (2009) Clinical Lipidology , 4: 6, 821-827. Seko, et al., Graefes Arch Clin Exp Ophthalmol. 2001 Jan; 239 (1): 47-52. Blue light-induced apoptosis in cultured retinal pigment epithelium cells of the rat Narimatsu, et al., Exp Eye Res. 2015 Mar; 132: 48-51. Blue light-induced inflammatory marker expression in the retinal pigment epithelium-choroid of mice and the protective effect of a yellow intraocular lens material in vivo). Blue light exists in ambient light such as sunlight and artificial light (for example, office lighting), as well as electronic display devices such as TVs, monitors, smartphones, notebooks and tablets (Moon, and others, Blue light effect on retinal pigment epithelial cells by display devices, Integr Biol (Camb). 2017, 22; 9 (5): 436-443. Doi: 10.1039 / c7ib00032d).
[239] [239] In this study, a cell viability assay found RPE atrophy in a BCD cell model. Exposure to light (blue) caused cell death significantly higher in iPS-RPE samples from patients with BCD than in control samples. Clinical BCD phenotype (ie, RPE atrophy) is evident in a BCD cell model. AAV.CYP4V2 demonstrated efficacy in rescuing RPE atrophy in a BCD cell model. D. BCD Cell Model Applications
[240] [240] In addition to the assessment of cell-level phenotype associated with BCD, the BCD Cell Model can be used for other applications of a disease model, including, without limitation, drug evaluation, agent development or therapeutic devices, determining dosage ranges, testing for safety and toxicity, testing different formulations for BCD or other conditions related to CYP4V2 or studying the functions and uses of CYP4V2, including, without limitation, development and evaluation of drugs comprising or expressing CYP4V2 protein, for example, gene therapy with CYP4V2. In addition, BCD patient-specific iPS-RPE (and other eye cells derived from BCD patient-specific stem cells, including, without limitation, iPS-photoreceptor cells, iPS-corneal cells) can be used as therapy cell, either in unmodified form or after genetic repair (for example, through gene transfer or genetic editing as described here). The Examples section provides examples of non-limiting examples of BCD Cell Model applications. E. Compound Evaluation Methods
[241] [241] Significantly, the iPSC-RPE cell lines described here may provide models of human cell disease (for example, BCD, retinitis pigmentosa, IRD). Such iPSC-RPE cells, iPSC-CEC cells or iPSC-PRC cells, which can be collectively referred to as “iPSC-ocular cells”, can be used for diagnosis, prognosis, prediction of disease onset, severity and rate progression of a patient with BCD or a patient with retinitis pigmentosa or a patient having another type of inherited retinal disease. For example, such iPSC-ocular cell lines can also be used to evaluate test compounds for those that would have therapeutic efficacy for treating or preventing diseases associated with genetic or epigenetic changes in a CYP4V2 nucleic acid (eg, BCD) .
[242] [242] The pluripotent cells described here, particularly those produced from an individual having a genetic or epigenetic change in CYP4V2 or an individual who has an eye disease (eg, BCD), can be used as a research tool on methods to identify compounds that are therapeutic candidates for treatment, diagnosis, prognosis or prevention of eye disease (eg, BCD). It should be understood that the test compounds can be any type of compound. They can be of natural origin or they can be produced through chemical synthesis. They can be a library of structurally defined chemical compounds, uncharacterized compounds or substances, or a mixture of compounds. It should be understood by a person that test compounds can be, without limitation, nucleic acids or analogues thereof, polypeptides or analogues thereof, antibodies, chemical agents and molecules.
[243] [243] The cells described here, in the presence or absence of a test compound, can be assessed for their ability to grow and function in an animal model (for example, in the eye of an animal model) and for their propensity, or lack of propensity, to form tumors. Various methods can be used to evaluate cells, including, without limitation, PCR techniques, immunoassays and / or analyzes of lipid / fatty acid metabolism. Methods and Compositions for Cellular Therapies
[244] [244] As discussed here, gene therapy with CYP4V2 has been shown to be effective in correcting biochemical abnormalities in BPS patient-specific iPS-RPE cells. However, a prerequisite for gene therapy to work in vivo is that the individual still has some of the remaining RPE and photoreceptor cells in the eye being treated. For patients with advanced stage BCD who have few or no RPE cells or photoreceptor cells remaining in the eye, cell therapy can be used as an alternative or in combination with gene therapy as a treatment option.
[245] [245] Cell therapy involves transplanting new cells to replace dead or degenerated cells. For BCD, the new cells can be RPE cells, photoreceptor cells (cone and / or base), photoreceptor progenitor cells, choroid cells, corneal epithelium cells, lens cells or other types of ocular cells. depending on which type of cells showed degeneration and needs a replacement in the individual. The description and Examples that are described here used iPS-RPE cells to illustrate the methods and processes. They can be applied to another type of eye cell.
[246] [246] Cell therapy for BCD and other types of eye diseases including, without limitation, inherited retinal diseases (IRD), retinitis pigmentosa (RP), macular degeneration (including age-related macular degeneration (AMD)), it can be categorized as follows. (1) Allogeneic transplant:
[247] [247] In one embodiment, RPE cells, PRCs, ECCs, CE cells and other eye cells derived from embryonic stem cells (ESC) or iPSCs from a healthy donor can be used in allogeneic transplantation as cell therapy for BCD. It involves differentiating a healthy ESC or iPSC from a healthy individual (ie, one without CYP4V2 mutations) in RPE cells and transplanting such ESC-RPE cell into the eye of a BCD patient. Methods for reprogramming iPSC and differentiating ESC or iPSC in RPE are provided here in the Examples section. In an earlier study, embryonic stem cell (ESC) derived from RPE cells was used to treat age-related macular degeneration (AMD), see, Schwartz et al., Investigative Ophthalmology & Visual Science April 2016, Vol.57, ORSFc1 -ORSFc9. The pros of an allogeneic allograft transplant are that it is less expensive than an autologous transplant because a common source can be used to treat multiple patients. However, it has a significant disadvantage such as immune rejection by the host individual that can significantly affect its effectiveness and duration. In addition, it requires long-term immunosuppressants that can lead to severe systemic side effects. Finally, the use of ESC can give rise to ethical questions. (2) Autologous transplant without genetic repair
[248] [248] In one embodiment, autologous cells can be used in cell therapy for BCD. Such an autologous source are iPS cells and iPS-RPE cells derived from a patient with BCD, which can be transplanted into the eye of such a patient with BCD. BCD is a relatively late-onset disease. Symptoms in patients with BCD are usually developed in the 2nd, 3rd or even 4th decade of life. Also, iPS reprogramming processes have some degree of “reset the clock” effect on iPS cells and cells derived from iPS cells. In this way, iPS-RPE cells and other iPS-ocular cells derived from a patient with BCD can be used as a cell therapy for transplantation of the patient with BCD even without any genetic repair of CYP4V2 mutations in iPS- cells. RPE. IPS reprogramming and RPE differentiation methods are provided in the Examples section here. As a precaution, entire genome sequencing can be performed to compare genomic DNA in iPS or iPS-RPE cells and genomic DNA in the source cell (eg, fibroblast or blood cell) if there are any disease-causing changes created during the iPS reprogramming process and RPE differentiation. (3) Autologous Patient Cells Genetically Repaired for Cell Therapy for BCD and other types of IRDs and RPs
[249] [249] The present description provides methods and compositions for generating genetically repaired autologous cells for cell therapy. As used herein, “genetically repaired” or “genetic repair” refers to the correction of CYP4V2 mutations through or by genetically editing the patient's genome (for example, directly on the chromosome using, for example, CRISPR / Cas9, CRISPR / Cpf1, Zinc Finger, TALEN) or via gene transfer from a healthy copy of the CYP4V2 gene (cDNA, RNA or otherwise) to the patient's cell, which typically does not integrate into the genome (for example, genetic therapy with CYP4V2 as described here) or correction or compensation for the defective mRNA in the patient's cell.
[250] [250] As a disease caused by genetic mutations, autologous cells for use in cell therapy for BCD or another IRD or RP should ideally have their genetic effects (ie, the changes
[251] [251] The present description provides compositions and methods for correcting a CYP4V2 mutation through genetic editing. The description in the Examples section here illustrates the compositions and methods using a CRISPR / Cas9 construct to correct the c.802- 8_810del17insGC mutation, the most common mutation among BCD patients. It can also be applied to other general editing methods
[252] [252] The most common CYP4V2 mutation among patients with BCD is c.802-8_810del17insGC (with reference to a deletion of base 17 with two bases (GC) inserted in place starting from 8 bases from the end of intron 6 of CYP4V2 gene, also referred to as IVS6-8 del / insGC, see SEQ ID NO: 46 showing the sequence of the genomic DNA region CYP4V2 and SEQ ID NO: 47 showing the corresponding wild type sequence. 8_810del17insGC is illustrated in the following sequence that shows junction of CYP4V2 intron 6 -exon 7. The sequence of intron 6 is shown in lower case and the exon sequence 7 in upper case. and the insertion of GC are in parentheses): caa aca gaa gca tgt gat tat cat tca aa (tca tac agG TCA TCG CT) (GC) GAA CGG GCC AAT GAA ATG AAC GCC AAT GA) (SEQ ID NO: 46) resulting in the leap of exon 7. (Xiao et al., Biochem Biophys Res Commun. 409: 181-6, 2011; Meng et al., 2014, Mol. Vis., 20: 1806-14; Wada et al., Am J Ophthalmol . 139: 894-9, 2005; Jiao et al., European Journal of Human Genetics (2017) 25, 461-471). A recent study estimated that the age of the c.802-8_810del17insGC mutation should be 1,040-8,200 generations in the Chinese population and 300-1100 generations in the Japanese. See Jiao et al., European Journal of Human Genetics (2017) 25, 461-471.
[253] [253] Cell therapy (also known as cell therapy or cryotherapy) can be used as described here to treat or prevent eye disease in an individual. As described here, BCD, certain RP, IRD and other eye diseases referred to here are associated with a genetic or epigenetic change in an acid sequence.
[254] [254] Cell therapy usually involves injection, implantation, transplantation or otherwise administering a composition that includes cells to an individual (eg, to a patient's tissue or organ (eg, an eye)). The methods described here are unique in that they allow for genetically repaired autologous cell therapy for an individual having an eye disease.
[255] [255] Methods that include obtaining cells from an individual having an eye disease (for example, associated with a genetic or epigenetic change in a CYP4V2 nucleic acid sequence) and repairing the ( s) mutation (s) within the CYP4V2 nucleic acid (for example, DNA or RNA) using, for example, genetic editing, or repair by administering a nucleic acid sequence encoding a functional CYP4V2 protein (for example, gene transfer). Cells can be made pluripotent (for example, by inducing pluripotency, for example, to produce iPSCs) and be differentiated into one or more eye cells (for example, iPS-RPE, iPS-CECs, iPS-PRCs) before administration back to the individual (for example, to the individual's eye). It would be understood that cells can be repaired genetically before or after becoming pluripotent or after being differentiated into eye cells. A. Origination of Cells
[256] [256] In some cases, autologous cells (eg, individual specific cells (eg, patient) can be used in the cell therapy methods described here. For example, such fibroblasts or peripheral blood mononuclear cells (PBMCs) can be obtained from an individual and used to produce iPSCs as described in the Examples section. Virtually all cell types can be used to generate iPSCs and in this way can be used
[257] [257] Methods of producing induced pluripotent stem cells (iPSCs) are known in the art. In short, iPSCs can be made by introducing a particular set of proteins (for example, nucleic acids encoding a particular set of proteins) into cells. It would be understood by the converse that an exemplary non-limiting method is through the introduction of one or more transgenes encoding OCT4, SOX2, KLF4, c-MYC (for example, the “Yamanaka factors”). In some modalities, reprogramming uses all four transcription factors. In some embodiments, one, two or three transcription factors can be used. Li et al., Stem Cells, 2009; 27: 2992–3000. Zhu et al., Cell Stem Cell 2010; 7: 651–655. In some modalities, iPSCs can be generated through the direct administration of reprogramming proteins. Kim et al., Cell Stem Cell. 2009; 4 (6): 472–6. The Examples section provides a method for producing iPSCs using non-integrating methods, for example, using the Sendai virus (Example 1) or through episomal methods (Example 2). Any method of producing iPSCs, however, is included within the scope of this description.
[258] [258] Various methods (for example, Sendai virus, episomal method, with or without small molecules) can be used to generate iPSCs, see section Examples, see also, for example, Hubbard et al., J. Vis. Exp., 2014, 92: 52009. In addition, methods of producing iPSCs from several different cell types are known in the art. See, for example, Hayashi et al., 2012, PLoS One, 7 (9):
[259] [259] Any type of stem cells can be used in the cell therapy methods described here including, without limitation, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), embryonic stem cells (ES) , mesenchymal stem cells, adult stem cells or tissue-specific stem cells. Stem cells for use in the methods described here can be pluripotent, multipotent or totipotent stem cells.
[260] [260] As used herein, the term “pluripotent” refers to a cell capable of at least growing into one of ecto-dermal, endodermal and mesodermal cells. In one embodiment, the term “pluripotent” refers to cells that are totipotent and multipotent. As used herein, the term “totipotent” cell refers to a cell capable of growing in all cell lines. As used here, the term “multipotent” refers to a cell that is not terminally differentiated. The pluripotent cells of the present invention can be any stem cells or produced from non-pluripotent cells, such as fibroblasts, using methods of induction, de-differentiation and nuclear transfer known in the art. The pluripotent cells described here, whether stem cells or produced from non-pluripotent cells, may be from an individual having BCD or having mutations in CYP4V2 or a healthy individual.
[261] [261] iPSCs can be characterized by one or more of the following: a. the unique morphology of iPSCs; B. one or more pluripotency markers, such as Oct-4, Sox-2, SSEA-4, TRA-1-60, TRA-1-81, Nanog and AP; ç. The ability to differentiate in the desired cell type (for example, RPE cells) and / or d. a teratoma assay. Not all of the above are necessary for the characterization of iPSCs and validation of pluripotency (for example, teratoma, see, for example, Bata et al., 2013, Stem Cell Res., 11 (1): 552–562). C. Genetic editing
[262] [262] Various genetic editing technologies can be used in the methods described here to repair a genetic or epigenetic change present in an individual's CYP4V2 nucleic acid. Genetic editing can be performed using any number of technologies including regularly interspaced grouped short palindromic repetition technology (CRISPR) (see, for example, US Patent Nos. 8,697,359; 8,889,418; 8,999,641; and US 2014/0068797), nuclease technology with transcriptional activator type effectors (TALEN) (see, for example, Li et al., 2011, Nucleic Acids Res., 39 (14): 6315-25) or nuclease technology zinc finger (see, for example, Wright et al., 2005, The Plant J., 44: 693- 705).
[263] [263] To perform genetic editing using CRISPR technology, nucleic acids encoding a nuclease (for example, often a Cas9 nuclease, but other nucleases (for example, other Cas nucleases, for example, Cpf1, or non-Cas nucleases) can also be used) can be incorporated into one or more vectors and delivered to an individual as described here. Simply by way of example, the cells described here (for example, cells of the individual before reprogramming into iPSCs, iPSCs of the individual before differentiation into RPE, corneal epithelial cells or photoreceptor cells, or after differentiation into RPE, corneal epithelial cells or photoreceptor cells (referred to here as “iPSCs-RPE,” “iPSC-CEC” or “iPSC-PRC”)) can be transduced or transfected with one or more constructs (for example, vectors, RNP, mRNAs) containing and / or encoding at least one guide RNA (gRNA), at least one CRISPR-associated protein (e.g., Cas9 or Cpf1) and at least one donor model nucleic acid. In some embodiments, donor model nucleic acid is not required, for example, when genetic repair is achieved through inactivation.
[264] [264] Similarly, to obtain genetic editing using TALEN technology, a nucleic acid encoding TALEN (eg, dimeric / nuclease transcription factor) can be incorporated into a vector and delivered to an individual as described herein. Likewise, to obtain genetic editing using zinc-finger nuclease technology, a nucleic acid encoding a personalized DNA endonuclease (for example, a heterodimer in which each subunit contains a finger-domain. zinc and a FokI endonuclease domain) can be incorporated into one or more vectors and administered to an individual as described here.
[265] [265] The components necessary to realize each of these technologies are commercially available and are customizable for the particular target sequence (s). See, for example, Caparou Biosciences; GenScript, CRISPR Therapeutics; Medicine Editas; Cellectis Bioresearch; Life Technologies; Sangamo BioSciences; or Sigma Aldrich Chemical Co.
[266] [266] Under the appropriate circumstances, genetic editing can occur so that the genetic or epigenetic change in the individual's CYP4V2 nucleic acid is repaired and as a result a functional CYP4V2 protein is expressed. A CYP4V2 nucleic acid sequence has been repaired when the presence of CYP4V2 nucleic acid (for example, CYP4V2 mRNA) is restored, the presence of the CYP4V2 protein is restored or the function of the CYP4V2 protein is restored. Similarly, “repaired” or “corrected” can refer to a restoration of the affected sequence (for example, genetic or epigenetic alteration) to the wild-type sequence or another
[267] [267] There may be some cases when it is desirable to introduce, using genetic editing, one or more mutations into a cell (for example, in the CYP4V2 nucleic acid). This is a way in which a cell model of disease (for example, BCD) can be created. For example, gene addition can be performed on embryonic stem cells (ES cells) to create cell lines with artificial CYP4V2 mutations, which can then be differentiated into RPE cells. Alternatively, genetic editing can be performed on iPS cell lines from a healthy individual (for example, a non-BCD individual) or on an RPE cell line (for example, ARPE-19 cell line) to create lines iPS or RPE mutant cells in CYP4V2.
[268] [268] In some cases, it is desirable to evaluate cells (for example, using entire genome sequencing) after the genetic editing steps are complete to confirm that the targeted mutation has been repaired and that no out-of-target editing significant event has occurred.
[269] [269] CRISPR and CRISPR-associated protein 9 (Cas9), known as CRISPR-Cas9, consisting of an RNA-guided nuclease (Cas9) and a guide RNA, generate site-specific DNA breaks, which are repaired by endogenous cellular mechanisms. Possible results of the approach include mutation of a specific site through non-homologous mutagenic end junction (NHEJ), creating insertions or deletions (indels) at the site of the break, and precise change of a genomic sequence through homologous recombination ( HR) using an exogenously introduced donor model. The CRISPR guide RNA is composed of two RNAs called CRISPR targeting RNA (crRNA, also referred to here as CRISPR RNA) and transactivating crRNA (tracrRNA). CrRNAs are typically about 20 nucleotides (nt) in length. It hybridizes to a target DNA sequence by pairing the Watson-Crick base and guides the Cas endonuclease to cleave the target genomic DNA.
[270] [270] To genetically repair the most common CYP4V2 mutation through genetic editing, several CRISPR correction constructs for the CYP4V2 mutation have been developed (see Examples section). CRISPR was used because it is simpler to implement and edits more efficiently than other forms of genetic editing, such as TALENs and zinc finger nucleases. CRISPR constructs contain optimized and validated in vitro gRNA sequences and different construct options that can be readily used to correct the c.802-8_810del17insGC mutation in BCD patient cell lines, resulting in genetically repaired cells that can be used in cell therapy, including, without limitation, autologous cell therapy, for BCD.
[271] [271] CRISPR gene editing therapy involves the use of a CRISPR-associated protein (Cas) which is a CRISPR nuclease and RNA guide. The role of the CRISPR guide RNA is to guide Cas to the sequence that is directed by the CRISPR guide RNA through a protospace element contained in the CRISPR guide RNA that is complementary (or specific to) the target sequence. For Cas (for example, Cas9 or Crf1) to bind to and cleave at or near the target sequence, a motif sequence adjacent to the protospace (PAM) must also be present. A PAM sequence is a short stretch of DNA (typically 2-6 nucleotides) that serves as a binding signal for Cas. Different cas may have different PAM and cleavage pattern. For example, for Cas9 of Streptococcus pyogenes (SpCas9), the canonical PAM sequence is NGG. For Staphylococcus aureus (SaCas9), the PAM sequence is NGRRT or NGRRN.
[272] [272] The CRISPR guide RNA for Cas9 typically comprises a CRISPR RNA (crRNA) and a transactivation crRNA (tracrR-NA). The crRNA comprises a sequence of a protospace element that is designed to be complementary (or specific) to a sequence targeted next to the gene targeted for correction, disruption or replacement, and a sequence that corresponds to a complementary region tracrRNA. The tracrRNA that comprises a region that is complementary to the corresponding region of the crRNA and a sequence that interacts with the protein associated with CRISPR 9 (Cas9). No tracrRNA is required for Cpf1.
[273] [273] The length of the protospace element is typically about 20 nucleotides. Longer or shorter protospacer sequence (about 16-24 nt) can also be used. The protospace element may be 100% complementary to the target sequence or may contain incompatibilities with the target sequence. In some embodiments, a "G" nucleotide can optionally be added at the beginning of the protospace element sequence.
[274] [274] After a DNA molecule is cleaved by Cas, it can be repaired in one of two ways. An error-prone non-homologous end junction repair (NHEJ) can result in an indelible mutation that can disrupt the function of the protein encoded by the gene. NHEJ can be used to create artificial mutations in a cell line. In some embodiments, it can be used to create mutations in the CYP4V2 gene (for example, an indel in an exon or a joining acceptor region) of a cell line (for example, an ES cell, an iPS cell or an AR cell line - PE-19) without any endogenous CYP4V2 mutations and in this way generating a cell model of disease (for example, a cell model of BCD). In addition, two or more CRISPR guide RNAs can be used together to inactivate a targeted region of a target gene or the entire target gene in this way, generating an inactivation model. In some embodiments, CRISPR-based gene silencing is used to disrupt (or silence) a defective gene, for example, in the treatment of a dominant genetic disease. During gene silencing, the cell tries to repair the broken DNA, but NHEJ often does so with errors that disrupt the gene in this way by silencing it effectively. In some embodiments, NHEJ can also result in correction of a mutation, for example, especially when the mutation is a variation of a single nucleotide or more than about 10 nucleotides. Alternatively, if a donor nucleic acid sequence is available, the DNA break can be repaired through homology-directed repair (HDR) to correct or replace the target gene. A donor nucleic acid sequence can be provided in the form of a single-stranded DNA (ssDNA) or a single-stranded oligo DNA nucleotide (ssODN) or a vector. In some embodiments, the donor nucleic acid sequence is no more than about 1kb, 800bp, 600bp, 500bp, 400bp, 300bp, 280bp, 260bp, 240bp, 220bp or 200pb for a donor nucleic acid sequence provided in an ssODN. In some embodiments, the sweet nucleic acid sequence is no more than about 25kb, 20kb, 15kb, 10kb, 9kb, 8kb, 7kb, 6kb, 5kb, 4.5kb, 4kb, 3.5kb or 3kb for one donor nucleic acid sequence provided in a vector. In some embodiments, a donor nucleic acid sequence is symmetrical. In some modalities, the donor nucleic acid sequence is asymmetric. In some embodiments, the length of a donor nucleic acid sequence can be adjusted to a higher HDR rate. In some modalities, if the Cas-targeted PAM used in adding the CRISPR gene is also present in the donor nucleic acid sequence, it can be mutated (switch to a different nucleotide) so that PAM does not there is more in the donor nucleic acid sequence to prevent the donor model or the DNA sequence repaired by the donor model from being cleaved and destroyed by Cas. In addition to correcting or replacing a mutated or defective gene or a portion of it, HDR can also be used to create artificial mutation (s) in the CYP4V2 gene (for example, inserting a mutation into an exon or an joining acceptor region) of a cell line (for example, an ES cell, an iPS cell or an ARPE-19 cell line) without any endogenous CYP4V2 mutations and thus generating a cell model of disease (for example , cell model of BCD).
[275] [275] CRISPR's guide RNA and Cas used in CRISPR gene editing therapy can be provided in a vector form (eg, a plasmid (eg, pX330, pX458, pX459), a vector of Recombinant AAV or a recombinant lentivirus vector) or an mRNA encoding such component (s) and / or RNA and protein.
[276] [276] The donor model can be provided in an ssDNA (for example, ssODN) or cloned in a plasmid or other types of vectors (for example, an AAV vector (for example, AAV2 or AAV6) for use in HDR .
[277] [277] Various compositions and methods can be used to improve target editing or repair efficiencies and / or decrease potential off-target occurrences. For example, different Cas (for example, Cas9 or Cpf1) or Cas of different species (for example, SpCas9, SaCas9, NMCas9) or variants (SpCas9, SpCas9 VQR) can be used to extend the PAM selections available for a target frequency, thereby increasing specificity. If a target sequence region does not have the PAM NGG site for SpCas9, but is rich in AT, then Cpf1 can be considered. Cas9 nickase (eg Cas9 D10A) generates only a single strand break in the target DNA and thus requires two CRISPS guide RNAs in pair to generate a double strand break. This condition dramatically increases the specificity of the target, since it is unlikely that two notches outside the target will be generated within proximity large enough to cause a DSB. Also, asymmetric donor model can increase the HDR rate. dCas9 catalytically inactive does not cut target DNA, but can still obtain a sequence replacement without any of the error-prone repair that normally accompanies the Cas9 cut. See, Richardson et al., Nature Bio-technology 34, 339–344 (2016).
[278] [278] Obtaining targeted gene correction and in the meantime avoiding or minimizing off-target editing are two goals of genetic editing. Previous research has revealed off-target mutations caused by genetic editing technologies, including, without limitation, CRISPR and TA-LEN, see, for example, Tsai et al., Nature Biotechnology 33, 187–
[279] [279] In this way, careful design, validation and refinements were employed in the development and validation of the CRYPR mutation constructs for CYP4V2 mutation: (1) Multiple gRNA candidates were generated based on the nucleic acid sequence of CYP4V2 mutant containing the c.802-8_810del17insGC mutation (2) The top 5 gRNAs were selected using the following criteria (SEQ ID NOs: 48-52, Table 5 and Figure 12): a. The proximity of the gRNA cleavage site to the modification site, and b. The profile outside the target of the gRNA; (3) The activity of the 5 best gRNAs was evaluated in the genetic DNA of a patient with BCD with homozygous c.802- 8_810del17insGC mutations (see Figure 13); (4) Based on (2) and (3), three gRNAs were selected. Each of the 3 gRNAs was cloned into a pX459 plasmid along with nucleic acid sequences encoding Cas9 and puromycin resistance gene (Pure) for selection of transfected cell using pure-mycine (See Figures 15 and 18). (5) Two donor models (both forward and reverse complementary) providing HDR donor nucleic acid sequence were generated. The ssODNs containing the donor model sequences were synthesized using IDT (see SEQ ID NOs: 56 and 57). (6) In addition to plasmid constructs, a CRISPR RNP construct was developed. An RNP construct offers certain advantages over other constructs. A detailed discussion is provided below and in the Examples section. (7) CYP4V2 CRISPR correction constructs are validated in iPS cells derived from a patient with BCD with homozygous c.802-8_810del17insGC mutations. (8) Whole genome sequencing is performed on unmodified cells and iPS cells genetically repaired by the CYP4V2 mutation CRISPR correction constructs to confirm the correction of the c.802-8_810del17insGC mutation and evaluate off-target edits.
[280] [280] Methods for determining the optimal conditions for transfection in iPSCs and selecting transfected cells are provided. See the Examples section for a detailed description. It is understood that these constructs can be used to treat not only specific iPS cells of patients with BCD in vitro, but also source cells (for example, fibroblasts or PBMCs) or iPS-RPE, iPS cells -PRC, iPS-CE or iPS-CEC cells or other ocular cells derived from patient-specific iCD cells with BCD in vitro, as well as in vivo in patients with the c.802-8_810del17insGC mutation. In one embodiment, the components of the constructs can be used directly. In some modalities, the components in the construct can be modified or cloned into a different vector to obtain greater transduction efficiency in vivo or greater specificity for the type of target cell or achieve other purposes. For example, Cas9 can be changed to Cas9 nickase (Cas9n D10A), which
[281] [281] Several improvements have been made to the CRISPR RNP construct. Instead of IVT sgRNA or a crRNA: tracrRNA duplex, a synthetic sgRNA was used. Synthetic gRNAs have greater purity than IVT sgRNAs and thus decrease the risk of off-target editing caused by impurities in sgRNA. In addition, chemical modification is applied to the sgRNA to protect the sgRNA from intracellular degradation, which can increase editing efficiency. See the Examples section for more details.
[282] [282] It is understood that, in addition to the plasmid constructs and CRISPR RNP constructs described herein, an mRNA construct comprising Cas9 encoding mRNA and a guide RNA oligonucleotide can also be used.
[283] [283] After BPS patient-specific iPS cells are transfected with the CYP4V2 mutation CRISPR correction constructs, the transfected cells are selected using pure-mycine. It should be understood that other markers, such as GFP, can be incorporated into the constructs and used as a marker in place of or in addition to puromycin. Following selection, single cell cloning is performed, after which some cells from the single cell clone are collected for sequencing. After sequencing results confirm successful gene editing on the target and any disease-causing gene edits are found, the remaining cells of the same clone are used to differentiate into the desired eye cell type, for example, iPS-RPE cells. D. Differentiation of iPSCs
[284] [284] iPS cells from genetically repaired BCD patients are differentiated into iPS-RPE cells (or another type of eye cell (for example, iPS-CEC, iPS-CE or iPS-PRC cells). - all for differentiating iPSCs cells into RPE or another type of eye cell (for example, CEC or PRC) are known. See, for example, Hayashi et al., 2012, PLoS One, 7 (9): e45435; Songs- tad, et al., Investigative Ophthalmology & Visual Science December 2015, Vol.56, 8258-8267; and Lamba et al., PLoS One. 2010 Jan 20; 5 (1): e8763. For example, induced pluripotent stem cells (iPSCs ) reprogrammed from cells can be produced and differentiated further into, for example, RPE cells (referred to here as “iPS-RPE”), corneal epithelial cells (referred to here as “iPS-CEC”), photoreceptor cells (or progenitor of photoreceptors; referred to here as “iPS-PRC”) or iPS-choroidal endothelial cells (CE) (referred to as “iPS-CE”).
[285] [285] Differentiated cells, for example, iPS-RPE cells, are tested for their biochemical functions (as described in the Examples section) to confirm that they have improved biochemical functions compared to the patient's iPS-RPE cells without genetic repair. co.
[286] [286] iPSC-RPE cell lines produced as described here exhibit morphology (for example, pigmentation and / or hexagonal shape) and / or express one or more biomarkers that are indicative of RPE cells. Biomarkers for RPE cells (and iPS-RPE cells) are known and include, without limitation, one or more of RLBP1 (tcc CRALBP), RPE65, BESTROPHIN-1, MITF, VINCULIN, LRAT, RDH5, PAX6, MERTK, TYR and / or ZO-1, and can be used to determine or confirm that RPE differentiation has taken place. Similarly, biomarkers for CECs (and iPS-CECs) and PRCs (and iPS-PRCs) are known and include, for example, cytokeratin 12 and cytokeratin 3 for corneal epithelial cells; and Crx for photoreceptors, recoverin for sticks and cones and Nrl for sticks. E. Administration
[287] [287] Genetically repaired iPS-RPE cells can be used in autologous transplantation to the patient from which iPS-RPE cells are derived. Patients with BCD or another eye condition due to CYP4V2 mutations can be treated using the cell therapy methods provided here. Similarly, the method can be used to provide an autologous cell therapy that is genetically repaired for eye diseases caused by one or more genetic mutations.
[288] [288] Methods of administering cells are known, and methods of administering cells to the eye are known. See, for example, Wert et al., J Vis Exp. 2012; (69): 4286; WO 2016/179496; Schwartz et al., Investigative Ophthalmology & Visual Science April 2016, Vol.57, ORSFc1-ORSFc9. In one embodiment, the eye cell can be transplanted by injecting cell suspension, for example, RPE cell suspension. In another embodiment, cells can be transplanted as part of a leaf or base, for example, into tissue in vitro using natural and / or synthetic bases to generate a polarized functional RPE monolayer.
[289] [289] The therapeutically effective amount of cells administered to the eye is known to those skilled in the art and will vary with the type of cells being transplanted, the maturity of the cells being transplanted and whether it is expected to divide post-transplant, the size of the cell. area or number of cells targeted for replacement, and the individual being treated (for example, age, sex, weight, stage of disease development and condition of the individual being treated); the route of administration; and the required regime. The therapeutically effective amount of cells used in ocular cell therapy can vary from about 1 * 10 ^ 3 to about 1 * 10 ^ 8 cells in a single administration.
[290] [290] While iPSC cell lines can be generated for individual subjects, an iPSC cell bank having common HLA haplotypes (or in which the HLA haplotype was genetically engineered) can be generated, which would be designed to obtain with - immunological patibility with a large portion of the patient population. See, for example, Turner et al., Cell Stem Cell, 13: 382– 384, 2013. In addition, an iPSC cell line can be generated, which is immunologically silent regardless of the individual's genotype (see, for example, Riolobos and others, Mol. Ther., 21: 1232–41, 2013). When combined with these methods, patient-specific iPS cells and iPS-ocular cells can be used not only in a strict autologous sense, but can also be used for transplantation to other patients.
[291] [291] Typically, the cell therapy administration stage occurs after the onset of symptoms of the disease or after the individual has shown signs of retinal degeneration or corneal dystrophy, as applicable. In one embodiment, eye cell therapy provided here can be used independently in the treatment of an eye disease (eg, BCD). In another embodiment, eye cell therapy provided here can be used in combination with one or more other treatment options, including, without limitation, therapy.
[292] [292] Similarly, administration can occur once, or a plurality of times (for example, over several weeks, months or years), and can be applied to the same eye or the contralateral eye. In addition, one or more cell types can be administered in single or separate administrations.
[293] [293] Post-treatment evaluation can use methods described in the section Gene therapy with CYP4V2 here, including, without limitation, through eye examinations such as visual function, for example, as measured by visual acuity, visual field, adaptation in the dark, visual function and / or Optical Coherence Tomography (OCT, for example, Spectral Domain-OCT (SD-OCT) and ERG. Methods of Use of CRISPR RNP in Ocular Cell Therapy and Gene Therapy
[294] [294] RNP CRISPR is a genetically edited ribonucleoprotein (RNP) complex that includes a guide RNA complexed with a Cas protein (eg, Cas9 proteins). The guide RNA is formed by two RNAs called CRISPR RNA (crRNA) and transactivation crRNA (tracrRNA). In one embodiment, the crRNA and tracrRNA are provided as two separate nucleic acid molecules. In another embodiment, the crRNA and tracrRNA can be combined into a single chimeric guide RNA (sgRNA). The sgRNA can be about 100 nucleotides (nt) in length, or shorter or longer as desired or necessary. Twenty nt at the 5 'end (crRNA) hybridize to a target DNA sequence through Watson-Crick base pairing and guide the Cas endonuclease to cleave the target genomic DNA, with the remaining double-stranded structure on side 3 'for Cas9 recognition.
[295] [295] CRISPR RNPs have pros and cons compared to
[296] [296] To assess the above hypothesis and prove whether RNP CRISPR constructs can achieve both the objectives of the desired gene edition in ocular cell therapy and gene therapy, two sets of constructs were designed. One construct is a plasmid construct and the other is an RNP construct. Both constructs use the same BPS patient iPS cells for transfection, which are subsequently sequenced to analyze genetic repair on the target and off-target editing of each construct. Off-target editions are determined by comparing against genomic DNA from unmodified fibroblasts from the same patient. Results from both plasmid construct and RNP construct can be compared.
[297] [297] Detailed description of RNP, methods for forming RNP and using the RNP construct to generate genetically repaired cells (iPS and iPS-RPE cells) for a patient with BCD, is provided in the Examples section.
[298] [298] It should be noted that a similar RNP CRISPR construct can be used to correct or inactivate other BCD mutations and mutations of other RP and IRDs. In one aspect, the crR-NA sequence used here is changed to another crRNA sequence specifically targeting a different target mutation sequence. In another aspect, a guide RNA or sgRNA in an RNP construct can be modified to increase the efficiency of genetic editing. See Hendel et al., Nat. Biotechnol. 2015 Sep; 33 (9): 985-989. In some modalities, the RNP CRISPR constructs can be transfected using electroporation. In some embodiments, the RNP CRISPR construct can be transfected using lipofection or nucleofection. In some modalities, the RNP CRISPR construct can be administered through microinjection.
[299] [299] In addition to genetically repairing and treating patient cells in vitro, RNP CRISPR constructs can also be used to treat an eye disease caused by genetic mutations in vivo and have advantages over other types of CRISPR constructs (for example, plasmids and / or mRNAs encoding CRISPR components) for in vivo applications. For example, RNP CRISPR constructs have higher potency, lower off-target risk and / or toxicity or lower innate immune response compared to Cas9 mRNA and srRNAs transcribed in vitro. In one embodiment, RNP CRISPR constructs comprised of a Cas9 protein complexed with a guide RNA targeting the region of the mutant DNA sequence can be injected directly into the individual's eye (for example, sub-retinal injection, intravitreal or corneal injection) . In another modality, engineered Cas9 variants with SV40 nuclear localization sequences (NLS) that showed increased editing efficiency in brain cells in vivo (Staahl et al., Nat Biotechnol. 2017 May; 35 (5 ): 431-434) can be used to
[300] [300] It would be understood that the ratio between the components of RNP CRISPR, for example, the guide RNA and Cas9 protein, can be adjusted and optimized by testing different ratios in patient cell lines in vitro (for example, patient-specific iPS cells with BCD or iPS-RPE cells) prior to in vitro or in vivo treatment. The RNP CRISPR construct can be used independently or in combination with another CRISPR construct, including, without limitation, a plasmid or vector encoding a CRISPR guide RNA or crRNA or a Cas protein or a combination thereof; a Cas9 encoding mRNA; a guide RNA oligonucleotide; another RNP CRISPR construct or a combination or hybrid thereof. In addition, RNP CRISPR constructs can be used to correct or inactivate one or more of a mutation related to one or more of an eye disease. Combination Treatment of Gene Therapy and Cell Therapy
[301] [301] The present description provides multiple treatment options for BCD and other eye diseases caused by CYP4V2 mutations, including, without limitation, CYP4V2 gene transfer therapy and CRYPR CYP4V2 gene editing therapy. Both CYP4V2 gene transfer therapy and CYP4V2 gene editing therapy can be used either in vivo or in vitro or both in vivo and in vitro. When applied in vivo, CYP4V2 gene transfer therapy and / or CYP4V2 CRISPR gene editing therapy can treat remaining eye cells affected by BCD as genetic therapy. When applied in vitro to patient cells or patient-derived cells, cells treated by CYP4V2 gene transfer therapy and / or by CYP4V2 CRISPR gene editing therapy can be transplanted into the patient to replace eye cells dead or degenerate as cell therapy. Significantly, gene therapy and cell therapy compositions and methods provided here can be combined to provide additional benefits to patients that cannot be obtained using gene therapy or cell therapy alone. “Combination treatment” can also expand the eligible patient base. For example, for patients at an advanced stage who have no or few remaining photoreceptor cells or RPE, gene therapy is not as effective as for patients at an early stage. In this case, cell therapy can benefit from the provision of new cells (for example, RPE or photoreceptor cells), while gene therapy can improve the effect of cell therapy by rescuing the remaining RPE or photoreceptor cells and / or by improving the conditions of choroidal cells whose health affects the conditions of ocular cells. The combination of the “rescue” and “replacement” effect of gene therapy and cell therapy, respectively, makes combination treatment an improvement of either gene therapy or cell therapy. This method of combination treatment can be applied to eye diseases caused by one or more genetic mutations. Methods and Compositions for CYP4V2 Gene Therapy
[302] [302] The present invention relates to various compositions comprising a nucleic acid molecule encoding a functional CYP4V2 protein and various methods using them for treating an eye cell and / or eye disease. In one embodiment, a functional CYP4V2 protein can be used directly to
[303] [303] In some modalities, the vector is a vector of well-associated recombinant virus (rAAV). In some embodiments, the vector is a plasmid. In some embodiments, the vector is another type of viral or non-viral vector. The treatment methods comprise administering an effective amount (or an effective concentration) of said vectors to the subject's eye and / or target cells. In one embodiment, the treatment is applied directly in vivo. In another embodiment, the treatment comprises ex vivo treatment in target cells (for example, an eye cell) and transplantation of the target cells treated in the subject (for example, to the subject's eye). The methods of treatment are aimed at eye diseases and other conditions associated with CYP4V2 mutations. In one mode, the eye disease is Bietti's Crystalline Dystrophy (BCD). A. Functional CYP4V2 Protein and Nucleic Acids encoding a Functional CYP4V2 Protein
[304] [304] CYP4V2 (Cytochrome P450, Family 4, Subfamily V, Polypeptide 2, (MIM 608614), synonym: CYP4AH1) is one of the proteins in the cytochrome P450 (P450) superfamily and a member of cytochrome P450 subfamily 4 ( CYP4). Cytochrome P450s (CYPs) are important heme-containing proteins, known for their roles as enzyme oxidases. The term P450 is derived from the spectrophotometric peak at the wavelength of the maximum absorption of the enzyme (450 nm) when it is in the reduced state and complexed with carbon monoxide. They are involved in the metabolism of xenobiotic and endogenous compounds, such as steroids and fatty acids. CYP enzymes have been identified in all realms of life: animals, plants, fungi, protists, bacteria, archaea and even viruses. However, they are not omnipresent; for example, they were not found in Escherichia coli.
[305] [305] P450 proteins share key elements in structure. For example, P450 proteins can be identified by their signature sequence element FXXGXXXCXG (SEQ ID NO: 30), where cysteine serves as an axial ligand for heme iron. Sequence identity is relatively low among P450 proteins, but its actual topography and structural folds are highly conserved. The conserved nucleus is composed of a spiral called 'meander', a bundle of four helices, J and K helices and two sets of beta sheets. These constitute the heme binding loop (with an absolutely conserved cysteine that serves as the 5th ligand for heme iron), the proton transfer groove and the conserved EXXR motif (SEQ ID NO: 31) at helix K. P450 proteins are mainly proteins associated with the membrane located either in the inner membrane of the mitochondria or in the endoplasmic reticulum of cells.
[306] [306] In addition to structural similarities, P450 proteins also share functional similarities. The most common reaction catalyzed by P450 enzymes is a monooxygenase reaction, for example, insertion of an oxygen atom in the aliphatic position of an organic substrate (RH) while the other oxygen atom is reduced in water: RH + O2 + NADPH + H + → ROH + H2O + NADP +
[307] [307] Many hydroxylation reactions (insertion of hydroxyl groups) use P450 enzymes. Many P450 enzymes have steroids and / or fatty acids as substrates.
[308] [308] The human CYP4V2 protein (NCBI RefSeq: NP_997235.3) has 525 amino acids (amino acid sequence shown in SEQ ID NO: 4). There are variants of human CYP4V2 protein, including pathological variants (ie, mutations) (see Table 1 here for a select list of CYP4V2 mutations among BCD patients) and non-pathological (ie, functional) variants.
[309] [309] In one aspect, a functional CYP4V2 protein is human CYP4V2 protein (SEQ ID NO: 4). In other respects, a functional CYP4V2 protein is a functional variant or fragment of human CYP4V2 protein, including, without limitation, one with an amino acid sequence as shown in SEQ ID NO: 5).
[310] [310] A functional CYP4V2 protein can also be a variant of another functional CYP4V2 protein. What follows is a discussion based on changing the amino acids of a polypeptide described here to create an equivalent, or even improved, second-generation molecule. For example, certain amino acids can replace other amino acids in a protein structure without appreciable loss of ability to interact interactively with structures such as, for example, binding sites on substrate molecules, for example, binding sites for fatty acids. Since its interactive capacity and the nature of a protein that define the biological functional activity of this protein, certain amino acid substitutions can be made in a protein sequence, and in its base DNA or RNA coding sequence, and not they nevertheless produce a protein with similar properties. It is then understood that several changes can be made to the amino acid sequence of a functional CYP4V2 protein or to the DNA or RNA sequences of genes or coding regions thereof without appreciable loss of their usefulness or biological activity, as discussed herein. For example, SEQ ID NO: 5 is the amino acid sequence of a CYP4V2 protein variant that has an amino acid change from the human CYP4V2 protein sequence shown in SEQ ID NO: 4.
[311] [311] Various techniques, algorithms, software and tools can be used to design or engineer derivatives, variants and / or functional fragments of a functional CYP4V2 protein, for example, the human CYP4V2 protein. For example, the structure and functions of the various polypeptides or changes can be modeled, resolved or predicted by NMR, X-ray crystallography or computer modeling, for example, ClustalW, SWISS-MODEL server, Swiss-Pdb Viewer, Polyphen -2, PROVEAN, SIFT, Condel, MutationAssessor and FatHMM.
[312] [312] A functional CYP4V2 protein can also be a fragment or derived from a fragment of a functional CYP4V2 protein. For example, the human CYP4V2 protein (SEQ ID NO: 4) and its variant (SEQ ID NO: 5) both have a transmembrane domain between more or less the 13th amino acid residue and more or less the 35th residue from the terminal N. The main structure of human CYP4V2 protein (SEQ ID NO: 4) is located between about 36-525aa. In this way, a functional CYP4V2 can be derived by deleting the first plus or minus 35 amino acids from the human CYP4V2 protein (SEQ ID NO: 6) and replacing it with an alternative transmembrane domain sequence. Another source of functional CYP4V2 protein is a splice variant of functional CYP4V2 protein.
[313] [313] The predicted CYP4V2 transmembrane segment resides near the N-terminus, followed by a globular structural domain typical of the CYP450 family. The CYP4V2 globular domain includes 18 helices and beta structural segments.
[314] [314] In addition to substrates and functions shared with the other proteins of the CYP4 subfamily, computational analysis revealed that CYP4VC2 was formed from the duplication of the ancestors of CYP46A (SEQ ID NO: 7), which was then duplicated to generate the CYP4 family entire. Pan et al., Int. J. Mol. Sci., 2016, 17 (7) pii: E1020. It hurts:
[315] [315] Also, the CYP4V2 gene (or CYP4V2 gene orthologists, for example, Cyp4v3 for mouse) is conserved in many species, including, without limitation, human, chimpanzees, Rhesus monkey, dog, cow, mouse, rat, chicken, frog, horse, rabbit and fruit fly (SEQ ID NOs: 19-29). Orthologists with the human CYP4V2 gene were found in 196 organisms.
[316] [316] A functional CYP4V2 protein can comprise or be engineered, engineered, or derived from, including, without limitation, the following: (i) the human CYP4V2 protein (SEQ ID NO: 4) (ii) a variant of (for example, amino acid change and / or a splice variant) of the human CYP4V2 protein or a functional CYP4V2 protein (for example, SEQ ID NO: 5), (iii) one or more fragments of a functional CYP4V2 protein (for example , SEQ ID NO: 6), (iv) a CYP4V2 protein (or orthologist) from other species, (v) another CYP or CYP46A1 protein, (vi) a polypeptide that can improve, treat or stop one or more biochemical abnormalities in one or more compounds listed in Table 2 in a patient cell (for example, a BCD patient's iPS-RPE cell), and / or
[317] [317] It is understood that the compositions and methods disclosed herein can be used to express any functional CYP4V2 protein as described above. In one embodiment, a functional CYP4V2 protein is a polypeptide comprising all or part of the amino acid sequence shown in SEQ ID NO: 4, 5 or 6. In some embodiments, a functional CYP4V2 protein is a polypeptide comprising all or part of an amino acid sequence selected from the group consisting of CYP4V2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4X1, CYP4Z1 and CYP4Z1 Rhesus, dog, cow, mouse, rat, chicken, frog, horse, rabbit and fruit fly (SEQ ID NOs: 19-29), and derivatives, hybrids, variants and / or fragments thereof. In some embodiments, a functional CYP4V2 protein may have at least 80% amino acid sequence identity (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) with any of the selected sequences from the group consisting of SEQ ID NOs: 4-29. In a fashion, a functional CYP4V2 protein is a polypeptide comprising sequence elements from FxxGxxxCxG and ExxR (SEQ ID NOs: 30 and 31).
[318] [318] In some embodiments, a functional CYP4V2 protein is a compound or agent that can improve, treat or stop one or more biochemical abnormalities in a patient cell (for example, the iPS-RPE cell of a patient with BCD).
[319] [319] In one embodiment, a functional CYP4V2 protein can be used directly to treat BCD, similar to protein-based drugs for other diseases. In another embodiment, a nucleic acid molecule encoding a functional CYP4V2 protein is used to express the functional CYP4V2 protein in the targeted cells. In one embodiment, the nucleic acid molecule is an RNA. In another embodiment, the nucleic acid molecule is DNA, including, without limitation, complementary DNA (cDNA), for long-term expression. The cDNA can be positive or negative, single or double stranded. In some embodiments, the nucleic acid encoding a functional CYP4V2 protein is operably linked with one or more regulatory sequences to form a CYP4V2 expression cassette. In some embodiments, such an expression cassette is packaged in a vector for increased administration and / or expression efficiency.
[320] [320] A codon consists of a set of three nucleotides and either encodes a specific amino acid or results in the completion of translation (that is, stop codons). Most amino acids (usually everything except methionine) are encoded by multiple codons. In this way, different nucleic acid sequences can be used to express the same protein. The sequence identity between two nucleic acid molecules encoding the same protein sequence can vary from 0% to more than 99%. For example, a nucleic acid sequence (SEQ ID NO: 1) and another nucleic acid sequence (SEQ ID NO: 2), both encoding the human CYP4V2 protein (SEQ ID NO: 4), share only one identity 77% sequence.
[321] [321] Codon optimization of nucleic acid sequences can improve and / or stabilize protein expression without changing the encoded amino acid sequence. Codon optimization replaces codons present in a nucleic acid sequence with preferred codons encoding the same amino acid, for example, preferred codons
[322] [322] It must be understood that, depending on the codon optimization methods, configuration, algorithms or software being used, different codon-optimized nucleic acid sequences co-complicating the same protein can be generated. However, codon optimization does not always lead to improved expression compared to an unmodified, wild-type nucleic acid sequence. See Alexeyev MF, Winkler HH: Gene synthesis, bacterial expression and purification of the Rickettsia prowazekii ATP / ADP translocase. Biochim Biophys Acta. 1999, 1419: 299-306. 10.1016 / S0005- 2736 (99) 00078-4; Curran KA, Leavitt JM, Karim AS, Alper HS: Metabolic engineering of muconic acid production in Saccharomyces cerevisiae. Metab Eng. 2013, 15: 55-66; Agashe D, Martinez-Gomez NC, Drummond DA, Marx CJ: Good codons, Bad transcript: large reductions in gene expression and fitness arising from synonymous mutations in a Key enzyme. Mol Biol Evol. 2013, 30 (3): 549-560.
[323] [323] A codon-optimized nucleic acid sequence (SEQ ID NO: 2) encoding the human CYP4V2 protein (SEQ ID NO: 4) is provided here. Both SEQ ID NO: 1 and SEQ ID NO: 2 encode the same human CYP4V2 protein (SEQ ID NO: 4). The codon-optimized nucleic acid sequence (SEQ ID NO: 2) has an improved codon adaptation index (CAI) of 0.95 with respect to CAI of 0.94 for the nucleic acid sequence shown in SEQ ID NO: 1. A CAI of 1.0 is considered to be perfect in the desired expression organism. It would be understood that the present description comprises all forms and types of the codon optimized nucleic acid sequence as represented by the cDNA sequence shown in SEQ ID NO: 2, including any RNA sequence or DNA sequence or other nucleic acid sequence corresponding such a cDNA sequence or derivative thereof, and may be in the form of single strand or double strand of, and / or positive, negative, anti or complementary to the sequence provided herein.
[324] [324] In addition to codon optimization, other methods can be used to improve translation performance. For example, Kozak string or Shine-Dalgamo string can be used to increase the efficiency of translation initiation. A different stop codon (for example, TGA) can be used to increase translation completion efficiency. In addition to the ORF sequence, a nucleic acid sequence encoding a functional CYP4V2 protein may also include one or more non-coding sequences such as RTU (s) and / or one or more introns to improve protein expression. A Kozak sequence (exemplary sequence shown in SEQ ID NO: 36) can be inserted immediately before a cDNA encoding CYP4V2 to increase expression.
[325] [325] As discussed here, it is understood that variants and / or functional fragments of the human CYP4V2 protein can be used. A nucleic acid sequence encoding a functional variant (SEQ ID NO: 5) of the human CYP4V2 protein (SEQ ID NO: 4) is provided in SEQ ID NO: 3.
[326] [326] In some embodiments, a CYP4V2 nucleic acid molecule is a polynucleotide molecule that encodes any functional CYP4V2 protein, including, without limitation, SEQ ID NOs: 4-30 or encoding a polypeptide with at least 80% identity of amino acid sequence with any of the sequences shown in SEQ ID NOs: 4-30. In some embodiments, a CYP4V2 nucleic acid molecule is a polynucleotide sharing at least 60% sequence identity with any one of SEQ ID NO: 1, 2 or 3.
[327] [327] A vector (for example, a viral or non-viral vector) and a CYP4V2 expression cassette as described here typically contain one or more CYP4V2 nucleic acid molecules or a fragment thereof. It would be understood that a nucleic acid molecule can take many forms including, without limitation, DNA or RNA, single-stranded nucleic acids (for example, ssDNA, ssRNA), double-stranded nucleic acids (for example, dsDNA, dsRNA), acids nuclei of more or less filament, complementary DNAs (cDNAs), genomic DNA, messenger RNA (mRNA), small interference RNA (siRNA) and / or RNA interference directed to DNA (ddRNAi)). Nucleic acid molecules may also include one or more nucleotide analogs or major structure modifications. Still, it would be understood that a cDNA can be synthesized from an mRNA model in a reaction catalyzed by a reverse transcriptase enzyme, or it can be designed and synthesized based on the protein it intends to encode, including, without limitation. , a codon with codon optimized, or can be synthesized from another nucleic acid molecule through mutagenesis. It would also be understood that a cDNA can contain only exons, or it can contain exons plus other sequences, for example, untranslated regions (RTU) and / or introns. In some cases, a CYP4V2 expression vector and cassette described here may include a nucleic acid molecule that has a sequence encoding the human CYP4V2 protein or a functional variant or fragment thereof.
[328] [328] A suitable nucleic acid sequence can be any nucleic acid sequence that encodes a functional CYP4V2 protein. Such a nucleic acid sequence may or may not contain non-coding elements, such as RTUs, introns or a Kozak sequence. It can include a wild type sequence or a synthetic or modified sequence (for example, a sequence with optimized code). A nucleic acid sequence encoding a functional CYP4V2 protein can be generated as described herein or by other methods known in the art.
[329] [329] A nucleic acid molecule with the sequence as shown in SEQ ID NO: 1 encoding the human CYP4V2 protein is referred to herein as "CYP4V2st". A nucleic acid molecule with a codon-optimized sequence as shown in SEQ ID NO: 2 encoding the human CYP4V2 protein is referred to herein as "CYP4V2op". A nucleic acid molecule with the sequence shown in SEQ ID NO: 3 encoding a functional variant of the human CYP4V2 protein is referred to herein as "CYP4V2fv". In some modalities, a nucleic acid sequence encoding a functional CYP4V2 protein has a sequence identity of at least 60% with one of SEQ ID NO: 1, 2 or 3.
[330] [330] A functional CYP4V2 protein and a nucleic acid molecule encoding such a functional CYP4V2 protein can be synthesized.
[331] [331] A polypeptide can be synthesized (for example, through recombinant protein expression or chemical synthesis) or isolated. As used herein, a "purified" polypeptide is a polypeptide that has been separated or purified from cellular components that naturally accompany it. Typically, a polypeptide is considered "purified" when it is at least 70% (for example, at least 75%, 80%, 85%, 90%, 95% or 99%), through dry weight, free of polypeptides and naturally occurring molecules with which they are naturally associated. Since a polypeptide that is chemically synthesized is, by nature, separate from the components that naturally accompany it, a synthetic polypeptide is "purified".
[332] [332] Polypeptides can be purified from natural sources (for example, a biological sample) using known methods such as DEAE ion exchange, gel filtration and hydroxyapatite chromatography. A polypeptide can also be purified by, for example, expressing a nucleic acid in an expression vector. In addition, a purified polypeptide can be obtained through chemical synthesis. The degree of purity of a polypeptide can be measured using any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis or HPLC analysis.
[333] [333] Polypeptides are typically detected using antibodies
[334] [334] An “isolated” nucleic acid molecule typically refers to a nucleic acid molecule that is free of sequences that naturally flank one or both ends of the nucleic acid in the organism's genome from which the molecule of Isolated nucleic acid is derived (for example, a cDNA or fragment of genomic DNA produced through PCR or restriction endonuclear digestion). Such an isolated nucleic acid molecule is usually introduced into a construct (for example, a cloning construct or an expression construct for use in gene therapy), usually for convenience of manipulation, to express a protein, to generate a fusion protein or for other purposes, including, without limitation, for packaging into a vector (for example, a viral or non-viral vector).
[335] [335] Nucleic acids can be isolated using routine field techniques. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology, site specific mutagenesis, polymerase chain reaction (PCR) and / or other genetic engineering methods. General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Nucleic acid techniques re-
[336] [336] Isolated nucleic acids can also be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.
[337] [337] Constructs containing a nucleic acid are known in the art. Constructs, including cloning constructs and expression constructs, can be custom made commercially or can be produced using routine recombinant DNA techniques in the field. A construct can have regulatory sequences operably linked to a nucleic acid to be expressed, and it can also include sequences such as those encoding a selectable marker (for example, an antibiotic resistance gene). Regulatory sequences are discussed here. A construct containing a nucleic acid can encode a chimeric or fusion polypeptide (i.e., a polypeptide operably linked to a heterologous polypeptide, which may be either at the N or C-terminus of the polypeptide). Representative heterologous polypeptides are those that can be used in purification or detection of the encoded polypeptide (for example, 6xHis marker, glutathione S-transferase (GST), CFP, Fc, FLAG, HA, Myc, RFP, Strep, VSV, GFP and YFP).
[338] [338] Constructs carrying a nucleic acid sequence can be introduced into a host cell. As used herein, "host cell" refers to the particular cell into which the nucleic acid is introduced and also includes the progeny of such a cell that carries the construct. A host cell can be any prokaryotic or eukaryotic cell. For example, host cells can be bacterial cells such as E. coli, or insect cells, yeast or mammalian cells (such as Chinese hamster ovary (CHO) cells, COS cells, HEK293 cells, HeLa, Vero, V27 cells , A549, K562, B50, WI38 and BHK). Other host cells include, without limitation, iPS cells, ES cells, RPE cells, iPS-RPE cells, iPS-photoreceptor cells, ES-RPE cells, ARPE-19 cells, corneal cells, photoreceptor cells, choroid cells, cells of the optic nerve, any other type of ocular cells discussed here, neuronal cells, epithelial cells, blood cells, fibroblasts, lymphocytes and stem cell-derived cells. Many methods for introducing nucleic acids or an expression vector or cassette carrying a nucleic acid transgene into host cells, both in vivo and in vitro, are well known to those of skill in the art and include, without limitation, electroporation, sonoporation, calcium phosphate precipitation, transformation of polyethylene glycol (PEG), thermal shock, lipofection, microinjection and viral-mediated nucleic acid transfer.
[339] [339] Nucleic acids can be detected using any number of amplification techniques (see, for example, PCR Primer: A Laboratory Manual, 1995, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and US Patent Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188) with an appropriate pair of oligonucleotides (e.g., primers). Several modifications of the original PCR have been developed and can be used to detect a nucleic acid. Nucleic acids can also be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sections 7.37-7.57, 9.47-9.57, 11.7 -11.8 and 11.45-11.57). Sambrook and others discuss Southern blot conditions suitable for oligonucleotide probes of less than 100 nucleotides (Sections 11.45-11.46) and Southern blot conditions for oligonucleotide probes greater than about 100 nucleotides (see Sections 9.47-9.54). B. Vectors
[340] [340] In some embodiments, the nucleic acid molecule encoding a functional CYP4V2 protein or fragment thereof is administered to eye cells in need of treatment by means of a vector. For administration to ocular cells, the therapeutic vector is desirably non-toxic and efficient in administering a nucleic acid molecule (for example, DNA, RNA) in target cells. Gene therapy vectors are known in the art and can be viral or non-viral vectors.
[341] [341] One approach for introducing nucleic acid in vivo into a cell is through the use of a viral vector containing a nucleic acid molecule, for example, a cDNA. Infection of cells with a viral vector has the advantage that a large proportion of the target cells can receive the nucleic acid molecule. Also, molecules encoded with the viral vector, for example, by a cDNA contained in the viral vector, are efficiently expressed in cells that have absorbed viral vectors containing the nucleic acid molecule.
[342] [342] Examples of viral vectors that can be used include, without limitation, adenovirus vectors, adenoassociated virus vectors (AAV), lentivirus vectors, herpes virus vectors (HV) such as herpes simplex virus vectors ( HSV), papillomavirus vectors, poxvirus vectors, human foamy virus (HFV) vectors, Epstein Barr virus (EBV) vectors, vaccinia virus vectors, Sendai virus vectors and retrovirus vectors. Plasmids can also be used to deliver a nucleic acid molecule to the target cell. In some cases, the viral vector is a recombinant viral vector such as a recombinant AAV vector (rAAV). It would be understood by one skilled in the art that certain vectors will integrate, or are more likely to integrate, into the genome of the host cell (for example, the individual's cell), while other vectors will not integrate, or are less likely to integrate, into the genome of the host cell. host cell (for example, extrachromosomal expression).
[343] [343] Recombinant AAV vectors (rAAV) are commonly used in gene therapy approaches. AAVs belong to the parvovirus family and each contains a single-stranded DNA. RAAV vectors are currently considered to be the safest and most efficient platform for gene transfer in mammalian cells (Salganik et al., 2015, Microbiol. Spectr., 3 (4): doi: 10.1128 / micro-biolspec.MDNA3-0052 -2014). So far, 12 AAV serotypes (AAV1 to AAV12) and more than 100 variants have been isolated from human and non-human primate tissue samples (see, for example, Gao et al., 2005, Curr. Gene Ther., 5: 285 –97) and other species. Both types of naturally occurring and modified AAV can be used in the methods described here.
[344] [344] Wild-type AAVs contain a linear single-stranded DNA genome within a capsid composed of three VP1, VP2 and VP3 proteins. In recombinant AAVs (rAAVs), the rep and cap genes of the wild-type AAV genome are typically replaced by a transgene expression cassette, flanked by the inverted terminal repeats (ITRs) of AAV required for pairing. As used herein, "rAAV vector" refers to a recombinant AAV vector containing one or more capsid elements from or derived from one or more AAV viruses.
[345] [345] Despite the advantages of AAV and other gene therapy mediated by oral vector, not all so-called viral vectors and not all types of AAV are suitable for the treatment of a particular disease. Two main challenges faced by gene therapy using viral vectors (for example, AAV vectors). First, sufficient transduction efficiency by the AAV vector in the cell type targeted for treatment is desired. Second, potential immune reactions triggered by the viral vector need to be considered. See Madsen et al., Adeno-associated virus serotype 2 induces cell-mediated immune responses directed against multiple epitopes of the capsid protein VP1. J Gen Virol 90, 2622–2633 (2009); Mingozzi et al., CD8 (+) T-cell responses to adeno-associated virus capsid in humans. Nat Med 13, 419–422 (2007). Although as compared to most other organs and tissues, the eye is considered an immunoprivileged organ in relation to many other organs and immune responses in AAV-mediated gene therapy in the eye can be controlled by the use of immunosuppressants, the role of immune responses such as neutralizing antibodies (NABs) in AAV transduction of the eye is unclear in large animals. In addition, intravitreal AAV administration is more susceptible to interactions with the immune system than subretinal administration. In this way, the viral vector used in eye gene therapy will trigger minimal or no immune response, in order to avoid potential side effects and ensure that the transduction / expression efficiency of viral vectors is not substantially reduced by immune reactions, for example , Pre-existing NABs in the individual, and / or decrease the dose of rAAV vectors.
[346] [346] Various compositions and methods with respect to AAV vector design and selection can be used to address these challenges. For use of gene therapy with CYP4V2 to treat BCD, a vector with sufficient transduction efficiency in RPE cells is desired when the cells targeted for treatment are mainly RPE cells. When treating the corneal cells of a patient with BCD, a vector with sufficient transduction efficiency in corneal cells is desired. In some embodiments, a vector with sufficient transduction efficiency in RPE cells is used. In some embodiments, a vector with sufficient transduction efficiency in corneal cells is used. In some modalities, a vector with sufficient transduction efficiency in RPE and photoreceptor cells is used. In some modalities, a vector with sufficient translation efficiency in RPE cells, photoreceptors and choroid is used. In some embodiments, a vector with sufficient transduction efficiency in retinal cells is used. In some embodiments, a vector with sufficient transduction efficiency in eye cells is used. In some embodiments, a vector with sufficient transduction efficiency in eye cells and / or blood cells is used. To address the potential immune response (eg, NAB and cell-based immune responses against gene therapy vectors), different AAV serotypes and variants, AAV vectors and / or modified immunosuppression protocols can be used.
[347] [347] An rAAV vector used here can be based on or derived from a wild-type AAV (for example, from one of AAV1 to AAV12 or other wild-type AAV variants isolated from humans or other species, including , without limitation, AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11 and AAV12) or a modified AAV. A modified AAV can be generated in many different ways, including, without limitation, a pseudo-typified AAV (for example, AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 4, AAV2 / 6, AAV2 / 7, AAV2 / 9, AAV2 / 12, AAV8 / 2), a chimeric AAV (for example, AAV-DJ), an AAV with modified capsid (for example, a mutant AAV in capsid (for example, AAV with YF, KR mutations) , TA, SA and / or TV and AAV-DJ / 8 or AAV-DJ / 9 which are mutant AAVs in AAV-DJ capsid), a variant AAV capsid (for example, AAV 7m8 and derivatives), a Ancestral AAV (eg Anc80) A recombinant AAV involving any change in the genome and / or capsid of a naturally occurring or variant AAV and any combination thereof. It would be understood that there are different ways to refer to a Modified AAV, including, without limitation, artificial AAV, modified, synthesized, reconstructed, engineered, developed, designed, derived or improved or AAV generated through rational design and / or directed evolution and / or shuffling of DNA or an AAV variant. The use of a modified AAV can have certain advantages over an unmodified AAV, including, without limitation, greater transduction efficiency, larger tissue or cell specificity, less immune reactions and / or more suitable for a certain type administration (for example, intravitreal injection or administration through the bloodstream).
[348] [348] In some embodiments, a modified AAV vector used here is a pseudo-typified AAV. AAV pseudotyping refers to the mixture of a capsid and genome of different viral serotypes. These serotypes are denoted using a slash, so that AAV2 / 5 indicates a virus containing the genotype (for example, ITRs) of serotype 2 packaged in the serotype 5 capsid. In some modalities, an AAV vector is a vector of AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 8, AAV2 / 6, AAV2 / 9, AAV2 / 4, AAV2 / 7, AAV2 / 10 or AAV2 / 12.
[349] [349] In some embodiments, a modified AAV vector used here is a chimeric AAV (sometimes also referred to as hybrid or scrambled) that is derived from different AAV serotypes, including different AAV serotypes isolated from species many different. In some modalities, an AAV vector is AAV-DJ, AAV-DJ / 8 or AAV-DJ / 9. AAV-DJ is a variant of AAV generated from the libraries of eight serotype AAV hybrids using the DNA scrambling method. Grimm, D. and others (2008). J. Virol. 82: 5887-5911. It is capable of efficiently transducing a wide range of cell types including eye cells. In addition, chimeric AAVs have more ability to evade immune neutralization than naturally occurring AAVs and can therefore efficiently deliver larger amounts of therapeutic transgene. A hybrid AAV can be modified further. For example, AAV-DJ / 8 and AAV-DJ / 9 were created by making point mutations in the heparin-binding domain (HBD) of AAV-DJ. Grimm, D. and others (2008). J. Virol. 82: 5887-5911.
[350] [350] In some embodiments, a modified AAV used here is an AAV with a mutant capsid. It involves creating one or more mutations (for example, spot mutation) in AAV capsid protein. Mutant capsid AAVs may have advantages over unmodified AAVs. For example, point mutation of tyrosine residues (Y) exposed to the protein surface of the AAV capsid has been reported as a simple and effective method to prevent phosphorylation and subsequent ubiquitination, leading to greater transduction efficiency both in vitro and in vivo (Zhong et al., Proc Natl Acad Sci US A. 2008; 105 (22): 7827–32: Markusic et al., Mol Ther. 2010; 18 (12): 2048–56; Li et al, Hum Gene Ther. 2010 Nov; 21 (11): 1527–1543). For example, site-directed mutagenesis of each of the seven tyrosine residues of the AAV2 capsid (Y252, Y272, Y444, Y500, Y700, Y704 and Y730) by substituting the phenylaniline residue leads to vector transduction and expression of transgene increased by bypassing phosphorylation of EGFR-PTK and the ubiquitin-proteasome pathway in human cells in vitro and murine hepatocytes in vivo (Zhong et al., Virology. 2008 Nov 25; 381 (2): 194-202). It was also reported that point mutations of the AAV capsid in tyrosine (Y), serine (S), threonine (T) and lysine (K) residues could lead to significant transduction improvement both in vitro and in vivo (Gabriel and others, Hum Gene Ther Methods. 2013; 24 (2): 80–93; Sen and others,
[351] [351] In some embodiments, a modified AAV vector is an AAV with variant AAV capsid proteins. Variant AAV capsid proteins are known in the art. In some embodiments, a non-naturally occurring capsid protein may include a selected AAV sequence (for example, a fragment of a vp1 capsid protein) in combination with heterologous sequences (for example, sequences obtained from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, from a non-AAV viral source or from a non-viral source). In some embodiments, a modified AAV vector includes one or more amino acid inserts (for example, from about 5 amino acids to about 11 amino acids) in the GH loop of the capsid protein. Variant AAV capsid proteins can confer increased retinal cell infectivity compared to retinal cell infectivity by a non-variant AAV (for example, wild-type AAVs). In some embodiments, a modified AAV is one that can deliver the transgene through the blood-eye barrier (BOB) which makes it suitable for delivery via the blood stream, offering an alternative route of administration from conventional (for example) , sub-retinal injection or intravitreal injection) used in eye gene therapy. In some modalities, an AAV with AAV capsid protein variants in a 7m8 AAV, or its derivatives or variants (Dalkara et al., Science Translation Medicine, 5: 189ra76, 2013; PCT Application No. PCT / US2012 / 034413, PCT Application No. PCT / US2014 / 039015, US Application No. 14 / 214,011 and US Application No. 13 / 899,481). In some modalities, an AAV with variant AAV capsid proteins is an AAV-PHP-B.
[352] [352] In some modalities, an AAV vector may be re-
[353] [353] In some embodiments, one or more AAVs and / or other viral vectors can be modified (for example, optimized for intravitreal administration, for increased transduction in target cell type (for example, RPE cells) or for administration through the bloodstream) using techniques known in the field including, for example, "directed evolution" and / or "rational design". See, for example, Asuri and others, Mol Ther. 20: 329-338, 2012 and Yang et al., Methods Mol Biol. 709: 127-139, 2011. Modified AAVs or other viral vectors can be described as, for example, “engineered”, “hybrids”, “developed”, “improved” or “designed”. Such modifications can, for example, improve targeting of the vector (for example, improving suitability for intravitreal administration or for administration through the bloodstream), transduction efficiency and / or lower immune reaction, resulting in, for example, a dose minor being required. In some embodiments, an rAAV vector is an AAV rh10 (EP 20100178940) or ShH10 serotype. In some embodiments, an rAAV vector is an AAV-PHP.B (US 20150079038).
[354] [354] In some modalities, an AAV vector can be generated and / or selected from a combination of more than one strategy declared here. For example, AAV-DJ / 8 and AAV-DJ / 9 were created by mutating by point in the heparin binding domain (HBD) of AAV-DJ, a hybrid AAV.
[355] [355] It is known in the art that certain AAVs may be more suitable for intravitreal administration than some other AAVs.
[356] [356] In some modalities, an autonomous AAV vector (scAAV) is used. Wild-type AAVs have a single-stranded DNA genome. A drawback of AAV is its single-stranded DNA genome. Because the single-stranded AAV genome depends on the cell's DNA replication machinery to synthesize the complementary strand, transgene expression is delayed and is not as robust as double-stranded DNA. For gene therapy with CYP4V2, the applicant developed a scAAV design (see Figure 7) to avoid a rate-limiting second-strand synthesis in conventional single-strand AAV vectors and facilitate robust transgene expression. ScAA.CYP4V2 comprises a self-complementary intra-molecular CYP4V2 DNA structure that eliminates the need for host cell DNA synthesis and results in faster and more robust expression when transducing. The self-complementary structure of a scAAV, however, reduces the scAAV vector packaging limit from from 4.7-5.0 kb for ssAAV to about 2.4-2.5 kb for scAAV. In this way, shorter-length regulatory sequences (eg, promoter, enhancer and / or polyA signal) are required in a scAAV project. To ensure that the expression cassette does not exceed the vector packaging limit and depending on the length
[357] [357] Several other vector designs can be used. For example, a double vector system (for example, an AAV-based double vector system, for example, hybrid double or trans AAV vectors) can be used to express a nucleic acid sequence (for example, example, a CYP4V2 nucleic acid sequence). See, for example, Colella, et al., Gene Ther. 21, 450–456, 2014. For example, a double vector system can include (i) a first AAV vector polynucleotide having an inverted terminal repeat at each end (5 'and 3' ends) of the polynucleotide, and among the inverted terminal repeats, a suitable promoter operably linked to a partial coding sequence that encodes an N-terminal part of the protein encoded by the nucleic acid sequence of interest; and ii) a second AAV vector polynucleotide having an inverted end repeat at each end (5 'and 3' ends) of the polynucleotide, and between inverted end repeats, a partial coding sequence encoding a C part -terminal of the protein encoded by the nucleic acid sequence of interest, followed by a polyadenylation signal sequence (pA).
[358] [358] Several rAAV vectors were designed and generated for the present study, including scAAV2 / 1, AAV2 / 2, AAV2 / 5, scAAV2 / 5, AAV2 / 8, scAAV2 / 9 and AAV2 / 2 (Y444F + Y500F + Y730F ) (see schematic drawings and notes in Figure 7 in this report). They demonstrate that rAAV vectors from various vector designs can be used in gene therapy with CYP4V2. In addition, the inclusion of multiple rAAV vectors as options can help reduce the potential immune response in gene therapy with CYP4V2 given the pre-existing neutralizing antibodies and another individual immune response against certain types of AAV among the patient population. It would also provide more options if a subsequent administration to the same eye or an administration to the contralateral eye of the same individual is desired.
[359] [359] Methods for producing viral delivery vectors, including production using an auxiliary-free system, are known in the art. See, for example, PCT / US2007 / 010055; Patent No. 6458587, Patent No. US 6428988 B1). Production of various vectors used in gene therapy, including, without limitation, AAV, adenovirus, lentivirus and retrovirus vectors, is also commercially available through contract research organizations (CROs) and contract manufacturing organizations (CMOs), for example. , Vector Biolabs (Malvern, PA) and Cell Biolabs, Inc., (San Diego, CA).
[360] [360] In some embodiments, a recombinant AAV vector useful in the methods described here can be generated by culturing a host cell (for example, a HEK293 cell) that contains a nucleic acid molecule encoding a serotype capsid protein from AAV or fragment thereof; a rep gene; a mini-gene comprising, at a minimum, inverted terminal repeats (ITRs) from AAV and a nucleic acid molecule of interest (for example, having a CYP4V2 nucleic acid sequence); and sufficient auxiliary functions to allow packaging of the nucleic acid of interest in the AAV capsid protein. The components required to be cultured in the host cell to package a nucleic acid in an AAV capsid can be provided in the host cell in cis or trans. Alternatively, any one or more of the required components (for example, nucleic acid molecule of interest, rep sequences, cap sequences and / or auxiliary functions) can be provided by a stable host cell that has been engineered to contain one or more of the required components. Any of these components can be selected from any suitable serotype. For example, rAAV vectors are generated by cotransfecting producer cells (eg HEK 293 cells) with (a) a plasmid (AAV cis-plasmid) containing a cloned recombinant AAV genome composed of the gene of interest (for example , a cDNA encoding CYP4V2) and other desired regulatory sequences flanked by two AAV ITRs, (b) a separate construct expressing the viral Rep and Cap genes in trans. (c) auxiliary adenovirus factors, which are provided either by adenovirus infection or transfection in cells producing a third plasmid that provides these adenovirus auxiliary factors. In addition to HEK293 cells, other cell lines can be used in the production of rAAV vectors, including, without limitation, HeLa, Veer, A549, B50, WI38 and BHK cells.
[361] [361] In some embodiments, the viral delivery vector is a rAAV2 virus, a rAAV2 / 5 virus, a rAAV2 / 8 virus, a rAAV2 / 1 virus, a rAAV2 / 4 virus, a rAAV2 virus / 6, a rAAV2 / 9 virus, a rAAV2 / 12 virus or an rAAV virus with capsid elements from one or more of AAV1, AAV2, AAV5, AAV8, AAV9 and / or AAV12 viruses. In one embodiment, the viral delivery vector is rAAV virus with one or more Y-F mutations, including, without limitation, AAV2 (Y444F + Y500F + Y730F) or AAV8 (Y733F).
[362] [362] In some embodiments, the viral delivery vector is a single-stranded rAAV virus (ssAAV). In some modalities, the viral delivery vector is an autocomplete rAAV virus.
[363] [363] In addition to AAV vectors, other viral vectors can be used in gene therapy with CYP4V2. For example, adenoviral vectors have also been shown to be useful for gene administration. For example, Mori et al., 2002. IOVS, 43: 1610-1615 reveal the use of an adenoviral vector that is an Ad type 5 with deleted E-1, partially deleted E-3 in which the transgene (protein fluorescent green) is driven by a CMV promoter. Peak expression levels were demonstrated when injecting 10 ^ 7 to 10 ^ 8 viral particles, with subretinal injection providing higher levels of expression than intravitreal injection.
[364] [364] In some embodiments, the delivery vector is a plasmid containing a nucleic acid molecule encoding the human CYP4V2 protein or a functional variant or fragment thereof.
[365] [365] Non-viral vectors can also be used in gene therapy with CYP4V2. Examples of non-viral vectors include, without limitation, naked nucleic acids, dendrimers, liposomes (eg, cationic or anionic liposomes), polymers (eg, polyplexes), lipid-polymer systems and nanoparticles (eg, inorganic or synthesized nanoparticles). For example, efficient non-viral ocular gene transfer was demonstrated by Farjo et al., 2006, PLoS 1: e38, who used compacted DNA nanoparticles as a system for transferring non-viral gene to ocular tissues . As a proof of concept, the expression construct pZEEGFP5.1 (5,147 bp) that encodes the transcriptively enhanced green protein (GFP) cDNA controlled by the CMV immediate-early promoter and enhancer. DNA nanoparticles were formulated by mixing plasmid DNA with CK3OPEG10K, a 30 mer lysine peptide with an N-
[366] [366] In addition, several patents have been issued for ocular gene transfer methods including, but not limited to, Pat. No. 7,144,870 which provides hyaluronic acid mediated adenoviral transduction methods; Pat. U.S. Nos. 7,122,181 and 6,555,107 that provide lentiviral vectors and their use to mediate ocular gene administration; Pat. No. 6,106,826 which provides herpes simplex viral vectors and their use to mediate ocular gene administration; and Pat. No. 5,770,580, which provides DNA expression vectors and their use to mediate ocular gene administration.
[367] [367] A method of evaluating and selecting vectors suitable for use in gene therapy with CYP4V2 of different vectors is provided in the Examples section here. The Examples used different AAV vectors to illustrate the method. It would be understood that such a method can also be used by the person skilled in the art to compare and select from different types of vectors, for example, non-viral, adenovirus vs. AAV, lentivirus vs. AAV, HSV vs. AAV, etc. C. CYP4V2 Expression Cassettes and Regulatory Sequences
[368] [368] The description also provides an expression cassette comprising a nucleic acid sequence encoding a functional CYP4V2 protein (for example, a nucleic acid sequence of SEQ ID NO: 1, 2 or 3) and a sequence of expression control operably linked to the nucleic acid sequence encoding CYP4V2. In addition to the nucleic acid molecule encoding a functional CYP4V2 protein, the other key elements of an expression cassette used in gene therapy with CYP4V2 include one or more regulatory sequences to control the expression of said nucleic acid molecule. In some embodiments, the expression cassette is packaged in an administration vector (for example, in an rAAV vector flanked by AAV ITRs) for increased administration, transduction and / or expression efficiency. Any AAV ITRs can be used in the methods described here. The ssAAV vectors described in the present Examples contain two AAV2 ITRs of about 141 bp each (exemplary sequences shown in SEQ ID NO: 40). The scAAV vector described in the Examples contains two AAV2 ITRs, one of which is truncated (exemplary sequences shown in SEQ ID NO: 41). An AAV2 ITR generally has a length of about 132 to about 167 bp depending on the parental vector being used.
[369] [369] As used herein, the term “regulatory sequence” refers to any genetic element (for example, polynucleotide sequence) that may have a regulatory effect on replication or expression (transcription or translation) of the nucleic acid, or otherwise directs, influences and / or regulates expression of the nucleic acid sequence. Common expression control sequences include promoters, polyadenylation (polyA) signals, enhancers, upstream regulatory domains, introns, RTUs, response elements or inducible elements, origins of replication, internal ribosome entry sites (IRES), transcription start sequences, termination sequences, RNA processing sequences such as binding and polyadenylation sequences (polyA), sequences that stabilize cytoplasmic mRNA, sequences that increase translation efficiency (ie, consensus sequence Kozak), sequences that increase protein stability or sequences that increase
[370] [370] Various promoter sequences can be used to target the expression of a nucleic acid coding sequence. Some promoters are constitutive promoters, which target expression in virtually all tissues and in most cell types. While other promoters are more controlled. Regulated promoters would act only on certain tissues or cells
[371] [371] In some cases, it may be desirable to use a constitutive (or ubiquitous) promoter. Exemplary constitutive promoters include, without limitation, the cytomegalovirus (CMV) promoter (Gray et al., Hum Gene Ther. 2011 Sep; 22 (9): 1143–1153; Norman et al., PLoS ONE 5 (8): e12413, Ag 2010 ), the chicken β-actin promoter, the hybrid CAG promoter (tcc CAGGS, CBA or CB) derived from chicken CMV / beta-actin / rabbit beta-globin (Miyazaki J, Takaki S, Araki K, Tasha F, Tominaga A, Takatsu K, Yamamura K. 1989. Expression vector system based on the chicken β-actin promoter directs efficient interleukin-5 production Gene 79: 269–277; Acland, GM and others MoI Then , 2005, 12: 1072-1082), the small CBA (smCBA) promoter (~ 953 pbs, see, Mah, et al. 2003, Hum. Gene Ther. 14: 143-152; Haire, et al. 2006 IOVS, 2006, 47: 3745-3753), the CBh promoter (~ 800 pbs, see, Gray and others, Hum Gene Ther. 2011 Sep; 22 (9): 1143–1153), the human β-actin (ACTB) promoter ( Norman et al., PLoS ONE 5 (8): e12413, Ag 2010), the factor 1 alpha stretching promoter (EF-1 alpha) (see, Gill et al., Gene Ther. 2001; 8 (20): 1539–1546; Norman et al., PLoS ONE 5 (8): e12413, Ag 2010), the phosphoglycerate kinase promoter (PGK, human or mouse) (Norman et al., PLoS ONE 5 (8): e12413, Ag 2010), the promoter Ubiquitin C (UBC) (Norman et al., PLoS ONE 5 (8): e12413, Ag 2010), the GUSB promoter (Glucuronidase Beta), the minimum GUSB promoter (hGBp) (Husain, Gene Therapy (2009) 16, 927– 932), the UCOE promoter, the 1α short elongation factor (EFS) promoter, the Simian 40 virus promoter (SV40). The Rous sarcoma virus (RSV) promoter. Saw-
[372] [372] In some cases, it is desirable to use a cell-specific or tissue-specific promoter that directs the expression of a nucleic acid coding sequence in a particular type of cell or tissue. Based on the present description, it would be understood that a cell-specific or tissue-specific promoter can be specific for an ocular cell or tissue or for lymphocytes. Ocular cell types include, without limitation, retinal cells, bipolar retinal cells, photoreceptor cells, rod cells and cone cells, ganglion cells, retinal pigment epithelium (RPE) cells, choroidal cells or corneal epithelial cells. Thus, a cell-specific promoter as described herein can be a retinal-specific promoter (for example, RPE-specific, receptor-specific (for example, cone-specific and / or rod-specific) and / or specific choroid) or a specific corneal promoter. Exemplary eye cell specific promoters include, without limitation, the receptor protein kinase coupled to human G protein 1 tcc rhodopsin kinase promoter 1 (GRK1) (Genbank Accession Number AY327580), a 292 nt fragment (positions 1793- 2087) of the GRK1 promoter (see Beltran et al., Gene Therapy 17: 1162-74, 2010), the human interphotoreceptor retinoid-binding protein (IRBP) proximal promoter, a 235 nt fragment of the hIRBP promoter, the proximal promoter RPGR, the red opsin promoter, the red-green opsin promoter, the blue opsin promoter, the mouse opsin promoter (both long and short versions, Le et al., Molecular Vision 2006; 12: 389-398; Beltran et al., Gene Therapy 17: 1 162-74, 2010), the rhodopsin (Rho) promoter (Mussolino et al., Gene Therapy, 18: 637-45, 2011); the alpha subunit of the cone transducin (Morrissey et al., BMC Dev, Biol, 11: 3, 2011); beta phosphodiesterase (PDE) promoter; promoter retinitis pigmentosa (RP1) (Nicord et al., J. Gene Vied. 9: 1015-23, 2007), the NXNL2 / NXNL1 promoter (Lambard et al., PLoS One, 5: el3025, 2010), the RPE65 promoter (Li and others, Investigative Ophthalmology & Visual Science, December 2002, Vol.43, 3640); the slow retinal degeneration / peripheral promoter 2 (Rds / perphZ) (Cai et al., Exp Eye Res, 91: 186-94, 2010), the VMD2 promoter (vitelliform macular dystrophy 2; tcc BEST1, Kachi and others, Human Gene Therapy, 20: 31-9, 2009), the IRBP / GNAT2 promoter (hIRBP promoter fused to alpha promoter of the transducin cone), the Rds promoter (slow retinal degeneration), the hPDE6b promoter or the promoter VEcad (VE-cadherin / Cadherin 5 (CDH5) / CD144 promoter). It would be understood that other promoters that are known in the art can be used in place of, or in addition to, any of the exemplary promoters provided here based on the reasoning and discussions provided here.
[373] [373] Exemplary inducible promoters include, without limitation, a calcium-sensitive promoter (for example, an NFAT promoter, see Gene Ther. 2013, Mar; 20 (3): 248-54), the inducible metallothionine promoter zinc (MX), the mouse mammary tumor virus (MMTV) promoter inducible by dexamethasone (Dex), the T7 polymerase promoter system; the acdisone insect promoter, the tetracycline repressible system, the tetracycline inducible system, the RU486 inducible system, the rapamycin inducible system, several commercial inducible promoters available and inducible promoters regulated by a specific physiological state , for example, temperature
[374] [374] A promoter may be a hybrid of either a truncated / shortened or modified version of or otherwise derived from another promoter and / or another regulatory sequence, for example, the CAG promoter is an immediate early enhancer hybrid of CMV, chicken beta-actin promoter and rabbit beta-globin gene, the smCBA promoter is a truncated version of the CBA promoter. A promoter can contain other elements, for example, an intron, exon and / or an enhancer, for example, the CAG promoter. More than one promoter can be used together on an expression cassette.
[375] [375] In some cases, it may be desirable to use an enhancer sequence in order to increase and / or stabilize expression above that which occurs due to the promoter. Representative enhancement sequences include, without limitation, a post-transcriptional regulatory element (eg, a post-transcriptional regulatory element of the hepatitis woodchuck virus (tcc WPRE) or a post-transcriptional regulatory element of the Hepatitis B virus (tcc HPRE) or HBVPRE, Donello et al., J Virol. 1998 Jun; 72 (6): 5085-92; Sun et al., DNA Cell Biol. 2009 May; 28 (5): 233–240), or several shortened WPREs, mutants or modified, for example, a shortened WPRE of ~ 247 bps containing minimal gamma and alpha elements from the WPRE (Choi et al., Mol Brain. 2014; 7: 17; Donello et al., J Virol. 1998 Jun; 72 (6) : 5085–5092; Zanta-Boussif et al., Gene Therapy (2009) 16, 605–619) or the 1RBP enhancer (Nicord et al., J. Gene Vied. 9: 1015-23, 2007), a trans element enhancer - constitutive bearing (CTE) (for example, Macon-Pfizer Monkey Virus CTE or Avian Leukemia Virus CTE), the
[376] [376] Polyadenylation of a transcript is important for nuclear export, translation and mRNA stability. Thus, the transcript polyadenylation efficiency is important for transgene expression. Representative PoliA signal sequences include, without limitation, an SV40 polyA signal, an SV40 late polyA signal, an SV40 early polyA signal, a bovine growth hormone polyadenylation signal (polyA bGH), a polyA or a sign of polyadenylation of human growth hormone (polyA hGH). In some cases, an upstream enhancement sequence (USE) can be used to increase the efficiency of a polyA signal, for example, 2xUSE of late SV40, USE of HIV-1 (human immunodeficiency virus 1), USE of GHV (Squirrel hepatitis virus), USE of adenovirus (L3) (Adenovirus), USE of hTGBH (Human prothrombin) or USE of hC2 (Gene of human complement C2) (Schambach A, Galla M, Maetzig T, Loew R, Baum C. Improving transcriptional termination of self-inactivating gamma-retroviral and lentiviral vectors. Mol Ther. 2007; 15 (6): 1167–1173).
[377] [377] Like promoter sequences, the other regulatory sequences used in an expression cassette can be a hybrid of, shortened / truncated, modified or otherwise derived from a regulatory sequence. For example, the shortened WPRE, the late SV40 2xUSE, the late SV40 polyA. In addition to the elements described here, an expression cassette can also contain other regulatory sequences, for example, introns, RTUs and similar sequences. The inclusion of a splicing site (ie, exon flanked by two introns) has been shown to be useful for increasing the expression gene of expression cassette proteins.
[378] [378] It is known in the art that it is common for a regulatory sequence or a hybrid regulatory sequence to have multiple versions and to have more than one name. For example, several promoters, enhancers and polyA signals have multiple versions, including, without limitation, the CMV promoter, EF1α promoter, WPRE enhancer and SV40 polyA signal. The CAG promoter has multiple alternative names including, without limitation, the CBA promoter, CB promoter or CAGGS promoter. Furthermore, it is also known in the art that a regulatory sequence can be shortened, modified or combined with other sequences to generate a derivative or variant, for example, the CAG promoter (tcc CBA, CB or CAGGS) is an immediate early enhancer hybrid of CMV, chicken beta-actin promoter and rabbit beta-globin gene, the smCBA promoter is a truncated CAG promoter. The CBSB promoter is a shortened CAG promoter, differing by about 152 bp at the 5 'end of the CMV immediate early enhancer. In addition, a regulatory sequence can be called differently, for example, a post-transcriptional regulatory element such as HPRE or WPRE can also be referred to as an enhancer. Any regulatory sequences described here comprise all variations, derivatives and / or hybrids of such regulatory sequence. Any exemplary sequence provided here with respect to a regulatory sequence is exemplary in nature and does not limit the definition or scope of such a regulatory sequence to that shown in the exemplary sequence.
[379] [379] In some embodiments, the microRNA (miRNA) technique can be used in the expression cassette design to obtain targeted expression specificity, for example, by suppressing off-target transgene expression. Just as an example and not a limitation, a target sequence for miR191 (a miRNA shown to be expressed exclusively in ganglion cells and internal retina) can be added immediately downstream of CYP4V2 cDNA to inhibit CYP4V2-mediated protein expression synthesis cassette in ganglion cells and internal retinal cells. Similarly, a target sequence for a miRNA that is exclusively expressed in certain cell types can be used to suppress cassette-mediated CYP4V2 protein expression in these cell types to obtain specific tissue or targeted cell expression. D. Efficient Expression Cassette Design and Administration Vectors for CYP4V2 Gene Therapy
[380] [380] A detailed discussion of the CYP4V2 expression cassette and delivery vector design method and several designs for these studies are provided in the Examples section here. Use of EFS promoter and / or Small PoliA Sign (SPA) in the treatment of an eye disease
[381] [381] As discussed here, a gene delivery vector has a packaging size limit. For example, single-stranded AAV vectors have a packaging limit of about 4.7-5.0 kb, with the overrun the efficiency of transduction and expression would drop significantly. For self-compliant AAVs (scAAVs), the packaging limit is halved to about 2.4-2.5 kb. In this way, size does not matter for vector-mediated gene administration and gene therapy. To see
[382] [382] The project using an EFS promoter and a SPA occupies only about 300 bps and together with the AAV ITRs it occupies a total of about 600 bps, thus leaving remaining packaging space of about 1.8- 1.9 kb for sequencing nucleic acid
[383] [383] An expression cassette layout comprising the EFS promoter and the SPA is provided in Figure 4b. The construct shown in Figure 7b includes a CYP4V2 cDNA. The CYP4V2 cDNA can be replaced by another gene of interest for the expression cassette to be used for another transgene expression.
[384] [384] The use of the EFS promoter and the SPA in an expression cassette and an administration vector to target an acid coding sequence to treat an eye disease was tested in this study. A scAAV2 / 1 vector containing the EFS promoter, a CYP4V2 cDNA and the SPAM called scAAV1.EFS.CYP4V2op.SPA, was generated. ScAAV1.EFS.CYP4V2op.SPA was applied to the BPS patient's iPS-RPE cells. ScAAV1.EFS.CYP4V2op.SPA showed fast and robust action on iPS-RPE cells from a patient with BCD in just 4 days despite the short lengths of the EFS promoter and the SPA (see Table 3). It demonstrates that the EFS and / or SPA promoter are small regulatory sequences that are very useful in an scAAV system for eye gene therapy. Furthermore, the robust expression of scAAV vectors makes the scAAv design also suitable for other routes of administration (for example,
[385] [385] The use of the EFS and / or SPA promoter is not limited to gene therapy with CYP4V2 or in a scAAV construct. They can be used for gene therapy involving other genes, where the size of the transgene and / or a scAAV project requires the use of a short-length polyA promoter and signal to target fast and sufficient protein expression. E. Treatment Options, Individual Selection and Administration
[386] [386] CYP4V2 gene therapy can be applied in multiple ways. In some cases, treatment can be applied in vivo to an individual (for example, a patient with BCD) through effective administration of administration vectors containing the CYP4V2 expression cassette to cells, tissues or organs targeted for treatment , for example, RPE, photoreceptors, choroid, cornea, lymphocytes, the retina or the eye, of the individual. In some cases, treatment may be applied in vitro to targeted cells (eg, patient's iPS-RPE cells, patient's iPS-photoreceptor cells, photoreceptor iPS-progenitor cells, iPS-CEC, lymphocytes). Then the treated cells can be transplanted to an individual in need (for example, a patient with BCD). In some cases, treatment can be applied by combining both in vivo and in vitro approaches. In some cases, gene therapy with CYP4V2 can be used independently. In some cases, gene therapy with CYP4V2 can be used with another treatment option.
[387] [387] Individuals who are candidates for the present treatment methods include those who are diagnosed with BCD. Individuals suffering from other clinically defined ophthalmological conditions (eg, inherited retinal degeneration (IRD), pigmented retinitis (RP) or corneal dystrophy) caused by mutations in the gene
[388] [388] Several mutations have been identified in the CYP4V2 gene and causing BCD, with at least one mutation in each of the gene's 11 exons. Genotype analysis showed that the most common CYP4V2 mutation among patients with BCD is c.802- 8_810del17insGC (with reference to a deletion at base 17 with two bases (GC) inserted in place starting 8 bases from the end intron 6 of GYP4V2 gene, also referred to as IVS6- 8del / insGC; this insertion-deletion mutation is at the junction of intron 6-exon 7 and the 17 bp deletion includes the exon 7 acceptor site, leading a deletion in structure of exon 7 coding for 62 amino acids) resulting in the jump of exon 7. (Xiao et al., Bio-chem Biophys Res Commun. 409: 181-6, 2011; Meng et al., 2014, Mol. Vis ., 20: 1806-14; Wada et al., Am J Ophthalmol. 139: 894-9, 2005; Jiao et al., European Journal of Human Genetics (2017) 25, 461-471). Various types of mutations have been found in CYP4V2 mutations associated with BCD, including, but not limited to, erroneous, union site, structure change, deletion, insertion, indel,
[389] [389] It should be noted that the human CYP4V2 mutations in Table 1 are not exhaustive. More CYP4V2 mutations may be identified in the future. It should be understood that not all changes to the reference sequence are mutations. Some variations are non-pathological. Methods for confirming whether a genetic variation is pathological, that is, a mutation, are known in the art, including, but not limited to, comparing the variation with previously known clinically identified mutations and / or determining whether a corresponding change in function exists. For example, a method for determining whether a genetic variation is a pathological variation (ie, a mutation) is to test the biochemical functions of the individual derived iPS-RPE cell line as described here and to assess whether there are any abnormalities compared with those of healthy control iPS-RPE cell line.
[390] [390] Patients with BCD or another ophthalmic condition due to CYP4V2 mutations that can be treated using a method described here preferably retain some photoreceptors and visual function, for example, as measured by visual acuity, visual field, visual function and / or Optical Coherence Tomography (OCT, for example, Spectral Domain-OCT (SD-OCT)).
[391] [391] Before administration, the final product will undergo a series of steps (for example, ultrapurification) to satisfy clinical grade criteria. Clinical-grade productions are commercially available through several GMP facilities, including, without limitation, facilities in the NIH Gene Therapy Resource Program (GTRP) and contract manufacturing organizations (CMOs).
[392] [392] Prior to administration, the individual can test for pre-existing neutralizing antibodies (NAb) against the type of AAV vector that the individual is about to receive the administration. In a modality, if the individual has pre-existing NAb against such a type of AAV, an alternative AAV vector with low cross-reactivity to the individual's pre-existing NAb or an AAV vector with modified capsid structure can be used for administration to such individual to decrease immune reactions and retain sufficient transduction efficiency by the AAV vector. Other methods for minimizing immune response are known in the art, including, without limitation, application of immunosuppression agents and protocols before, during and / or post-treatment.
[393] [393] Viral or non-viral vectors, or combinations thereof (for example, hybrid vectors), can be administered to an individual's eye cells using one or more physical means. Other cells as used herein refer to, without limitation, retinal pigment epithelium (RPE) cells, photoreceptor cells, corneal epithelial cells, retinal cells, bipolar retinal cells, rod cells, cone cells, ganglion cells, ganglion cells, choroidal and / or lens cells. In addition to, or alternatively, vectors may be administered to surrounding or neighboring cells or cells that may come into contact with targeted cells, including, without limitation, cells in the brain or cells in the optic nerve or blood cells.
[394] [394] In vitro treatment can use any method or combination of methods and / or agents that effectively deliver a vector to the targeted cell for treatment (for example, a cell
[395] [395] Cells treated in vitro can then be transplanted into the individual's eye. For example, iPS-RPE cells that are genetically repaired from a patient with BCD can be transplanted into the patient through sub-retinal injection. Methods, agents and devices used in cell transplantation to the eye are known in the art, see, for example, Wert et al., J Vis Exp. 2012; (69): 4286; WO 2016/179496; Schwartz et al., Investigative Ophthalmology & Visual Science April 2016, Vol.57, ORSFc1-ORSFc9.
[396] [396] For in vivo treatment, the expression vector and / or cassette can be administered to cells targeted for in vivo treatment (for example, by administration to the eye of an individual in need of treatment for administration. cells targeted for treatment). Methods of administering a nucleic acid molecule, an expression cassette, a vector to a target eye cell in vivo are known in the art. For example, administration to the eye can use any method (or a combination of methods and / or agents) that effectively deliver a vector to the retina, the sub-retinal space, the choroid, or generally the posterior segment of the eye, the cornea, lens or vitreous, depending on the right cells
[397] [397] In addition to conventional administration to RPE cells using subretinal injection, one aspect of the methods discussed here is intravitreal administration of the nucleic acid molecule (for example, having a non-mutant CYP4V2 nucleic acid sequence) for treatment or prevention of an eye disease. Some vectors (for example, AAV2 (quadY-F + T-V) and AAV 7m8) show particular promise for efficient transduction in the retina through cross-administration. In addition, AAVs or other viral vectors can be modified using techniques known in the field including, for example, "directed evolution" or "rational design" to improve or optimize their suitability as vectors for gene administration to one or more cell types or tissues (for example, intravitreal injection) other than through conventional sub-retinal injection. ScAAV vectors can also be used in intravitreal administration in addition to subretinal administration due to their immediate and robust expression profile. Due to the fact that CYP4V2 is almost ubiquitously distributed with particularly high expression on the retina, genetic and epigenetic changes of CYP4V2 are particularly suitable for repair through intravitreal administration of one or more vectors. Current gene therapy methods generally require subretinal administration of the vector. In this way, one of the technical advances achieved by the materials and methods disclosed here is the intravitreal administration of a nucleic acid sequence (for example, a wild-type or non-mutant nucleic acid sequence or a nucleic acid sequence encoding genetic editing polypeptides ) and / or a polypeptide for the treatment and prevention of eye diseases associated with genetic or epigenetic changes in the CYP4V2 nucleic acid sequence.
[398] [398] Certain techniques and agents can be used to facilitate administration processes. Non-limiting examples including the use of a lubricating agent so that adherence of the vector to the delivery vehicle (for example, a needle) is avoided. In addition, the use of immunosuppressive drugs before, during and / or after the administration process can increase the efficiency of infection or transduction.
[399] [399] A vector can be formulated to deliver to an individual's eye cells using various pharmaceutically and / or physiologically acceptable excipients, diluents and / or carriers. Examples of excipients, diluents and / or carrier carriers suitable for administration to the eye, which may be referred to as pharmaceutically acceptable carriers, include sterile, pyrogen-free water, and sterile, pyrogen-free, buffered saline (for example , buffered saline using phosphate or other buffers such as HEPES to maintain pH at appropriate physiological levels), isotonic sodium chloride solution, balanced saline,
[400] [400] Methods of determining the most effective means of administration and therapeutically effective dosages are known to those of skill in the art and will vary with the vector, its capsid structure, the design of the vector (eg ssAAV vs. scAAV ), the composition of the expression cassette, the expression levels of the vector, the promoter, other regulatory sequences or the nucleic acid molecule, the vector title, the type of target cell, the expression levels- target, the size of the area or number of cells targeted and the individual being treated (for example, age, sex, weight, stage of development of the disease and condition of the individual to be treated and potential immune reactions); the route of administration; the location of cells targeted for treatment (for example, retina vs. cornea); the nature and level of expression of the relevant gene in wild-type cells and / or tissue; and the required regime. Therapeutically effective doses can be determined and evaluated in disease models (eg, BCD cell model (eg, iPS-RPE cell line from patients with BCD) or an animal model, and confirmed or refined by clinical tests. cells in vivo, the dose is usually expressed as MOI and then multiply the MOI by the number of cells being treated.The MOI generally ranges from about 1 x 10 ^ 3 GC to about 1 x 10 ^ 6 GC per cell or an infectious MOI of about 100 to about 10,000 GC per cell (GC: genome copies, measurement genome containing AAV particles (tcc vector genome (vg) or genome particles (gp)). For in vivo treatment, in addition to the factors described above, the actual dose administered can also be affected by individual situations specific to each patient during administration, for example, a reduced dose during sub-retinal administration for patient 6 in the case Choroidemia described below O. In this way, the therapeutically effective dose for a single in vivo administration can be on the order of from about 1 x 10 ^ 6 to 2 x 10 ^ 13 GC, inclusive (for example, a high dose range of about from 1 x 10 ^ 11 GC to about 1 x 10 ^ 12 GC, an average dose range of about 1 x 10 ^ 10 GC to about 1 x 10 ^ 11 GC, a low dose range of about 1 x 10 ^ 9 GC at about 1 x 10 ^ 10 GC, a very low dose range of about 1 x 10 ^ 6 GC at about 1 x 10 ^ 9 GC and a very high dose range of about 1 x 10 ^ 12 GC at about 2 x 10 ^ 13 GC), or any dose within these ranges that is sufficient to provide the desired effect. In one embodiment, the composition is administered at a dose of about 1 x 10 ^ 6 to 2 x 10 ^ 13 GC. In another embodiment, the dose administered in vivo is determined by multiplying the number of cells targeted for treatment by the target MOI (for example, 1 x 10 ^ 3 GC to about 1 x 10 ^ 6 GC per cell)) . The volume of the agent containing the rAAV vectors in any single administration to the eye can vary from about 1 µL (0.001 ml) to about 1000 µL (1 ml).
[401] [401] The compositions as described here can be formulated as a single dose or a plurality of doses. Similarly, administration can occur once or a plurality of times (for example, over several weeks, months or years) and can be applied to the same eye or the contralateral eye. Under circumstances of multiple administrations, the same or different AAAV serotypes and / or route (s) of administrations can be considered. Administration can also be applied to treat different tissues and cells, for example, one administration targeting RPE and another administration targeting the cornea.
[402] [402] Methods of viral vector generation, GMP production, purification, formulation and doses for use in gene therapy (including eye gene therapy) are known to those of skill in the art, and methods of preparing viral vectors can be performed. - through any number of companies and methods as demonstrated in several ACL group gene therapy studies below. Expression cassettes provided here can be inserted into any of the exemplary viral vectors listed below. Alternatively, viral vectors can be generated based on the examples provided below. See Bainbridge et al., 2008. N Engl J Med. 358: 2231-9; Maguire et al., 2008. N Engl J Med. 358: 2240-8 ;; Hauswirth et al., Hum Gene Ther. 2008 Oct; 19 (10): 979–990.
[403] [403] For example, in the Bainbridge study, the vector tgAAG76, a recombinant adenoassociated virus vector of serotype 2, was used for gene administration. The vector contains the human RPE65 coding sequence directed by a human RPE65 promoter and terminated by the bovine growth hormone polyadenylation site, as described elsewhere. The vector was produced by Targeted Genetics Corporation according to Good Manufacturing Practice guidelines using a B50 packaging cell line, an adenovirus-associated adenovirus-hybrid hybrid vector containing the genome of the tgAAG76 vector and a virus adenovirus helper 5. The vector was filled into a buffered saline solution in a titre of 1 x 10 ^ 11 vector particles per milliliter and frozen in 1 ml aliquots at -70º C.
[404] [404] Maguire used the recombinant AAV2.hRPE65v2 viral vector which is a replication-deficient AAV vector containing RPE65 cDNA that has been documented to provide sustained, long-term (> 7.5 years, with ongoing observation) function restoration visualization in a canine model of LCA2 after a single sub-retinal injection of AAV2.RPE65. The cis plasmid used to generate AAV2.RPE65 contains the kanamycin resistance gene. The virus was manufactured by The Center for Cellular and Molecular Therapeutics after triple transfection of HEK293 cells and was isolated and purified by microfluidization, filtration, cation exchange chromatography (POROS 50HS; GE Healthcare, Piscataway, NJ), ultracentrifugation- density gradient and diafiltration in PBS. This combination provides optimum purity of the AAV vector product, including efficient removal of empty capsids and residual cesium chloride. A portion of the product was supplemented with PF68 NF Prill Poloxamer 188 (PF68; BASF, Ludwigshafen, Germany) to provide subsequent vector losses for product contact surfaces. The purified virus, with or without PF68, was then passed through a 0.22 µm filter using a sterile 60 ml syringe and syringe filter and frozen stored (-80º C) in sterile tubes until use. An injection of 1.5x10 ^ 10 of AAV2.hRPE65v2 vector genome in a volume of 150 µl of phosphate buffered saline supplemented with Pluronic F-68 NF Prill Poloxamer 188 was administered to the subretinal space.
[405] [405] The viral vector used by Hauswirth was a serotype 2 (rAAV2) recombinant virus associated vector, altered to carry the human RPE65 gene (rAAV2-CBSB-hRPE65), which had previously been shown to restore vision in models animals with a deficiency in RPE65. The RPE65-LCA viral vector was administered via sub-retinal injection (5.96x10 ^ 10 vector genomes in 150 µl).
[406] [406] Methods and protocols for administering therapeutic agents (eg, protein, nucleic acid molecule, expression cassettes, gene therapy vectors, cells) including, without limitation, to the eye, and other procedures and protocols (including, without limitation
[407] [407] In the Hauswirth study, administration was performed as follows. After mild intravenous sedation, the surgical eye received retrobulbar anesthesia and was then prepared and covered in a standard sterile manner. A standard 23-gauge 23-gauge core and peripheral vitrectomy were performed. The conjunctiva in the right lateral sclerotomy was dissected with Westcott scissors and 0.3 forceps. Hemostasis was maintained with rubber tip cautery. Sclerotomy was increased with a 20-gauge MRV slide so that the subretinal cannula could be easily inserted into the eye. The vector was sucked into a 39-gauge injection cannula (Synergetic, O'Fallon, MO) and was introduced into the sub-retinal space. At the end of the procedure, the sclerotomy sites were secured with Vicryl 7.0 sutures and the conjunctiva was closed with interrupted sutures. Antibiotics and subconjunctival steroids were administered. Topical antibiotics and steroids were used for 20 days after surgery. See, Hauswirth and others, Hum Gen Ther. 2008 Oct; 19 (10): 979–990.
[408] [408] For gene therapy treatment with CYP4V2 in vitro, post-treatment evaluation can compare cell morphology and / or biochemical dysfunctions of the patient's cells, for example, by comparing the levels of compounds that showed abnormalities in cells iPS-RPE of patient with BCD (or iPS-RPE or iPS-CEC cells, if applicable) before and after treatment, to assess whether cell morphology and / or biochemical function improved after treatment.
[409] [409] For treatment of gene therapy with CYP4V2 in vivo, post-treatment evaluation may use eye and retinal examinations (and corneal tests, if applicable) known in the art for retinal and corneal diseases, including, without limitation, adaptation to dark, contrast sensitivity, visual field test, visual acuity test, color vision tests, ERG, OCT, background image, corneal examination,
[410] [410] A viral vector-mediated gene therapy challenge is immune responses from the individual receiving gene therapy. In addition to conventionally associated risks for the individual, immune responses can significantly reduce the transduction efficiency of viral vectors and / or result in a failure to establish long-term transgene expression. Mingozzi F, Meulenberg JJ, Hui DJ, Basner-Tschakarjan E, Hasbrouck NC, Edmonson SA, Hutnick NA, Betts MR, Kastelein JJ, Stroes ES, High KA, AAV-1-mediated gene transfer to skeletal muscle in humans results in dose -dependent activation of capsid-specific T cells. Blood. 2009 Sep 3; 114 (10): 2077-
[411] [411] Perhaps, partly due to the unique immune environment of the eye, the immunological effects of several recombinant viral vectors (eg, AAV, lentivirus, adenovirus) in eye gene therapy appear to be quite benign. Nevertheless, a significant cell-mediated immune response can develop after intraocular administration of adenovirus. Neither AAV nor lentivirus, however, elicits a cell-mediated response and are therefore promising vectors for the treatment of chronic (retinal) eye diseases. J Bennett, Immune response following intraocular delivery of recombinant viral vectors, Gene Therapy (2003) 10, 977–982. doi: 10.1038 / sj.gt.3302030. On the other hand, however, a previous study showed that intravitreal administration of AAV vectors resulted in an increase in levels of anti-AAV antibodies in both viral fluid as well as serum from non-human primates. Furthermore, the presence of pre-existing neutralizing antibody titers in the serum of monkeys was
[412] [412] Historically a common practice for companies in the field of gene therapy has been to use AAV serotype vectors. It typically uses a type of vector with good transduction efficiency and a large amount of safety data in animal studies and / or clinical tests of other gene therapy. For example, AAV2 is the most commonly used AAV serotype for eye gene therapy in clinical trials. However, the best serotype for one patient is not always the best for another patient due to individual differences in the immune system, for example, pre-existing anti-AAV antibodies. For example, the prevalence of pre-existing anti-AAV neutralizing antibodies against specific AAV serotypes is different between countries and populations. Still, immune reactions can significantly reduce the efficiency of transduction, which can reduce the effectiveness of the gene therapy being applied and / or require that a larger dose be administered.
[413] [413] A method is provided here to reduce immune responses to viral vectors, conserve transduction efficiency, decrease the dose of viral and / or immunosuppressive vectors and / or maximize the therapeutic effect for patients other than the same genetic disease, in genetic therapy mediated by viral vector, comprising: (a) establishment of a group of more than one recombinant viral vector (for example, rAAVs) with sufficient transduction efficiency;
[414] [414] Potential benefits of this method include reducing the use of immunosuppressants, a lower dose of rAAV vectors, greater transduction efficiency and long-term transgene expression and / or a higher percentage of patients eligible for gene therapy.
[415] [415] It would be understood that this method can be used in connection with other viral vectors. In addition, this method can be used in all types of ocular gene therapy and non-ocular gene therapy, whether related to the CYP4V2 gene or other gene (s).
[416] [416] Methods of detecting pre-existing anti-AAV antibodies are known in the art. It is important to say that anti-AAV antibodies include both neutralizing antibodies and non-neutralizing antibodies. Methods for detecting pre-existing anti-AAV neutralizing antibodies and other immune responses to AAVs are known in the art. Melvin Y Rincon et al., JMIR Res Protoc. 2016 Apr-Jun; 5 (2): e102; Hauswirth et al., Hum Gene Ther. 2008 Oct; 19 (10): 979–990. Although the effect is more significant with neutralizing antibodies, even non-neutralizing antibodies can trigger vector elimination by the immune system. Non-neutralizing antibodies can be detected by ELISA. Boutin S, Monteilhet V, Veron P, Leborgne C, Benveniste O, Montus MF, Masurier C, Prevalence of serum IgG and neutralizing factors against adeno- associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther. 2010 Jun; 21 (6): 704-12.
[417] [417] Unless otherwise defined, all the technical and scientific terms used here have the same meaning as they are commonly understood by a common verse in the technique to which the methods and compositions of matter belong. In addition to the definitions of terms provided here, definitions of common terms in molecular biology can also be found in Glossary of Genetics:
[418] [418] Representative methods and materials are described here; other suitable methods and materials known in the art can also be used. The methods and materials are illustrative only and are not intended to be limiting. EXAMPLES
[419] [419] The invention is further described in the examples that follow, which do not limit the scope of the invention or the claims.
[420] [420] The studies were initiated, planned, organized and sponsored by Reflection Biotechnologies Limited (“ReflectionBio”), a biotechnology company founded and run by a patient and family living with a rare retinal disease. Patients with a rare disease bear the inevitable probabilities of genetic mutations for humanity, but are often ignored by society and little supported by public resources. A patient-led bio-technology company, ReflectionBio, applies a “For Patients, For Patients” approach for patients to join forces and play a more active role in conducting scientific and medical R&D for rare diseases and other challenging diseases .
[421] [421] Patients diagnosed with BCD and having different biallelic CYP4V2 mutations (including homozygous CYP4V2 mutation or compound heterozygous CYP4V2 mutations) were included in this study. In particular, a patient (hereinafter referred to as Patient 1, P1 or RB001) has a c.802-
[422] [422] Informed consent was obtained. Procedures followed the guidelines of the Declaration of Helsinki and were approved by an Institutional Review Board. Examples of BCD Human Cell Disease Model
[423] [423] Clinically, BCD is associated with RPE atrophy, which in turn causes photoreceptor death and loss of vision. In this way, it is critical to create and use a human RPE model to study BCD and develop treatment for BCD. Example 1 - Generation and Characterization of Induced Pluripotent Stem Cells (iPSCs) Derived from Patients with BCD
[424] [424] In the study, free integration methods were used to generate iPSCs from patients with BCD. Traditional technologies used for reprogramming iPSC (eg, lentivirus, retrovirus) integrate into the genome of target cells. The resulting iPSCs and cells differentiated from these iPSCs will contain foreign DNA and could be insecure and problematic for use in cell therapy and drug development applications. In addition, integration could occur in a critical region of the genome, causing problems with unrelated developmental processes. Compared to traditional reprogramming methods, integration-free reprogramming methods generate iPSCs that do not contain detectable vectors or transgenes, thus making them more suitable for cell therapy and drug development applications.
[425] [425] In the study, two different integration-free reprogramming methods were used to generate iPSCs from patients with BCD, one employing Sendai virus, the other employing episomal vectors. Two different types of samples were used, one is a skin sample (skin fibroblasts) and the other is a blood sample (peripheral blood mono-onuclear cells (PBMCs)). Any method can be used to generate specific BPS patient iPSCs from skin, blood or other samples, such as urine and hair samples. A. Reprogramming iPSC from a skin sample
[426] [426] Skin biopsy was performed in patients with BCD, and human fibroblast cells were obtained from the biopsy. BCD patient-specific fibroblast cells were then reprogrammed into iPS cell lines using Sendai virus, a footprint-free RNA virus that carries no risk of altering the host's genome. Other vectors, including, without limitation, lentivirus, can also be used in iPS reprogramming, but with a risk of integrating into the host cell genome. See Figure 1 for pictures of iPS cells derived from patients with BCD. Fibroblast cells from healthy individuals were also reprogrammed in the same way to generate wild-type (or control) iPS cell lines.
[427] [427] To generate iPSCs, 5 x 104 fibroblasts were plated and cultured in a 12-well plate until the cells became adherent (for about 12 hours) and were around 70% -80% convergent. The culture medium was removed, and the cells were transfected with a Sendai virus expressing Oct3 / 4, Sox2, Klf4 and c-Myc (CytoTune ™ -iPS 2.0 Sendai Reprogramming Kit, A16517, Life Technologies) in a MOI of 5 : 5: 3: 5 in 500 µl of fibroblast culture medium. The cells were incubated at 37º C and 5% CO2 overnight, after which the virus-containing medium was removed and replaced with KO-DMEM medium (KnockOut DMEM, 15% KnockOut serum replacement, L-glutamine 1, non-essential amino acids 1, penicillin-streptomycin 1, β-mercaptoethanol 0.1 mM, basic fibroblast growth factor (bFGF) 10 ng / ml). The transfected cells were incubated for about 7 days, with the medium changed every day.
[428] [428] The transfected cells were washed in PBS, exposed to trypsin (eg, TrypLE Express at 37 ° C for 4 min) and resuspended in 2 ml of KO-DMEM medium containing 10 µm ROCK inhibitor. The cells were then plated on a MEF feeder layer treated with mitomycin-C and returned to 37º C and 5% CO2. After 24 hours and every subsequent day, the medium was removed and replaced with KO-DMEM medium (without ROCK inhibitor). Colonies were visible 7-14 days after passage. Each iPS colony was microdissected into pieces of about 100-150 cells, following a rapid treatment with KO-DMEM medium with 10 uM ROCK inhibitor, and then cultured again in KO-DMEM medium at 37º C and 5% CO2 by one more week.
[429] [429] iPSC characterization was performed using primary antibodies from pluripotent markers: OCT4 Santa Cruz sc-9081 Rbbit poly, SOX2 R&D Systems 245610 Mouse IgG, TRA-1-60 Millipore (Chemicon) MAB4381, Mouse, IgM, SSEA4 Millipore (Chemicon) MAB4304 Mouse IgG, Nanog R&D Systems AF1997 Goat poly. Typically for characterization using markers, cells were washed, blocked (for example, with 3% serum and 0.1% Triton X), exposed to a primary antibody (1: 200) and incubated at room temperature for 2-3 hours. The cells were washed again, exposed to a secondary antibody and incubated at room temperature for 60 min. The cells then went through
[430] [430] See Figure 1 (a) for iPSCs generated from fibroblasts from patients with BCD and characterization through Oct-4, Sox-2, SSEA-4, Nanog and Tra-1-60. B. Reprogramming iPSC from a blood sample
[431] [431] In addition to skin biopsy samples, iPSCs were also generated from blood samples from a patient with BCD and healthy control. IPSCs were generated from peripheral blood mono-onuclear cells (PBMCs) using an episomal method. The protocol is described as below.
[432] [432] T cell activation: a) Frozen PBMCs were thawed and approximately 0.5 million viable cells were subjected to T cell activation using Dynabeads (Human T activator, CD3 / CD28, Thermo Fisher, Cat # 11132D) according to the manufacturer's protocol. b) Activated T cells were then expanded in blood cell culture medium for 10-14 days.
[433] [433] Reprogramming: a) To generate iPSC strains, activated T cells were disassociated from dynabeads and electroporated with Episomal iPSC Reprogramming Vectors (No. Cat.A14703, Invitrogen, Carlsbad, CA, USA) using Neon Transfection System (Cat. No. MPK10096, Invitrogen) according to the manufacturer's instructions. b) The two sets of electroporated cells were plated on two sets of 35 mm plates pre-cultured with CEF CF1 feeders (#Cat: (ASF-1213, Applied StemCell, Milpitas, CA, USA) The cells were fed daily with human iPSC growth medium c) After 2-3 weeks, human ESC-like iPSC colonies were taken and transferred to 24 well plates coated with matrigel for expansion. d) Patient-specific human iPSC strains were then cultured and passed on Matrigel (Cat. No. Corning 354277) on Human iPSC Feeder-Free Growth Medium (mTeSR ™ 1, Cat. No. 05850, StemCell Technologies Inc., Vancouver , Canada) for 2 - 3 more passes until sufficient cell number obtained before cryopreservation.
[434] [434] Alkaline phosphatase: a) For dyeing with alkaline phosphatase (AP), iPSCs were fixed and then dyed with alkaline phosphatase dyeing solution (Naphthol / fast red violet, Sigma). b) Cell images were captured using an Olympus microscope (IX51, Olympus, Tokyo, Japan).
[435] [435] See Figure 1 (b) for iPSC phase contrast images generated from mono-onuclear peripheral blood cells (PBMC) from blood samples from a patient with BCD and healthy control and staining results with AP.
[436] [436] See Figure 1 (c) for iPSC karyotype images derived from a patient with BCD showing apparently normal human karyotype. Example 2 - Differentiating iPSCs from Patients with BCD in Retinal Pigment Epithelium (RPE) Cells
[437] [437] iPSC differentiation started in passages 3 through 6 for all iPSC strains from patients with BCD and healthy controls. For differentiation, iPS colonies were cultured for confluence on 6-well culture plates (Costar, Corning, Corning, NY) pre-treated with 1:50 diluted Matrigel (CORNING, 356230) in differentiation medium consisting of Knock-Out ( MO) DMEM (Thermo Fisher Scientific, 10829018), serum replacement KO 15% (Thermo Fisher Scientific, 10829028), non-essential amino acids 1% (Thermo
[438] [438] RPE cells differentiated from BPS patient iPSCs were observed under light microscopy and RPE pigment and distinct hexagonal cell shapes were seen (see Figure 2). In addition to morphological distinctions, RPE cells derived from iPS from patients with BCD were also validated by the presence of specific RPE, RPE65, CRALBP and MITF markers. See Figure 2 (b) for results of RPE markers from iPS-RPE cells from patients with BCD, showing the presence of specific RPE, RPE65, CRALBP and MITF markers.
[439] [439] Multiple protocols can be used to differentiate iPSCs in RPE cells. The RPE differentiation protocol described here is an extended protocol that usually takes more than 3 months. Other protocols take less time, for example, less than 2 months. Although both shorter and extended protocols can differentiate iPSCs in RPE cells, there may be differences in terms of the risk of tumorigenesis between iPSC-RPE cells generated by different protocols. The risk of tumorigenesis associated with iPSC differentiation is attributed to a portion of the remaining iPS cells that are not differentiated or not fully differentiated at the end of the protocol, and the extended protocol in the same way contributes to the lack of formation tumor because iPSCs are totally differentiated in mature RPE cells. The longer term protocol was used to ensure the purity of the iPS-RPE cell lines generated for biochemical and other assays and functional studies, and to support the safety of iPSC-RPE cells for cell therapy including, without limitation, autologous transplantation. Example 3 - Biochemical Assays, Cell Viability and others for BCD Cell Model and Functional Studies of CYP4V2 Lipid Assays:
[440] [440] Previous studies on BCD and CYP4V2 enzyme function focused on fatty acids. In this study, further lipid assays including not only fatty acids, but also ceramides (Cer), sphingomyelin (SM) and sphingosine and sphinganine (SOSA), were used to analyze the abnormalities / biochemical phenotype in a doping model. ences BCD and analyze the biochemical functions of the CYP4V2 protein.
[441] [441] Biochemical assays in free fatty acids (FFA), ceramides (Cer), sphingomyelin (SM) and sphingosine and sphinganine (SOSA) were conducted at Columbia University's Biomarkers Core Laboratory (New York, NY, USA) based on their relevant trials and protocols.
[442] [442] Free fatty acids (FFA), ceramide, sphingosine and sphinxine were extracted using chloroform: methanol. In short, about 1 million iPS-RPE cells were homogenized in 150 μL of water. 100 μL of homogenate was mixed with 3 mL of chloroform: methanol (v: v = 2: 1) containing internal standards (Palmitic acid-D31, ceramide C12, ceramide C25, sphingosine C17, sphinganine C17). The sample was vortexed well and 0.5 ml of water was added to allow phase separation. The mixture was vortexed again and centrifuged at 3,000 g for 10 minutes at 4 ° C. The lower organic phase was transferred to a second clean glass tube using a Pasteur pipette. Two ml of chloroform was added to the residual aqueous phase, followed by vortexing and centrifuging again to extract any remaining lipids. The lower organic phases were grouped and evaporated under nitrogen at 37º C. The extracted lipids were reconstituted in 50 µl of methanol: acetonitrile (v: v = 1: 1) and transferred to LC self-sampling vials for injection. Sphingomyelin was also extracted using chloroform: methanol like other lipids, but only 2 µL of cell homogenate was used for sample preparation for sphingomyelin. All tests were performed on a Waters Xevo TQ MS ACQUITY UPLC system (Waiters, Milford, MA, USA). FFA was eluted by a 100 mm Waters AC-QUITY UPLC HSS C18 column. Ceramide, sphingosine, sphinganine, sphingomyelin were separated on a 100 mm Waters ACQUITY UPLC BEH Phenyl column. FFA was monitored using negative SIR method and others through positive MRM acquisition.
[443] [443] A list of compounds tested in the biochemical assays is provided in Table 2 below. Certain chemical compounds were purchased for use as standards in this study (as written in Table
[444] [444] Additionally, LC-MS / MS was used to detect hydro-fatty acids in iPS-RPE cells, including 16-HEPE, 17-HEPE, 18-HEPE, 19-HEPE, 20-HEPE, 17-HDHA, 18 -HDHA, 19-HDHA, 20-HDHA, 21- HDHA, 22-HDHA, 19 (20) -EpDPA (formal name: (±) 19,20-epoxy- 4Z, 7Z, 10Z, 13Z, 16Z-docosapentaenoic acid , tcc (±) 19,20 EDP, (±) 19,20-epoxy docosapentaenoic acid, (±) 19,20-epoxy DPA, (±) 19,20- EpDPE) and 19 (20) -DiHDPA (formal name : (±) 19,20-dihydroxy-4Z, 7Z, 10Z, 13Z, 16Z-docosapentaenoic acid, tcc: (±) 19,20-DiHDoPE). HDHA compounds are DHA hydroxy metabolites and HEPE compounds are EPA hydroxy metabolites, respectively. 19 (20) - EpDPA is a metabolite of DHA oxygenase, derived through the epoxidation of the double bond ω-3 of DHA. 19 (20) -DiHPDA is also a metabolite of DHA. DHA is an important fatty acid and the most abundant ω-3 fatty acid for the brain and retina. Previous research has indicated that CYP4V2 is a hydroxylase for ω-3 fatty acids, particularly DHA.
[445] [445] Materials: hydroxy fatty acid standards (±) 18-HEPE (Item No. 32840), (±) 20-HDHA (Item No. 33750), (±) 19 (20) -EpDPA (Item No. 10175) and (±) 19 (20) -DiHDPA (Item No. 10007001) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Internal standard deuterated palmitic acid (C16-D31 fatty acid) was purchased from C / D / N Isotopes Inc. (# D-2002, Quebec, Canada).
[446] [446] It should be understood that in addition to the LC-MS or LC-MS / MS methods described above, the species and chemical compounds tested in the study can also be detected and / or quantified using other methods. For example, there are GC-MS or GC-MS / MS methods for FFA with methylation pretreatment. For Cer and SM, FIA-MS / MS or GC-MS / MS can be used. Cell Viability Test:
[447] [447] Exposure to blue light: iPS-RPE cells were seeded in 3.5 cm plates and 4-well chamber plates. After 2 months, they were exposed to light (blue) 430 ± 20 nm at 1.5mW / cm2 for 1 hour in PBS (+) containing 10 µg / ml of glucose. The sample seeding density was used for all cell lines. After exposure to blue light, treated cells were fed with fresh RPE medium and recovered in a 5% CO2 incubator and 37ºC overnight.
[448] [448] In addition to 1 hour, shorter or longer light exposure durations can be used, for example, no exposure, 30 minutes, 45 minutes, 75 minutes, 90 minutes or 120 minutes, etc. Similarly, light exposure of a different wavelength or a broader spectrum can also be used. In addition, iPS-RPE samples from different culture days (eg 2 months, 3 months, 4 months, 5 months or 6 months in RPE culture) can be used, for example, to study the effect of aging.
[449] [449] Cell viability assay: living / healthy cells were stained with Calcein AM cell permeation dye (Thermo Fisher Scientific, catalog number: C3099, USA) at a final concentration of 3 µmol / ml PBS (+) (1 ml for each 3.5 cm plate or 200 µl for each chamber) and dead / diseased cells were marked with Propidium Iodide (PI) (Thermo Fisher Scientific, catalog no .: P3566, USA) at a concentration final of 2 µg / ml PBS (+) (1 ml for each 3.5 cm plate or 200 µl for each chamber) at room temperature for 1 hour. Since PI is DNA-binding and does not permeate living cells, it is commonly used to detect dead cells in a population. Then after washing with PBS (-), cellular fluorescent levels were observed and photos were taken by an inverted fluorescent microscope (Nikon Eclipse Ts2R) in 20-fold magnification. Frog-
[450] [450] In addition to biochemical assays and cell viability testing, RPE function tests can be performed on iPS-RPE cells from patients with BCD such as phagocytic activity, transepithelial resistance. CYP4V2 expression:
[451] [451] Experiments were performed to detect and compare CYP4V2 expression levels in control and specific BPS patient-specific iPS-RPE cells. Expression of CYP4V2 in cell lines can be evaluated either through anti-CYP4V2 antibody (Western Blot) or through quantitative PCR.
[452] [452] CYP4V2 Western Blot: 45 µg of whole cell protein from each iPS-RPE sample was processed on a 7.5% SDS page gel, then transferred to a membrane wet. The membrane was blocked with 5% BSA in PBST for 1 hour at room temperature, then incubated with primary antibody (Anti-CYP4V2 produced in rabbit, # Sigma Aldrich catalog: SAB 1410565, USA) in a concentration of 1: 1000 in BSA 5% overnight at 4ºC. Washing was done for 3 x 10 minutes with PBST. The membrane was then incubated with secondary IgG goat anti-rabbit HRP antibody (# Santa Cruz catalog: sc-2004, USA) in a concentration of 1: 3000 in BSA 5% for 4 hours at 4ºC. Final washing was done for 3x10 minutes with PBST. GAPDH was used as a loading control.
[453] [453] CYP4V2 western blot detected expression of CYP4V2 protein in control iPS-RPE samples, but not in BPS patient iPS-RPE sample. After treatment by AAV.CYP4V2, CYP4V2 protein was detected in specific BPS patient-specific iPS-RPE samples.
[454] [454] Real-time PCR and relative mRNA quantification:
[455] [455] Results: Quantitative PCR was performed to test the expression of CYP4V2 and CYP4V2op in iPS-RPE cell samples. The CYP4V2 and CYP4V2op transcript levels were normalized by a patient sample and a control sample, respectively. For CYP4V2, all non-patient control samples expressed similar levels of CYP4V2, several hundred times greater than the level of CYP4V2 expression in the patient sample. After treatment with AAV.CYP4V2, the level of expression of CYP4V2 in the patient sample increased more than a hundred times to a level compared with the control samples of non-patient (Figure 3). For CYP4V2op, all samples treated with AAV expressed much higher levels compared to untreated samples (Figure 4). These results demonstrated that AAV vectors were capable of delivering the CYP4V2 cDNA to iPS-RPE cells from patients with BCD and the expression cassettes were capable of expressing the gene. Example 4 - Phenotype in BCD Cell Model and Findings on CYP4V2 Functions Lipid Test Results:
[456] [456] To determine whether and what biochemical defects / abnormalities (ie, phenotype) exist in the BCD cell model (eg, BPS patient iPS-RPE cells), the biochemical assays described in Example 3 were used to detect and quantify fatty acids, ceramides, sphingomyelin, sphingosine, sphinganine and hydroxy fatty acid in iPS-RPE cells derived from patients with BCD compared to those in iPS-RPE cells derived from healthy controls.
[457] [457] Before testing, cells were collected as follows. Approximately 1 million iPS-RPE cells derived from a BCD patient were washed twice with PBS, then released from the plate by a plastic cell filter and transferred to a 1.5 ml Eppendorf tube using a 1 ml pipette. ml. The Eppendorf tube was placed in a -80 ° C freezer before testing. Healthy iPS-RPE control cells were collected in the same way. Results of biochemical assays are shown in Table 3 below: Table not checked - Please return after formatting Table 3 Results of the fatty acid test WT P1 P1 AAV2.op P2 P2 AAV2tri.op P2 AAV2.op P2 AAV8. fv P2 total fatty acids AV1.op C12 0.2%, 1% 0.1% 0.1% 0.1% 0.0% 0.0% 0.1% C13 0.0% 0 , 0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% C14: 1 Isom- 0.1% 0.1% 0.1% 0.0% 0, 0% 0.0% 0.1% 0.1% ro 1 C14: 1 Isome- 0.0% 0.1% 0.0% 0.1% 0.0% 0.0% 0.0% 0 , 1% ro 2 C14 Acid 1.0% 0.6% 0.6% 0.7% 0.8% 0.8% 0.8% 1.1% myristic C15 1.3% 0.5% 0 , 7% 0.5% 0.5% 0.6% 0.6% 0.7% C16: 1 n7 cis 2.7% 2.4% 1.7% 2.3% 2.9% 2, 9% 2.8% 3.8% C16: 1 n9 cis 0.9% 0.9% 0.8% 1.0% 1.0% 1.1% 1.2% 1.6% C16: 1 n7 0.4% 0.5% 0.4% 0.5% 0.5% 0.4% 0.5% 0.4% trans C16 Acid 20.3% 14.2% 12.7% 14, 8% 17.0% 17.2% 17.9% 17.4% palmitic C17 1.1% 0.5% 0.8% 0.5% 0.4% 0.5% 0.5% 0, 4% C18: 3 n3 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Alpha
[458] [458] The results showed that iPS-RPE cell samples from a patient with BCD have a different fatty acid profile than that of the control. In particular, BCD patient samples have much higher levels of DHA (22: 6 n3) and total omega-3 fatty acids (ω-3 or n3) (sum of C18: 3 n3 Alpha, C20: 5 n3 EPA, C22: 6 n3 DHA and C22: 5 n3 DPA) than those of control. This confirmed suggestions from a previous study that CYF4V2 affects omega-3 fatty acid metabolism.
[459] [459] Surprisingly, in addition to abnormalities in n3 fatty acid levels, iPS-RPE cells from a patient with BCD also showed higher levels of C20: 4 n6 (arachidonic acid or AA). Abnormal AA level has not been reported in previous studies related to BCD.
[460] [460] Interestingly, abnormalities in n3 fatty acids (including DHA) and n6 fatty acids (including AA) were not found in previous research that tested serum fatty acid levels in patients with BCD. The different fatty acid profiles of iPS-RPE cells from patients with BCD and serum support the hypothesis that CYP4V2 function is replaced by other enzymes in non-retinal or non-RPE cells, many of which are expressed in other organs and tissues together with CYP4V2 except that CYP4V2 is the only CYP4 enzyme with a relatively high level of expression in RPE cells.
[461] [461] In addition to fatty acids, iPS-RPE cells from a BCD patient may have a phenotype in other compounds or classes of compound. Experiments were conducted to evaluate phenotype in other classes of compound, including, without limitation, corticosteroids, sphingolipids and phospholipids, including sphingomyelin, ceramide, sphingosine and sphinganine, and in lipid signaling. Also, experiment analysis and isotopic tracking proteomics (for example, proteomic analysis based on mass spectrometry) is performed on iPS-RPE cells of patients with BCD.
[462] [462] Still, previous research has found that CYP4V2 is a ω-3 fatty acid hydroxylase (DHA and EPA). Interestingly, hydroxy-DHAs or hydroxy-EPAs described in Example 3 were not detected either in healthy iPS-RPE control cells or in BCD patients using LC-MS / MS. It is possible that the enzymatic functions of CYP4V2 are different in living cells vs. in a chemical reaction outside living cells that was conducted in the previous research, either the hydroxy fatty acids are intermediates that are quickly converted into other compounds or forms in living cells, or the hydroxy fatty acids are at a trace level that can be detected only when a sample contains a very large number of cells. Cell Viability Test Results:
[463] [463] Clinically, BCD is associated with RPE atrophy, which in turn causes photoreceptor death and loss of vision. Cell viability assay (as described in Example 3 above) revealed atrophy of RPE in iPS-RPE cell samples from patients with BCD. See Figures 5 and 6 for comparison of cell viability between iPS-RPE samples from control and BCD patients (Figure 5 - without exposure to blue light; Figure 6 - after 1 hour of exposure to blue light).
[464] [464] Significantly, these images revealed that:
[465] [465] BCD patients differ widely in age of disease onset and progression. BCD onset varies from the beginning of adolescence to the 3rd decade of life or even beyond the 3rd decade; leading to legal blindness during the 3rd decade to the 6th decade of life. Still, sibling patients with BCD with the same mutation in CYP4V2 may have a material difference in age of disease onset and progression. Previously, there was no explanation for these differences. The difference in RPE atrophy levels between different BPS patient iPS-RPE samples provides guidance at the cellular level regarding the difference in disease onset and progression among BCD patients.
[466] [466] Multiple phenotypes (both molecular-level phenotypes such as biochemical abnormalities (eg, lipid) and cell-level phenotypes such as cell viability) were found in the BCD Cell Model in this study, including the clinical BCD phenotype (ie , atrophy of RPE). Example 5 - Use of iPS and iPS-RPE Cells from a BCD Individual to Evaluate Drug Candidates and Dosage Range, Study BCD and
[467] [467] Like the human cell model of BCD disease, iPS and iPS-RPE cells from patients with BCD have a wide range of applications, including, without limitation, studying BCD and CYP4V2 function (see Examples 3 and 4 above, for example); evaluate drug candidates and dosage range for BCD and related diseases (see the present Examples).
[468] [468] Methods and examples for using BCD cell model (for example, patient-specific iPS-RPE cell line with BCD or iPS-RPE cell lines with artificially generated CYP4V2 mutations) are described in detail in the present Examples, which are related to the use of the BCD cell model in gene therapy and cell therapy. In addition to testing gene therapy and as a cell basis for cell therapy, such a cell model of BCD can be used to evaluate and test the efficacy and / or safety of other therapeutic agents (eg drug candidates) and dosage, formulation and vector (viral or non-viral vectors) of the same or administration devices or mechanisms for the treatment of BCD, IRD or RP, in the same or similar manner as that described in detail in the present Examples.
[469] [469] Using a BCD cell model, the effectiveness of a therapeutic agent can be assessed by comparing the levels of compounds in the various species and atrophy of RPE described in Examples 3 and 4 above and other Examples here before treatment and post-treatment by such therapeutic agent and assess whether abnormalities in the levels of these compounds and RPE atrophy in the BCD cell model improve post-treatment. Similarly, doses, formulations (for example, formulation for chemical compounds, active pharmaceutical ingredients or type of vector and / or capsid for gene therapy or type of vector for gene editing) or different key constructs (for example, a promoter or another regulatory sequence in a gene therapy expression cassette) of a therapeutic agent can be compared using the BCD cell model. In addition, a cell model of BCD can be used to test the efficacy of a medical device or method, including, without limitation, in the administration of therapeutic agents to ocular cells or in improving the efficiency of transduction or transfection. It would be understood that treated cells can be compared with untreated cells or with the same cells prior to exposure to the compound. Different dosages can be used to determine the effective dosage range (measured per cell, per 1 million cells per 0.5 million cells, etc.). Data regarding levels of compounds other than fatty acids and other compounds and RPE atrophy reported in Examples 3 and 4 above, in iPS-RPE cells from patients with BCD (post-treatment vs. untreated) compared to those in cells from Healthy control RPE or iPS-RPE can be used to assess therapeutic effect and effective dosage range.
[470] [470] Also, patient-specific iPS cell lines with BCD, iPS-RPE cell lines, and other iPS-derived cell lines can be used to assess such individual patient responses to a therapeutic agent, dose, or device. Patient-specific iPS cells, iPS-RPE cells and other cells derived from iPS have specific characteristics for each patient, including, without limitation, immune response (for example, intracellular immunity, RPE immunity), genotype (for example , different mutations between patients that may result in a different response). Such an application can be used to develop and evaluate individualized therapeutic agent (for example, different AAV vector serotypes or capsid mutations) or optimal personalized dosage for different patients of the same disease. This approach can be used for other diseases, including, without limitation, other eye diseases.
[471] [471] Since iPS-RPE specific to patients with BCD revealed individual differences in patients with BCD, it can be used to assess optimal individual dosing and develop personalized medicine. For example, as seen in the Examples of gene therapy below, at the same dosage of 1x10e5 MOI, AAV2.CYP4V2op achieved different rescue levels (that is, different efficacy levels) of RPE atrophy between P1 and P2 iPS-RPE . This is an advantage that the BCD cell model has over animal models.
[472] [472] BPS patient-specific iPS-RPE cells (ie, BCD cell model) can be used to evaluate and suggest effective therapeutic dosage for in vivo treatment by multiplying the optimal dosage level (for example, indicated as MOI for in vitro gene therapy) determined in a BCD cell model in vitro by the estimated number of ocular cells (for example, RPE cells) targeted for in vivo treatment to reach the dose level of gene therapy vectors for use in vivo (e.g. GC or gp). Such a vector dose level is adjusted by a multiplier (for example 1 to 10) (for example, 1 to 5 for sub-retinal injection or 5 to 10 for intravitreal injection); the other factors affecting the multiplier to be applied include the size of the targeted area and the individual being treated (for example, age, weight, stage of development of the disease and condition of the individual to be treated and potential immune reactions (ie , Preexisting NAbs); the location and density of cells targeted for treatment) to suggest the effective therapeutic dose range for in vivo treatment, which can be further confirmed or refined by clinical tests. This method can also be used to evaluate or suggest an optimal personalized dose for in vivo treatment for an individual patient. Example 6 - BCD Cell Model with Artificially Created CYP4V2 Mutations
[473] [473] Because BCD is a rare disease, patient samples can be difficult to obtain. To overcome this difficulty, a BCD cell model can be generated using genetic editing technologies such as CRISPR to create artificial mutations in the CYP4V2 gene in non-BCD patient cells such as an individual's embryonic stem cell lines (ES) or iPS cells without BCD.
[474] [474] For example, as demonstrated in the present Examples, sgRNA1, sgRNA2, sgRNA3, sgRNA4 and sgRNA5 (see SEQ ID NOs: 48 to 52 for the protospace element sequence in each of sgRNA1, sgRNA2, sgRNA3, sgRNA4 and sgRNA, respectively; Vi- of SEQ ID NO; 55 and 59 for additional sequence for IVT sgRNAs) were used in combination with protein SpCas9 to create cleavage in a region of the CYP4V2 gene in genomic DNA of the patient with BCD containing the c.802 mutation -8_810del17insGC, the most common CYP4V2 mutation among patients with BCD. Among them, sgRNA 3, sgRNA 4 and sgRNA 5 are not specific for the c.802-8_810del17insGC mutation sequence and thus can create a double-stranded DNA (DSB) break in the CYP4V2 gene of a healthy cell ( for example, an ES or iPSC without a CYP4V2 mutation). In particular, after transfection, sgRNA4 and Cas9 can create a DSB in exon 7 of the CYP4V2 gene, which can result in a mutation in exon 7 (in one or both alleles) when the DNA is repaired through non-homologous end junction ( NHEJ) in cells, for example, an indel error created by NHEJ can result in a structure change mutation. As a result, some cells may have artificially created CYP4V2 mutations and can be used as a model of BCD cell disease or used to generate BCD cell model (for example, differentiating ES or iPS cells in RPE cells to generate CYP4V2 mutation containing ES-RPE-iPS-RPE cells). Similarly, two sets of gRNAs designed to create DSBs in different regions of the CYP4V2 gene can be used to generate a large deletion or inactivation mutation within the CYP4V2 gene or inactivate the entire CYP4V2 gene in cells, thereby generating a cell model of BCD containing a CYP4V2 mutation (s). A more detailed discussion of how to use the CRISPR system to cut and / or correct a target sequence and how to validate the results is provided in the Examples and the present description.
[475] [475] The BCD cell model with artificially created CYP4V2 mutations can be used to mimic a specific cell model of a BCD patient in a study of BCD and CYP4V2 functions, as well as in related applications as discussed here, including, but not limited to, testing and comparing drug candidates, determining the dosage range and testing a medical device or method of administration.
[476] [476] The same method can be used to generate models of cell disease with artificially created mutations for one eye disease or another, including those associated with the mutation or genetic defect in one or more gene (s) shown in Table 4. Example 7 - Generation and Use of Isogenic Control for Eye Diseases
[477] [477] An isogenic patient-specific iPS cell line with corrected mutation and / or other cell lines derived from the same (for example, iPS-RPE cells, iPS-RPCs, iPS-CECs, iPS-CE cells or other iPS-eye cells ) can be used as an isogenic control in the study of a disease and / or in the implications of
[478] [478] An isogenic control can be compared with a patient-specific cell disease model to identify phenotype, biochemical abnormalities and other structural and functional defects associated with the genetic mutation and / or the related defective protein. A specific non-limiting example and discussions of how to use bioassays to identify biochemical / phenotype abnormalities between patient cell lines and controls are provided here in Examples 3 and 4 above, including, without limitation, lipidomic, proteomic and isotopic scans. Discussion on the Human Model of BCD Cell Disease
[479] [479] Since BCD is a rare disease, it is impractical to obtain human RPE cells manifesting the disease from patients with BCD through biopsy. The lack of a human model of BCD disease has limited previous research on BCD to the use of non-BCD disease-causing cells (eg, fibroblasts and lymphocytes, which are not part of the eye) and serum from patients with BCD as objects of study. The results of these studies were centered on fatty acid anabolism.
[480] [480] In the study described here, iPS cell lines derived from patients with BCD were successfully generated and used to generate patient-specific BCD disease RPE cells, which carry the BCD disease phenotype in vitro. The BCD phenotype was identified directly in patient-specific iPS-RPE cells with BCD, the main cell type affected in BCD. Before the present study, it was not known whether iPS cell lines and cell lines
[481] [481] Biochemical testing has shown that iPS-RPE cells from patients with BCD have abnormal levels of fatty acids compared to those from healthy control iPS-RPE cells, including those that have not been reported in previous BCD studies. The in vitro phenotype of BCD disease-specific iPS-RPE cells provides further insight into CYP4V2-regulated pathways and BCD pathogenesis, and provides valuable insights into BCD pathogenesis and BYC4V2 protein function, and further supports the use of iPS-RPE cell lines from patients with BCD as a viable and robust human disease model for BCD.
[482] [482] iPS cell lines, iPS-RPE cell lines and other iPS-ocular cell lines from patients with BCD have additional applications, such as use for drug evaluation, development of new therapeutic agents or determination of dosage ranges, as well as use in cell therapy.
[483] [483] In addition to iPS, iPS-RPE and other BCD patient-specific iPS-ocular cell lines, a human BCD disease cell model can be developed through genetic editing to create pathological CYP4V2 mutations artificially in other strains cell derived from ES cells or iPS cells from non-BCD individuals, including, without limitation, ES cell lines, iPS cell lines and RPE cell lines.
[484] [484] In addition, methods to generate isogenic controls for eye diseases are provided. Isogenic controls have no individual differences from a patient-specific disease model. In this way, an isogenic control has its advantages in the study of eye diseases in relation to conventional controls. Gene therapy with CYP4V2
[485] [485] Three cDNAs were used in this study. The cDNA with the sequence shown in SEQ ID NO: 1 (hereinafter referred to as CYP4V2st) and the cDNA with the sequence shown in SEQ ID NO: 2 (hereinafter referred to as CYP4V2op) both encode the human CYP4V2 protein ( amino acid sequence shown in SEQ ID NO:
[486] [486] SEQ ID NO: 5 is the amino acid sequence of a functional variant of human CYP4V2 protein (SEQ ID NO: 4). Both proteins (SEQ ID NO: 4 and SEQ ID NO: 5) are functional CYP4V2 proteins as defined herein. The functional CYP4V2 protein shown in SEQ ID NO: 5 has an amino acid change from the human CYP4V2 protein shown in SEQ ID NO: 4. The cDNA shown in SEQ ID NO: 3 encoding the functional CYP4V2 protein (SEQ ID NO: 5) has two nucleotide differences from the cDNA shown in SEQ ID NO: 1 encoding the human CYP4V2 protein (SEQ ID NO: 4). Both the codon-optimized cDNA shown in SEQ ID NO: 2 and cDNA shown in SEQ ID NO: 1 encode the human CYP4V2 protein (SEQ ID NO: 4) and share a 77% sequence identity.
[487] [487] A codon optimized cDNA (CYP4V2fv-op) encoding the functional CYP4V2 protein of SEQ ID NO: 5 is provided here, which comprises the CYP4V2op cDNA sequence (SEQ ID NO: 2), except that CYP4V2fv-op sequence retains one or two nucleotide differences between SEQ ID NOs: 1 and 3. In addition to CYP4V2op and CYP4V2fv-op, other codon-optimized cDNAs or nucleic acid sequences encoding the human CYP4V2 protein or one protein
[488] [488] As described here, an expression cassette and an administration vector comprise several elements. Results can vary significantly based on different projects. Given the large number of options in each of the important elements including, but not limited to, the ones listed below and various combinations of them, a serious design of expression cassettes and efficient administration vectors is necessary for the success of the gene therapy with CYP4V2. In addition, the project process needs to take into account the phenotype and characteristics of the disease (for example, types of cells and / or tissues targeted for treatment) and safety (for example, toxicity, immune response). Finally, a project needs to be tested and verified against an important disease model. (a) Type of administration vector; (b) Vector serotype and design / selection of the capsid; (c) Additional vector design, for example, ssAAV vs. scAAV;
[489] [489] For (a), a viral vector was chosen to obtain high transduction efficiency in target cells (eg, human RPE). Among several types of viral vectors, AAV vectors were chosen due to their safety profile and the size of the nucleic acid encoding CYP4V2 (for example, a CYP4V2 cDNA) fits within the AAV vector packaging limit. Vectors with a higher packing limit, for example, an HSV vector, a lentivirus vector, a Baculovirus or adenovirus vector, can also be used for gene therapy with CYP4V2. In addition to viral vectors, non-viral vectors, for example, nanoparticles, including, but not limited to, liposome nanoparticles, solid lipid nanoparticles, liposome / DNA protamine lipoplex (LPD), can also be used to gene therapy with CYP4V2.
[490] [490] For (b), due to the fact that RPE cells are the main cell type for treatment in gene therapy with CYP4V2 for BCD, an AAV serotype with sufficient transduction efficiency in RPE cells is preferred. In addition, the following factors were considered. Because CYP4V2 expression has been observed widely in various human tissues and organs, for example, heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, retina, RPE, cornea and lymphocytes, and in addition to RPE, BCD also affects choroid, photoreceptors and, in some patients, the cornea, and that abnormalities have previously been reported in skin fibroblasts, lymphocytes and serum from patients with BCD, AAV serotypes and capsid structures that do not restrict transduction of AAV only in cell
[491] [491] For (c), due to the fact that the full-length CYP4V2 cDNA is 1578 bp (including start and stop codons), both ssAAV and scAAV designs can be used in CYP4V2 gene therapy. SsAAV and scAAV projects each have their own pros and cons as described here. Compared to ssAAV, an ssAAV design offers rapid expression and increased DNA stability. However, its packaging limit (around 2.4-2.5 kb) restricts the use of larger and potentially more active regulatory sequences (for example, promoter, PoliA signal). Also, depending on the size of the promoter used, an ssAAV project may need to shorten or continue without some optional regulatory sequences (for example, an enhancer). Both ssAAV and scAAV vectors were designed and generated for use in CYP4V2 gene therapy. Several pseudotyped AAVs containing the AAV2 genome (for example, the AAV2 ITRs (SEQ ID NOs: 42 and 43) and a capsid from each of the types of AAV described in (b) above were generated. one of the two AAV2 ITRs has been truncated / mutated (SEQ ID NO: 44).
[492] [492] For (d), as discussed here, there are multiple functional CYP4V2 proteins. In addition, several nucleic acid sequences can encode the same protein. Three (3) cDNAs were generated in the study; the first (SEQ ID NO: 1, referred to as CYP4V2st) encoding the human CYP4V2 protein (SEQ ID NO: 4), the second is a codon with codon optimized (SEQ ID NO: 2, referred to as CYP4V2op) encoding the protein Human CYP4V2 (SEQ ID NO: 2) and the third (SEQ ID NO: 2, referred to as CYP4V2fv) encoding a functional variant of the human CYP4V2 protein (SEQ ID NO: 5). A Kozak sequence (exemplary sequences shown in SEQ ID NO: 37 or 38) was inserted before the cDNA start codon.
[493] [493] For (e), similar to the reasoning in (b), the promoter needs to work well to direct expression in target cells (for example, RPE cells when the type of target cell for treatment is RPE, cells from the cornea when the target cell type is corneal cells). The promoter is a major element in the gene therapy vector expression cassette. Optimal promoter selection can increase target specificity and gene expression. Depending on the type of cell or tissue targeted for treatment, the promoter used in gene therapy with CYP4V2 can be either a constitutive promoter or a cell specific promoter (for example, a specific promoter for RPE cells , a specific promoter for both RPE and photoreceptors, a specific promoter for RPE cells and choroid cells, a specific promoter for RPE, photoreceptor cells
[494] [494] For (f), a polyA bGH was used (exemplary sequence shown in SEQ ID NO: 34) for the expression cassette design used in ssAAVs and a shorter polyA signal, small polyA (SPA ) (exemplary sequence shown in SEQ ID NO: 36) for the expression cassette design used in scAAVs. The SPA was also used in an expression cassette for ssAAVs. Other polyA signals (including derivatives or variants) can also be used instead, including, without limitation, an SV40 polyA signal, a late SV40 polyA signal (exemplary sequence shown in SEQ ID NO: 39 ) or other polyA signals as described herein, including, without limitation, polyA signal used in combination with an upstream enhancer (USE).
[495] [495] For (g), a WPRE enhancer was used (exemplary sequence shown in SEQ ID NO: 33) for the expression cassette used in ssAAVs. For the expression cassette design used in scAAVs, given the size limit, an enhancer was not included. It should be noted that an enhancer is optional in both cases.
[496] [496] In some cases, the CYP4V2 expression cassette includes a promoter (for example, a CAG promoter (tc CBA, CAGGS, CB), an EF-1 alpha promoter, a smCBA promoter, a CBh promoter, a an EFS promoter, a human beta-actin promoter, a CMV promoter, a VMD2 promoter or an RPE65 promoter), a nucleic acid sequence encoding a functional CYP4V2 protein (for example, a cDNA encoding the human CYP4V2 protein or a variant- functional fragment or fragments thereof), optionally linked with an enhancer sequence (for example, a WPRE enhancer, an HPRE enhancer or a shortened WPRE or HPRE enhancer) and a polyA signal (for example, a bGH polyA, a SPA or an SV40 PoliA, or a dou fragment derived therefrom, for example, a late SV40 polylA) and other regulatory sequences (for example, a Kozak sequence). See SEQ ID NOs: 1-41 for exemplary strings.
[497] [497] It should be understood that (i) the exemplary sequences of various regulatory sequences provided in the SEQ section are exemplary in nature and there are different versions of these regulatory sequences that can achieve the same or a similar function and (ii) there are different variants, fragments and / or derivatives of these sequences that can also be used, for example, a truncated CAG promoter, a shortened WPRE enhancer, a late SV40 polyA.
[498] [498] Based on the design approach described above, multiple CYP4V2 cDNAs, CYP4V2 expression cassettes and rAAV vectors for use in CYP4V2 gene therapy were generated, including: (1) Three CYP4V2 cDNAs as shown in SEQ ID NOs: 1, 2 and 3, respectively.
[499] [499] When packaged in an rAAV vector, the expression cassette was flanked by two AAV2 ITRs (SEQ ID NOs: 42 and 43). For scAAV, one of the AAV2 ITRs was truncated / mutated (SEQ ID NO: 44). It would be understood that the non-AAV2 genome, including non-AAV ITRs, can also be used to package the expression cassette. A Kozak sequence (SEQ ID NO. 37 or 38) was inserted just before the CYP4V2 cDNAs. See Figure 7 for schematic drawings showing the design of these expression cassettes. It would be understood that a CYP4V2 cDNA can be packaged in different expression cassettes and that a CYP4V2 expression cassette can be packaged in different AAV vectors. For example, the CYP4V2op cDNA can be used in both the CAG-CYP4V2-WPRE-polyA bGH expression cassette and the EFS-CYP4V2-SPA expression cassette. Either the CAG-CYP4V2-WPRE-polyA bGH expression cassette or EFS-CYP4V2-SPA expression cassette can be packaged in any suitable AAV vector, including, but not limited to, AAV1, AAV2, AAV2 (Y444F + Y500F + Y730F), AAV5, AAV8, AAV8 (Y733F), AAV9, AAV6, AAV7, AAV4, AAV12, AAV-PHP.B and other vectors. A scAAV design can be used in any AAV vector to create a recombinant scAAV vector, for example, scAAV1, scAAV2, scA-AV2 (Y444F + Y500F + Y730F), scAAV5, scAAV8, scAAV8 (Y733F),
[500] [500] It would be understood that a similar design process can be used in the design of other vectors (for example, a Lactivirus vector or a plasmid) for gene therapy with CYP4V2. Depending on the type of vector, certain elements described above may not be necessary or may need to be adjusted according to, for example, a promoter sequence.
[501] [501] In addition to the cDNAs, regulatory sequences and AAV types and designs specified here, other design options for each key element of the CYP4V2 expression cassette and administration vector can also be used. An example of how to compare transduction efficiency of various types of AAV and the resistance of different promoters in one type of target cell is provided here in the Examples section. Similar methods can be used to evaluate and compare design options for other key elements of the expression cassette and delivery vector, for example, cDNA, enhancer, polyA signal, ssAAV vs. scAAV, AAV vs. HSV, etc. Also, as provided here, the efficiency of a CYP4V2 expression cassette and delivery vector can be evaluated and compared by testing on a BCD cell model, for example, iPS-RPE cells from ECB patients, with methods described here and / or other methods to assess biochemical abnormalities, function or atrophy of RPE.
[502] [502] The CYP4V2 cDNAs, expression cassettes and delivery vectors described above were tested on patient-specific human iPS-RPE cell lines with BCD and the results are shown and discussed in the Examples that follow.
[503] [503] In addition, the junction / linker sequences between various regulatory sequences (including, without limitation, between ITR and a promoter, between an enhancer and a polyA signal, or between a power signal
[504] [504] Exemplary sequences of certain regulatory sequences and TIR sequences discussed in this Example are provided as if- gue: SEQ ID NO: 32 (CAG promoter 1715 bp) GACATTGATTATTGACTAGTTATTAATAGTAATCAAT- TACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA- CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGA- CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAG- TAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATT- TACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATG- CCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG- CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC - TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGG- TCGAGGTGAGCCCCACGTTCTGCTTCAC- TCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATT- TATTTTTTAATTATTTTGTGCAGCGATGGGGG- CGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGG- CGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGG- CGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTA- TGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCG- CGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTG- CCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGAC- TGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTT- CTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCG- TTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGA- GGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCG- TGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTG- CCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTG- TGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGG- TGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTG- CGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGG- CGCGGCGGTCGGGCTGTAACCCCCCCCTGCAC- CCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTG- CGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTG- CCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGG- CGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCG- CGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAG- CCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCA- GGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAATCTGGGA- GGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTG- CGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTG- CGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGG- CTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCA- GGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAG- CCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAG- CTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGG-
[505] [505] The BCD cell model (eg, patient-specific iPS-RPE cell lines with BCD or ES-RPE, iPS-RPE or RPE cell lines with artificially generated CYP4V2 mutations) can be used in drug evaluation and dosage. Samples of BPS patient-specific iPS-RPE were used to test, compare and evaluate various components and dosages for CYP4V2 gene therapy, including vector type (eg, AAV sero types and capsid structure) , promoter, enhancer, polyA signal and other sequences in the CYP4V2 expression cassette and CYP4V2 cDNA, as well as the total efficiency of a vector and dosage levels. Phenotype rescue was used to test and compare effectiveness.
[506] [506] Vectors of different serotypes (for example, AAV1, AAV2, AAV5, AAV8, AAV9) or capsid (for example, AAV with mutation (s) in the capsid, for example, AAV2 vs AAV2tri (YF)) or structured - ra (for example, scAAV vs. ssAAV) can be tested, compared using different vectors with the same expression cassette. For example, AAV2.CYP4V2op, AAV2tri (Y-F) .CYP4V2op and AAV5.CYP4V2op all have the same expression cassette, but are different in AAV serotype / capsid. scAAV1.CYP4V2op, scA- AV5.CYP4V2op and scAAV9.CYP4V2op all share the same expression cassette, but are different in AAV serotype. Phenotype rescue results can be used to test and compare difference in AAV serotype / capsid efficiency (for example, AAV2 vs AAV2tri (YF) vs AAV5) and structure (for example, scAAV5 vs ssAAV5) in transduction and administration of the CYP4V2 cDNA in human
[507] [507] The same method can be used to test and compare activity level of different expression cassette, cDNA or regulatory sequences or other sequences (for example, junction / union sequences) through phenotype rescue effectiveness test of rAAV vectors of the same construct (except for the element being tested and compared).
[508] [508] Also, as described in the present Examples, different dosages (for example, 1x10e4 and 1x10e5) of the same vector (for example, rAAV vector, for example, scAAV1.CYP4V2op) can be applied to samples of iPS-RPE of the same patient to assess the effective therapeutic dosage range (measured by cell MOI).
[509] [509] Also, given that the BCD cell model exhibited individual differences, it can also be used to assess and find the optimal dosage and vector construct customized for each individual patient.
[510] [510] See Related Examples and this description for more related discussion. Example 11 - Generation of Multiple Recombinant Adenoassociated Virus Vectors (rAAV) Loading a Functional CYP4V2 Coding Nucleic Acid Sequence and Expression Cassette
[511] [511] Various AAV.CYP4V2 vectors designed for this study (see Examples here), including AAV2.CYP4V2op, AAV2tri (Y- F) .CYP4V2op, AAV5.CYP4V2st, AAV5.CYP4V2op, AAV8.CYP4V2fv and scA-AV1 .CYP4V2op, were made to order by Vector BioLabs (Malvern, PA, USA). Recombinant AAV vectors from Vector Bio-Labs are helper free. The production process involves: (1) cloning a cis plasmid of pAAV, which is a plasmid containing AAV2 ITR that includes the relevant CYP4V2 cDNA (ie,
[512] [512] Different CYP4V2 expression cassette sequences (including ITR and junction / ligand sequences) packaged in various AAV.CYP4V2 vectors for the study are listed as follows. SEQ ID NO: 60 - CYP4V2 expression cassette sequence in AAV2.CYP4V2op, AAV2tri (YF) .CYP4V2op and AAV5.CYP4V2op .: Left ITR: 1-141 CAG promoter: 237-1951 cDP CYP4V2op: 2002-3579 Potent : 3736-4324 poliA bGH: 4350-4574 ITR Right 4659-4799 1 CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGG-
[513] [513] To assess the difference in efficacy between CYP4V2st and CYP4V2op cDNAs in CYP4V2 gene therapy, two AAV4 vectors with the same promoter (CAG), potentiating (WPRE) and polyA (bGH-polyA) sequences and the same junction / ligands, one carrying the CYP4V2st cDNA (AAV5.CYP4V2st (SEQ ID NO: 61)) and the other carrying the CYP4V2op cDNA (AAV5.CYP4V2op (new) (SEQ ID NO: 63)) regarding the efficacy of RPE atrophy in iPS-RPE derived from a patient with BCD using the cell viability assay described here.
[514] [514] To assess whether different junction sequences / ligands used in SEQ ID NO: 60 and SEQ ID NO: 63 affect the expression of CYP4V2 cDNA or expression cassette, two AAV5 vectors (AAV5.CYP4V2op (SEQ ID NO: 60) and AAV5.CYP4V2op (new) (SEQ ID NO: 63)) with the same promoter (CAG), enhancer (WPRE) and polylA (bGH-polyA) and the same CYP4V2 cDNA (CYP4V2op (SEQ ID NO : 2)) but different junction sequences / ligands are compared for efficacy in rescuing RPE atrophy in iPS-RPE derived from a patient with BCD using cell viability assay described here.
[515] [515] It should be understood that different CYP4V2 cDNAs (SEQ ID NOs: 1, 2 3 or others) can be used in any CYP4V2 expression case described here in place of the CYP4V2 cDNA contained in the expression cassette sequences provided here for use in CYP4V2 gene therapy. It should also be understood that each CYP4V2 expression cassette described here can be packaged in rAAV vectors of various serotypes / capsids for use in CYP4V2 gene therapy, including those other than those used in this study (for example, AAV1,
[516] [516] iPS-RPE cells derived from patients with BCD were infected with several AAV.CYP4V2 vectors described above in serum-free RPE medium. After 1 day, the virus-containing medium was replaced with RPE medium containing fresh serum to continue RPE culture. To assess therapeutic effects of different dosage, multiplicity of different infection (MOI, genomic copies (GC) / cell) was tested. Example 13 - Assay to Assess the Effect of Gene Therapy with AAV.CYP4V2
[517] [517] After infection by AAV.CYP4V2, iPS-RPE cells from patients with BCD were cultured in RPE medium for at least 4 days for scAAV or at least 10 days for ssAAV before the cells were collected for testing. Cell collection protocols and sample preparation protocols were followed as previously described.
[518] [518] The biochemical tests described in the Examples here for detection of fatty acids, ceramides (Cer), sphingomyelin (SM) and sphingosine and sphinganine (SOSA) were performed on iPS-RPE cells from patients with BCD treated with AAV.CYP4V2 and the same biochemical test protocol using LC-MS was followed. Table 3 above shows the results in healthy iPS-RPE control cells, iPS-RPE cells from patients with BCD without treatment with AAV.CYP4V2 and post-treatment AAV.CYP4V2.
[519] [519] The results demonstrated that phenotype in iPS-RPE cells of a patient with BCD (eg, abnormal fatty acid levels (eg, DHA, AA and total n3 fatty acids) compared to control) was improved or corrected by gene therapy with AAV.CYP4V2. This established the efficacy of gene therapy with AAV.CYP4V2 in iPS-RPE cell lines derived from a patient with BCD. Due to the fact that BCD is mainly caused by RPE degradation, the efficacy of AAV.CYP4V2 gene therapy in BCD patient-specific iPS-RPE cell lines established the efficacy of AAV.CYP4V2 gene therapy for patients with B C D.
[520] [520] Significantly, treatment with scAAV1.CYP4V2op achieved the most significant improvement in a very short time (only 4 days after treatment). This proved that scAAV is fast-acting because it does not require cellular machinery to synthesize a complementary DNA strand. For the same reason, it is expected that a longer window of time between treatment with AAV.CYP4V2 and cell collection for testing can generate more significant improvements in results, particularly for gene therapy with CYP4V2 packaged in ssAAV vectors.
[521] [521] The rapid and robust results obtained by the scAAV vector in human RPE cells have established that scAAV vectors can be particularly useful in rescuing a human patient with early-stage and / or advanced stage RPE diseases or retinal degenerations . In addition, the robust expression profile of scAAV vectors makes it suitable for intravitreal administration for administration to the retina. Rescue of RPE atrophy by AAV.CYP4V2
[522] [522] Samples of iPS-RPE derived from a patient with BCD were exposed to blue light for 1 hour, so the cell viability assay was performed on the samples the next day as previously described here.
[523] [523] Cell viability images comparing iPS-RPE samples from patient without vs. with treatment with AAV.CYP4V2 are shown in the Figures here.
[524] [524] Each treatment of AAV2.CYP4V2op and scA-AV1.CYP4V2op showed rescue of RPE atrophy in BPS patient-derived iPS-RPE samples compared to untreated patient samples (Figure 8, MOI = 1x10e5 GC / cell). Interestingly, rescue efficacy by AAV2.CYP4Vop and scA-AV1.CYP4V2op in 1x10e5 MOI is higher in iPS-RPE P2 than in iPS-RPE P1. This suggests that optimal dosage for using AAV gene therapy.CYP4V2 to treat BCD may vary based on individual differences between patients and that BPS patient specific iPS-RPE is a useful tool in dose assessment great personalized for different patients.
[525] [525] Each treatment with AAV5.CYP4V2op, AAV5.CYP4V2st and AAV8.CYP4V2fv rescued RPE atrophy in BPS patient derived iPS-RPE samples compared to untreated patient sample (Figure 9, MOI = 1x10e5).
[526] [526] Each treatment with AAV5.CYP4V2op, scA-AV1.CYP4V2op and scAAV5.CYP4V2op rescued RPE atrophy in a BPS patient-derived iPS-RPE sample compared to an untreated patient sample (Figure 10, MOI = 1x10e4 ).
[527] [527] Treatment with scAAV.CYP4V2op rescued RPE atrophy in an iPS-RPE sample derived from a patient with BCD compared to an untreated patient sample (Figure 11. MOI = 1x10e5 2 weeks after treatment).
[528] [528] Significantly, treatment with AAV.CYP4V2 at a lower dose (MOI = 1x10e4) in P2 samples obtained similar results or better than a higher dose (MOI = 1x10e5 GC / cell) of treatment by the same vector in samples P1. This demonstrated at the cellular level that to obtain the same efficacy or similar efficacy in rescuing RPE atrophy, different patients may need a different dosage. In other words, a vector and a similar dose level for all patients with the same disease may not be the most medically or economically efficient approach to genetic therapy. BCD cell model and similar cell models for other eye diseases can provide personalized optimal guidance.
[529] [529] Other AAV.CYP4V2 vectors are also tested and show improved RPE atrophy in a BCD patient iPS-RPE sample, including treatment with AAV2tri (YF) .CYP4V2op (1x10e4 MOI) and AAV5.CYP4V2op ( new) (SEQ ID NO: 63) at different MOI levels (1x10e4 and 1x10e5 GC / cell). In addition, cell viability images were processed by ImageJ (Fiji) to count the number of dead and living cells in the iPS-RPE samples. Four different areas / images from each sample were used to count and the ratio of dead / living cells from multiple images of the same sample were averaged. Dead / live cell ratios demonstrated atrophy of RPE cell rescued by treatment with AAV.CYP4V2 in iPS-RPE derived from patient with BCD. For example
[530] [530] These results demonstrated that: (1) Several AAV.CYP4V2 vectors, CYP4V2 expression cassettes and cDNAs rescued RPE atrophy in BCD; (2) Self-supplementing AAV vector (scAAV) is quick to achieve rescue effectiveness; (3) Effectiveness can be achieved at different dosage levels. Example 14 - Safety of AAV.CYP4V2 Vectors and GMP Manufacturing for Clinical Use
[531] [531] Previous studies have shown that CYP4V2 is almost ubiquitously expressed in human organs and the level of expression within the eye is high in the retina. In addition, the safety of AAV vectors has been established in studies of gene therapy and clinical tests for other diseases. Thus, it is reasonable to expect that AAV.CYP4V2 vectors are safe to use in gene therapy.
[532] [532] In this study, several AAV.CYP4V2 vectors were used to treat human iPS-RPE samples at a high dose (for example, 1x10e5 MOI). No material difference in cell death between untreated and AAV.CYP4V2-treated samples was observed, except that AAV.CYP4V2 rescued RPE atrophy in iPS-RPE samples derived from a patient with BCD as described in the Example above. This established the safety of AAV.CYP4V2 vectors and demonstrated that high levels of expression of the transduced CYP4V2 coding gene can be obtained without evidence of significant toxicity.
[533] [533] In addition to testing on cell lines, the safety of gene therapy with AAV.CYP4V2 can also be tested on animals, for example, on mice, rats or non-human primates and / or through clinical tests on humans. Various manufacturing methods and platforms are available to produce recombinant AAV vectors for human clinical use. For example, and without limitation, GMP manufacture of rAAV vectors may use a 2 plasmid transfection method or a 3 plasmid transfection method, may use mammalian cell lines such as HEK293, A459 or 293T, or insect cell lines such as the baculovirus / Sf9 cell platform, can use adherent cell culture or suspension. Also, several methods, processes and / or platforms, including, without limitation, production system based on herpes simplex virus (HSV), single-use bioreactors (for example, iCELLis), HYPERStaks, roller bottles and column chromatography , can be used to increase yield or titer or improve purity and / or avoid potential contamination. These methods, processes, techniques and platforms for clinical production of rAAV vectors are known in the art and are commercially available from contract manufacturing organizations (CMOs) or academic GMP facilities, for example, Lonza (USA), Cobra Biologics (UK), Nationwide Children's Hospital (NCH. Ohio, USA), Children's Hospital of Philadelphia (CHOP. USA), WuXi Biologics (China and USA). AAV.CYP4V2 vectors for human clinical use can be manufactured using any one or more of the GMP methods, processes, techniques, platforms and facilities mentioned here and / or others known in the art or to be developed in the future. Example 15 - Individual Selection and Administration of AAV.CYP4V2 in vivo to Treat BCD
[534] [534] An eligibility criterion of exemplary individual for human clinical trial of AAV.CYP4V2 is listed as follows:
[535] [535] Individuals are eligible to participate in the study if they meet all of the following inclusion criteria:
[536] [536] Individuals are not eligible to participate in the study if they meet any of the exclusion criteria that follow.
[537] [537] For use in AAV.CYP4V2 to treat BCD, the patient must have a confirmed genetic or molecular diagnosis of BCD, that is, confirmation of mutation of biallelic CYP4V2 through genetic testing (single gene test or gene panel test multiple if medically necessary). Because BCD is sometimes diagnosed as inherited retinal disorder (IRD), retinal degeneration (RD) or retinitis pigmentosa (RP), AAV.CYP4V2 can also be used to treat a mutated IRD, RD or RP patient of genetically confirmed biallelic CYP4V2.
[538] [538] For treatment with AAV.CYP4V2 in vivo, the patient must have viable retinal cells as determined by optical coherence tomography (TOC) and / or ophthalmoscopy. Preferably, the patient should have some remaining vision (for example, better corrected visual acuity (BCVA) better than or equal to 20/200 (0.1 tenth in the eye to be treated).
[539] [539] Various means / route of administration can be used to deliver AAV.CYP4V2 vectors to target cells (eg, retinal or corneal cells) in vivo, including, without limitation, administration to the retina can be performed by sub-retinal injection, intravitreal injection (using AAV vectors suitable for intratrip administration, for example, AAV2 (Y444F + Y500 + Y730F), AAV 7m8 or its derivatives) or administration through the blood stream (using AAV that can penetrate the blood-retinal barrier (for example, AAV9 or AAV-PHP.B). Furthermore, AAV.CYP4V2 vectors can also be encapsulated in a device to be implanted intravitreally as a way of administration.
[540] [540] Surgical / administration methods related to gene therapy, as well as certain techniques to improve the efficiency of administration / transduction (for example, stripping of the inner limiting membrane (ILM) and vitrectomy (VIT)), are known in the art. Immunosuppressants, for example, corticosteroids, can be used before, during and / or after administration of AAV to minimize immune responses.
[541] [541] In addition to in vivo patient treatment, CYP4V2 gene therapy (including AAV.CYP4V2 gene therapy) can also be used to treat target cells (for example, RPE cells derived from iPS of a patient with BCD , retinal cells, corneal epithelial cells or corneal cells) in vitro and then transplant such cells to the patient as a cell therapy. Methods of using AAV.CYP4V2 vectors to treat BCD patient iPS-RPE cells are provided in the present Examples and description. Cell implantation / transplantation methods, for example, to the retina or cornea, are known in the art. For example, the same methods or similar methods and surgical techniques for transplanting ES-RPE cells to the retina can be used to transplant iPS-RPE cells from the patient with BCD.
[542] [542] Therapeutically effective doses can be determined and evaluated in disease models (eg, BCD cell model (eg, iPS-RPE cell line derived from patients with BCD) or an animal model, and confirmed or refined by testing For in vitro cell treatment, the dose is usually expressed as MOI and then multiply the MOI by the number of cells being treated.
[543] [543] See “E. Treatment Options, Individual Selection and Administration ”and another description here for further reporting. Example 16 - Post-treatment evaluation
[544] [544] Since clinical symptoms of BCD are similar to those of many other types of IRDs, RDs and RP, for example, loss in visual acuity, restricted visual fields, night blindness, reduced adaptation to dark, sensitivity to contrast and color vision, changes in the retina (and cornea for some patients) and decreased responses on electroretinogram (ERG), related measurements can be used to assess the disease status and progression before and after treatment of a patient with BCD, in this way evaluating the result of the treatment. Such measurements and related examinations and tests are known in the art for retinal and corneal diseases. For example, and without limitation, best corrected visual acuity (using visual acuity plot) can be used as the primary outcome measure for gene therapy for BCD, with one or more of the following as secondary outcome measures: microperimetry ( change in sensitivity), background autofluorescence (AF) (change in AF), optical coherence tomography (OCT) (ellipsoid zone and retinal thickness), contrast sensitivity (Pelli-Robson graph), color vision (tests Farnsworth-Munsell 100) and ERG (changes in ERG). In addition, functional tests such as mobility testing can also be used as a measure of primary or secondary outcome. Assessments can be carried out at different time points
[545] [545] Viral vector-mediated gene therapy can trigger cellular, local or systemic immune responses, which can offer safety risks. Immune reactions can also decrease transduction efficiency and thus decrease the effect of treating viral vector-mediated gene therapy. Immune responses can occur in the form of the humoral response (or antibody-mediated response) recognizing antigens or pathogens that are in the lymph or blood and / or cell-mediated immunity. To minimize immune responses, immunosuppressants such as corticosteroids are often used in connection with administration of gene therapy. Immunosuppressive drugs have effects, for example, that can cause increased intraocular pressure, cataracts and other adverse events (for example, prolonged use of immunosuppressants can increase the risk of cancer). In addition to the immune response, other individual differences exist between patients, for example, in response to different types (for example, different serotype or different capsid mutation / structure) of vectors, or in response to the same vector in the same dose.
[546] [546] A method for reducing immune responses to viral vectors, preserving transduction efficiency, decreasing the dose of viral and / or immunosuppressive vectors and / or maximizing therapeutic effect for patients with
[547] [547] Specifically, several rAAV vectors including five different AAV serotypes (AAV1, AAV2, AAV5, AAV8 and AAV9) and one AAV with capsid mutation (AAV2.tri (YF)) were generated and tested to assess differences between different patient cell lines in this study. Example 18: Use of scAAV in Rapid Rescue of Retinal Diseases and Use of EFS and / or SPA in a scAAV vector or an AAV vector in Eye Disease Treatment
[548] [548] As demonstrated in Example 13, treatment with scA-AV.CYP4V2 achieved robust rescue of biochemical phenotype in iPS-RPE cells from patient with BCD in a very short time (only 4 days). In addition, scAAV.CYP4V2 showed rescue of RPE atrophy in iPS-RPE cell line from a patient with BCD two weeks after treatment with AAV (see Figure 11). Rapid and robust expression in human iPS-RPE cells directed by the EFS promoter (exemplary sequence shown in SEQ ID NO: 35) and SPA (exemplary sequence shown in SEQ ID NO: 36) in a scAAV vector demonstrated the suitability of EFS promoter and / or SPA in the direction of expression of a transgene in human eye cells and treatment of human eye diseases. The rapid rescue obtained by scAAV vectors with the EFS and SPA promoter makes them particularly useful in the treatment of rapidly progressing diseases or patients with advanced disease stage.
[549] [549] In addition, the study proved the rapid and robust expression of a scAAV design in human retinal cells. This makes scAAV-mediated gene therapy particularly useful in treating a patient with early retinal disease or in treating an advanced-stage patient who needs a quick rescue. Discussion on CYP4V2 Gene Therapy
[550] [550] BCD is a rare blinding eye disease for which there is currently no approved treatment available. In clinical research involving the use of iPS-RPE cell lines specific to patients with BCD, the efficacy of several AAV.CYP4V2 vector designs and expression cassette in rescuing the phenotype in specific iPS-RPE cells BCD patient was proven in this study as assessed through fatty acid and lipid assays. In addition, different doses (MOI) were tested, which serve as a guide for determining the dose range for in vivo treatment. Finally, there is no significant evidence of toxicity associated with gene therapy with AAV.CYP4V2. Examples of Cell Therapy and CRISP Gene Editing Therapy Example 19 - Use of iPSCs, iPS-RPE or iPS-ocular Cells from a BCD Individual in Cell Therapy
[551] [551] BCD is a relatively late-onset disease. Symptoms in patients with BCD are usually developed in the 2nd, 3rd and even 4th decades of life. Also, the iPS reprogramming process may have a bit of a “reset clock” effect. In this way, iPS-RPE cells and other iPS-ocular cells derived from a BCD patient can be used as a cell therapy for transplantation to the BCD patient even without any genetic repair of CYP4V2 mutations in iPS-RPE cells .
[552] [552] Alternatively, iPSCs, iPS-RPE cells, iPS-PRCs, iPS-CE cells, iPS-CECs and / or other iPS-eye cells derived from a patient with BCD can be genetically repaired prior to cell therapy transplantation . Genetic repair can also be achieved or through CYP4V2 gene therapy as described in the Examples.
[553] [553] Cells derived from patient-specific iPSC (eg, iPS-RPE cells, iPS-CECs, iPS-CE cells, iPS-PRCs or iPS-eye cells) can be used as a source of autologous cells for transplantation in therapy for eye diseases, including, without limitation, retinal and corneal diseases. Compared with cells generated from allogeneic sources, such as ES cells (for example, ES-RPE, ES-CEC or ES-PRC cells and tissues formed from such ES-derived cells) or iPS cells from one another individual, such patient-specific iPS-derived autologous cells and tissues made from such cells generally require little or no patient immunosuppression and have no ethical issues related to the use of ES and ES-derived cells.
[554] [554] However, iPSCs generated from patient source cells (for example, fibroblasts and blood cells) and cells and tissues derived from such patient-specific iPSCs (for example, patient-specific iPS-RPE cells patient, iPS-PRCs, iPS-CECs, iPS-CE cells and iPS-ocular cells) still have disease-causing mutations and related phenotype. To generate healthy patient-derived cells and / or tissues, pathological mutations can be genetically repaired or corrected with genetic editing technology, including, without limitation, regularly interspersed grouped short palindromic repetitions (CRISPR), which can be designed to correct a target mutation in a patient's cell. These healthy genetically repaired iPSCs can then be used to generate various types of cells (for example, iPS-RPE cells, iPS-CECs, iPS-CE cells, iPS-PRCs or other iPS- cells
[555] [555] Furthermore, this proof of concept study demonstrates that these iPSCs with corrected gene and / or cells derived from iPS with corrected gene (for example, iPS-RPE cells) no longer have the phenotype (for example, biochemical profile abnormal as assessed by bioassays, for example, lipidomic and proteomic) as seen in patient-derived iPS (uncorrected) cells. In this way, these cells with the corrected gene serve as a source of genetically repaired, regenerative autologous cells, which can be used as replacement cells in cell therapy. Compositions and methods regarding autologous cells of the patient with the corrected gene are described in detail here and in the Examples below.
[556] [556] Another type of genetically repaired patient cells are patient iPSCs or iPS derived cells (for example, iPS-RPE cells, iPS-PRCs, iPS-CE cells, iPS-CECs and iPS- cells eyepieces, neuron iPS- cells) treated by gene supplementation therapy (for example, gene therapy with CYP4V2) as described above. After treatment with gene therapy, the patient's specific cells have a healthy copy of the mutated gene (for example, a cDNA) and / or express a functional protein encoded by the healthy transgene. In addition, patient-specific cells treated with gene therapy demonstrate an improved or normalized biochemical profile or other phenotype seen in untreated patient cells. In this way, they can also be used as a source of genetically repaired autologous cells for use as replacement cells in cell therapy, for example, iPS-RPE cells, iPS-PRCs, iPS-CECs, iPS-CE cells and iPS- cells specific eyes of a BCD patient treated with CYP4V2 gene therapy as autologous cells from a genetically repaired patient
[557] [557] Autologous cell replacement for ocular and retinal degenerative diseases associated with genetic mutations depends on the ability to repair a patient's pathogenic mutation by genetically correcting the mutation through genetic editing or repairing or mitigating the consequence of the mutation (eg example, by administering a healthy copy of a transgene relative to the disease gene, for example, gene therapy with CYP4V2) prior to transplantation. Here, patient-specific iPSCs from a patient with BCD with the most common CYP4V2 mutation (c.802-8_810del17insGC) were generated and the CRISPR genetic editing components (CRISPR guide RNA and donor model) and various constructs (plasmí - deo and RNP) to correct this mutation have been developed. Although CRISPR / Cas9 is used here as a means for genetic editing, it is anticipated that another CRISPR system (eg, Cpf1) and other genetic editing techniques including, but not limited to, TALEN as well as emerging genetic editing techniques and future ones such as CRISPR / Cpf1 can be used to obtain the same or similar results. It is also expected that genetic editing can be applied not only to iPSCs, but also to the original source cells that will be used to generate the iPSCs, as well as to the cells generated from the iPSCs, to correct the mutation (s) pathogen (s) in such cells.
[558] [558] Although iPS-derived cell lines are generated on a patient-specific basis, their application in cell therapy does not have to be. A key factor limiting the widespread use of
[559] [559] The following describes methods on how to generate genetically repaired patient-specific autologous cells, how to assess the effect of genetic repair on cells, and how to use them in cell therapy. The examples provided here are related to the generation of autologous patient cells genetically repaired from a BCD patient with the c.802-8_810del17insGC mutation in the CYP4V2 gene, the most common mutation among BCD patients. The same methods can be used to generate autologous cells from a genetically repaired patient from a patient with a different mutation in CYP4V2 or a patient with a mutation in another gene associated with an eye disease or a patient with a mutation in an associated gene with other types of diseases, including, without limitation, any gene contained in Table 4. Table 4 List of ABCA4, ABCC6, ABHD12, ADAM9, AHI1, AIPL1, ALMS1, ARL13B, ARL6, ARMS2, ATXN7, BBS1, BBS10, BBS12, BBS2, BBS4, BBS5, BBS7, BBS9, BEST1, C1QTNF5, C2, C2orf71, C3, C5orf42, C8orf37, CA4, CABP4, CAC-NA1F, CACNA2D4, CAPN5, CC2D2A, CDH23,
[560] [560] Using BCD, a disease with mutations in CYP4V2, as an example, iPSCs were generated from patient-specific cells carrying the patient-specific mutation with BCD. Patient-specific iPSCs are transfected with CRISPR guide RNAs (gRNA), Cas9 endonuclease and a donor homology model. Copies of CYP4V2 gene show correction of mutation and conversion in the wild type allele. The corrected iPSCs are then used to generate iPS-RPE cells with the corrected gene. The corrected gene iPS-RPE cells are then tested to confirm that they no longer have a phenotype (eg, abnormal biochemical profile (eg, fatty acid profile)). These genetically repaired autologous patient cells can be transplanted (either directly (for example, cell suspension) or in other forms, such as part of a layer, leaf, matrix, base or tissue) to the same patient as an autologous cell therapy for BCD. (i) Generation of Patient-Specific iPSC Strains with BCD:
[561] [561] iPSCs were generated from patient-specific cells from a patient with BCD carrying a homozygous c.802- 8_810del17insGC mutation in the CYP4V2 gene as described herein. See Example 1 for methods to generate patient-specific iPSCs. The mutation of the patient with BCD was identified through sequencing (ii) Design, Evaluation and Selection of CRISPR genetic editing components and constructs targeting the mutation:
[562] [562] See the Examples here on CRISPR gene addition therapy for a detailed description.
[563] [563] (CRISPR gRNAs were selected to minimize off-target editing and maximize specificity with a target sequence directly centered on the mutation site. Multiple gRNAs with high specificity for the region containing the patient-specific CYP4V2 mutation were The candidate gRNAs were separately inserted into an expression vector also containing the Cas9 endonuclease responsible for mediating target DNA cleavage and transfected into a 293 cell line. The patient's genomic DNA was amplified by PCR using primers for the CYP4V2 region, and the PCR products were analyzed for DNA cleavage activity.A research trial was used to assess which gRNA candidate has relatively high activity at the mutation site. higher cut is used for genetic editing). (iii) Genetic editing in iPSCs:
[564] [564] See Examples below on CRISPR gene addition therapy for a detailed description.
[565] [565] For genetic recessive diseases such as BCD, correction of a gene in an allele or mutation is sufficient. Multiple CRISPR constructs targeting different mutations can be used to correct multiple mutations carried by a cell. (iv) Generation of iPS-RPE cells or other iPSC ocular iPSC cells with Corrected Gene:
[566] [566] After confirming accurate correction of the pathogenic mutation with no or minimal off-target editing through sequencing, genetic editing-corrected iPSC is used to differentiate into and generate iPS-RPE cells or the other type of iPS-eye cells (for example, iPS-PRCs, iPS-CECs or iPS-CE cells) affected by the relevant disease as described herein. Corrected iPS-RPE cells derived from a patient with BCD then undergo the same confirmation of RPE destination (eg, distinct RPE morphology (eg, pigment and / or hexagonal shape) and / or specific RPE markers ). (v) Bioassays to confirm phenotype-free
[567] [567] Bioassays are used to confirm that these gene-corrected iPSCs and / or iPS-derived cells with corrected gene (for example, iPS-RPE cells) no longer have the phenotype as seen in iPS-derived (uncorrected) cells than patient. Bioassays can be any type of biological assay that can identify and assess the cell and / or molecular phenotype in patient cells since it refers to a specific disease. For example, they may include, without limitation, lipidomic, proteinomic, protein expression and / or other biochemical tests. For BCD, the bioassay includes tests for fatty acids and ceramides as described in the present Examples. The results indicate that these iPS-RPE cells with corrected gene derived from the patient with BCD no longer have the relevant biochemical defect / dysfunction as seen in uncorrected iPS-RPE cells derived from patients with BCD. This proves that iPS-RPE cells, with a corrected gene, are phenotype free and thus are a source of replacement cells suitable for cell therapy. (vi) Transplant:
[568] [568] These genetically repaired autologous patient cells (for example, iPS-RPE cells, iPS-PRCs, iPS-CE cells, iPS-CECs and other iPS-eye cells) can be transplanted (either directly or as part of a layer, a sheet, a matrix, a base or a tissue) for the same patient as a cell therapy for BCD. Example 21- Specific Example of CRISPR Gene Editing Therapy for an Eye Disease
[569] [569] CRISPR / Cas9 is highly specific when gRNAs are designed correctly, but specificity and off-target editing is still a major concern, particularly since CRISPR is being developed for clinical use. The Example that follows describes in detail methods for developing CRISPR gene editing therapy constructs with high target specificity and low off target editing risk for use in treating eye disease. Furthermore, the c.802-8_810del17insGC mutation represents one of the most challenging mutations to correct among all known CYP4V2 mutations and other genetic eye diseases. Most CYP4V2 mutations are single nucleotide change, insertion or deletion (see Table 1: Selecting CYP4V2 Mutations from BCD Patients), while the c.802- 8_810del17insGC mutation involves a 17 bp deletion and a 2 bp insertion and both an intron and an exon.
[570] [570] Several sets of CRISPR gene editing therapy constructs to correct the most common pathological CYP4V2 mutation (c.802-8_810del17insGC mutation) have been designed and built. What follows is a detailed description of the design, compositions and methods of using these CYP4V2 CRISPR Gene Edit constructs to correct the mutation and treat BCD. (a) Mutation Analysis
[571] [571] The c.802-8_810del17insGC mutation involves both an intron and an exon, both a deletion and an insertion, and affects a joining acceptor site.
[572] [572] The c.802-8_810del17insGC mutation refers to a deletion of 17 bases with two bases (GC) inserted in place beginning 8 bases from the intron 6 end of the CYP4V2 gene, also referred to as IVS6-8 del / insGC; see SEQ ID NO: 46 showing sequence of the CYP4V2 genomic DNA region comprising the c.802-8_810del17insGC mutation and SEQ ID NO: 47 showing the
[573] [573] To obtain a good repair rate using CRISPR, a cleavage generated by Cas as close as possible to the mutated sequence is desired. The CYP4V2 genomic DNA region containing the c.802-8_810del17insGC mutation has multiple SpCas9 (NGG) PAM sites. In this way, regular SpCas9 can be used to correct this change. Alternatively, Cas9 from other species, a mutated Cas9 or another CRISPR nuclease (for example, Cpf1) with a different PAM (for example, TTTN for Cpf1 that is present in the mutated sequence) can be used to correct the mutation c. 802- 8_810del17insGC and / or other mutations. (b) CRISRP gRNA design and selection
[574] [574] Based on the various MAP sites present in the c.802-8_810del17insGC mutation region of the CYP4V2 gene, multiple related protospacer element sequences (hereinafter referred to as gRNA, typically 20 nt in length, but can be be of different length, for example, 17-22 nt for use with Cas9) were evaluated using DeskGen software. Five (5) gRNA candidates were selected using the following criteria: a) the proximity of the gRNA / Cas9 cleavage site to the target correction site; and b) the predicted out-of-target profiles of the gRNA (see Table 5 and Figure 12; see SEQ ID NOs: 48 to 52 for gRNA sequences). Table 5. Sequences of gRNA candidates Classification Outside gRNA Target sequence score CYP4V2 g1 87 5'-TGA TTA TCA TTC AAA GCG AA CGG-3 'CYP4V2 g2 98 5'-GAT TAT CAT TCA AAG CGA AC GGG-3' CYP4V2 g3 73 5'-GAT AAT CAC ATG CTT CTG TT TGG-3 'CYP4V2 g4 70 5'-TTC ATT GGC GTT CAT TTC AT TGG-3' CYP4V2 g5 32 5'-CAC ATG CTT CTG TTT GGA CT TGG-3 '
[575] [575] The PAM site corresponding to each gRNA candidate is highlighted in bold. To avoid confusion, PAM sequence is not part of the gRNA (protospacer element) sequence. (c) Validation of gRNA using Patient Genomic DNA
[576] [576] Genomic DNA from a patient with BCD (P1) with c.802-8_810del17insGC mutations was used to select and validate the gRNAs. DNA amplicons flanking a CYP4V2 region containing the mutation site and several target sites were prepared using primers (see Table 6 and Figure 12). DNA amplicons, single guide RNA (sgRNA) prepared by in vitro transcription (IVT) (each comprising one of gRNA1, gRNA2, gRNA3, gRNA4 or gRNA5) and SpCas9 protein were mixed and incubated at 37º C for 1 h. Active sgRNA mediated Cas9 protein to create double-strand breaks in Amplicons and exhibit various fragment patterns (Table 7). The reactions were loaded and fragments of DNA were again determined on 1.5% agarose gel.
[577] [577] To confirm that the fragments are in fact originated from amplicon, samples of untreated amplicon DNA (Figure 16, top panel) and the smaller fragment treated with g2 (Figure 16, middle panel) were subjected to Sanger sequencing (Figure 14). All 5 gRNAs showed predicted cleavage activities.
[578] [578] In addition to or in place of validation on a patient's genomic DNA carrying the mutation, gRNA activities can also be validated in patient cells, including, without limitation, somatic cells, stem cells, iPSCs or derived cells of a stem cell. (d) Construction of gRNA expression vectors
[579] [579] Three gRNAs (g1, g2 and g3) with the highest off-target activity and highest classifications were cloned into the pX-U6-CBh-Cas9-Pure gRNA expression vector by inserting an oligo cassette double strand of each active gRNA. Each cassette was synthesized based on one of the gRNA sequences of g1, g2 and g3. Schematic illustrations showing the expression vector construct and the gRNA insertion site are provided in Figure 15 and Figure 16. See Figure 17 for a more detailed illustration (using g1 as an example) showing the entire IVT sgRNA sequence ( SEQ ID NO: 55 (not including the sequence of the protospace element or the optional "G")) following the U6 promoter. The “G” nucleotide (SEQ ID NO: 59) inserted at the beginning of each protospacer element (gRNA) sequence is optional. It is mainly to increase the transcription efficiency of the U6 promoter. It is not necessary if the sequence of the protospace element starts with a “G” residue or if a non-UT promoter is used (for example, H1 promoter). All gRNA constructs were verified through both restriction enzyme digestion and sequencing.
[580] [580] Three plasmids each expressing a higher RNA (g1, g2 or g3) and coexpressing hSpCas9 and genes for pu- romicin resistance, namely pX459-hSpCas9-2a-Pure, have been developed (Figures 15 and 16) and included as one of the constructs (see Table 8 below) for gene correction of the c.802-8_810del7insGC mutation.
[581] [581] It would be understood that the guide RNA, Cas protein and / or selection marker (e.g., puromycin resistance gene and / or GFP, EGFP or RFP) can be packaged in a plasmid or in a separate plasmid. In addition, when more than one gRNA is used (either to correct multiple mutations or to correct the same mutation, for example, a pairing gRNA for use with Cas9 Nickase), they can be packaged in the same or separate vectors.
[582] [582] In addition to the plasmid vector described here, several other vectors can be used to package CRISPR genetic editing components (guide RNA and / or Cas protein), and / or selection marker, including, without limitation, plasmid pX458 vector, adeno-associated virus (AAV) vectors and / or lentivirus vectors. In addition to DNA constructs encoding the components of CRISPR, guide RNA, Cas protein and / or selection markers, they can be used directly or in an mRNA construct or RNP construct. (e) Construction and validation of CRISPR RNP
[583] [583] In addition to a DNA construct in a vector (for example, a pX459 plasmid as described above), a CRISPR ribonucleoprotein (RNP) construct was developed for each of g1, g2, g3, g4 and g5 (see Tables 5 and 8). Each RNP construct comprises (i) a simple chimeric guide RNA (sgRNA) comprising the relevant protospace element (see Tables 5 and 8 and this detailed description); and (ii) a SpCas9 protein forming a ribonucleoprotein (RNP) complex. The cleavage activities of various RNP constructs (sgRNA1: Cas9, sgR-NA2: Cas9, sgR-NA3: Cas9, sgRNA4: Cas9, sgRNA5: Cas9) at the target site of the CYP4V2 gene were validated in patient genomic DNA (see Figures 12, 13 and 14) as described in paragraph (c) above.
[584] [584] A sgRNA is typically about 100nt in length, but can vary in lengths comprising a 17nt-22nt protospace element sequence. A sgRNA can be derived or synthetic IVT. IVT sgRNAs corresponding to g1, g2, g3, g4 and g5 were generated and validated as described above. synthetic sgRNAs corresponding to g1 and g2 were ordered from Synthego (Sicicon Valley, CA, USA) as described below.
[585] [585] In place of sgRNA, a duplex of crRNA (exemplary sequence in SEQ ID NO: 53) and tracrRNA (exemplary sequence in SEQ ID NO: 54) can be used together with a Cas protein (for example, Cas9) to form a CRISPR RNP complex (crRNA: tracrRNA: Cas9). When using a Cpf1 protein, no tracrRNA is needed.
[586] [586] A sgRNA or crRNA: tracrRNA can be chemically modified to protect against intracellular RNA degradation. For example, a chemically modified synthetic RNA may contain 2'-O-methyl analogs and 3 'phosphorothioate internucleotide bonds at the three terminal bases 5' and 3 'of the gRNA (Synthego (Silicon Valley, CA,
[587] [587] In a homology-directed repair (HDR), a donor model is used to provide the donor nucleic acid sequence necessary to correct the mutated sequence of the target gene. Two separate donor models for HDR were generated in the form of single-stranded Oligo Deoxynucleotide (ssODN). The first, referred to as the CYP4V2 or CYP4V2 ssODN1 donor model (SEQ ID NO: 56), contains the 17 bp correction and has the sequence as follows: 5’- AGA AAA ATA AAT GAA AGA AAC TAG CAT ATT TTA TAA GAA AAT GTG TTA ACT AGG GTG CAT CCA AGT CCA AAC AGA AGC ATG TGA TTA TCA TTC AAA TCA TAC AGG TCA TCG CTG AAC GGG CCA ATG AAA TGA ACG CCA ATG AAG ACT GTA GAG GTG
[588] [588] Any of the donor models can be used with any gRNA or sgRNA (g1, g2, g3, g4 or g5) described above, and a Cas9 protein to generate homology-directed repair (HDR) to correct the target CYP4V2 mutation (c.802-8_810del17insGC).
[589] [589] The donor models provided here are 200nt in length. Donor models of various lengths can be used. A donor model can be symmetrical or asymmetric with respect to the target site. A donor model can be provided by an ssDNA, ssODN or a vector (for example, a plasmid or AAV vector) containing or encoding the donor nucleic acid sequence. If the donor model has an intact sequence complementary to the protospace element in the CRISPR guide RNA and the PAM sequence directed by the Cas protein, to prevent this donor model from being degraded by the Cas protein (for example , Cas9) in cells, mutations can be made in the donor model, for example, to mutate the “NGG” PAM Cas9 in the donor model and change it to “NGT” or another non-PAM sequence. However, if the PAM mutation intended to be introduced by the donor model is within the coding region, care must be taken to ensure that it is a silent mutation.
[590] [590] Donor models can be made synthetically and are commercially available. For example, DNA oligos of a given sequence can be ordered (Ultramer® DNA Oligonucleotides, Integrated DNA Technologies (IDT), Coralville, Iowa, USA). (g) Cas Protein and Selection Marker
[591] [591] CRISPR-associated proteins / nucleases (Cas) (eg, Cas9 or Cpf1) are commercially available, including, without limitation, encoded by a plasmid or as a recombinant protein for use in an RNP construct. A Cas protein can also include one, two or more nuclear localization (NLS) sequences (for example, Catalog No.: 1074182, Integrated DNA Technologies (IDT), Coralville, Iowa, USA; Catalog No.: A034a-a - 1000, Feldan (Quebec, Canada); Cpf1: Catalog No.: 1076158 (IDT)) and can also be fused with a selection marker (eg, EGFP-fused SpCas9 protein, Catalog No.: PR-137211- E (Novatein Biosciences, Woburn, MA, USA).
[592] [592] When transfecting a CRISP gene editing construct in vitro into cells, a selection marker can be used to assess the transfection rate and / or assist in picking up the cells for processing in the next step. Various selection markers including, without limitation, fluorescence (eg GFP, EGFP, RFP) and / or puromycin), can be used in the process. A selection marker can be integrated with any component of a CRISPR construct or can be provided separately in a transfection process. For example, a fluorescence marker can be combined with tracrRNA (IDT) or Cas9 protein (Novatein Biosciences, Catalog No. PR-137211-E) for convenient manual imaging and classification or FACS of transfected cells. A puromycin resistance gene can be provided in a vector that is cotransfected with the CRISPR construct for selection using puromycin. Selection using puromycin is illustrated in the Examples. Selection markers of various types such as antibiotic selection markers (eg, puromycin) or fluorescence marking are commercially available and can be integrated into the CRISPR component (eg, Cas9 protein or CRISPR guide RNA) ) or provided separately (for example, an expression plasmid expressing the puromycin resistance gene), including, without limitation: IDT, Sigma Al-drich, Novatein Biosciences, Clonetech Laboratories and InvivoGen. (h) Constructs and recommended protocol
[593] [593] The following table (Table 8) shows the CRISPR genetic editing constructs (plasmid and RNP) generated for each of the 3 gRNAs (gRNA1, gRNA2 and gRNA3). They contain three constructs of gRNA plasmid or respective gRNA, two donor models (complementary advanced and reverse) and protein SpCas9. Table 8. Plasmid and RNP constructs for CRISPR gene correction therapy for CYP4V2 mutation (c.802- 8_810del17insGC) 1
[594] [594] The following protocols are for administering CYP4V2 mutation CRISPR gene repair constructs to the patient's iPSCs through electroporation and nucleofection. Other methods, including, without limitation, lipofection, viral vector transduction (eg, lentivirus vector) or AAV (eg, use of AAV6 to administer the donor model) or microinjection can also be
[595] [595] 1. Use an ice bucket. Defrost a sgRNA (Item No. 4 or 5 or 6; do not combine gRNAs), a ssODN donor model (or Item No. 7 or 8) and SpCas9 protein (Item No. 9), as well as the expression vector of Cas9-Pure on ice. The Cas9-Puro expression vector is used as a selection marker. It is a pX459-hSpCas9-2A-Pure plasmid and has a structure shown in Figure 15, except that it has not cloned into gRNA.
[596] [596] 2. Clearly mark 1.7 ml Eppendorf tubes and 6-well plates. For each sample, prepare an Eppendorf tube and 1 well. Add 3 ml of culture medium (TeSR-E8 from StemCell Technologies (Cat. No. 05940) to each well.
[597] [597] 3. Prepare a 10 cm plate with 25 ml of PBS to wash the Neon® tip.
[598] [598] 4. Prepare a 6-well plate for plating the electroporated cells. Add 3 ml of culture medium to each well.
[599] [599] 5. In each Eppendorf tube, add 4 µg (4 µl of stock) of sgRNA (Item 4, 5 or 6. Do not combine the sgRNAs) and 10 µg (10 µl of stock) of SpCas9 protein (Item No. 9), leave the tube at room temperature for at least 10 min.
[600] [600] 6. Add 5 µg (5 µl of stock) of ssODN (or item No. 7 or 8) and 2.5 µg (2.5 µl of stock) of Cas9-Pure expression vector to each tube.
[601] [601] 7. Resuspend cells in appropriate Neon® EP buffer to final density 1x107 cells / ml.
[602] [602] 8. Aliquot 105 µl of cell suspension and add each Eppendorf tube with a mixture of RNP CRISPR.
[603] [603] 9. Add 3 ml of Buffer E2 to the Neon® pipette and place the Neon® pipette in the Neon® pipette station.
[604] [604] 10. Use 100 µl of Neon® tip. Aspirate 100 µl of EP mixture from each Eppendorf tube and insert into a Neon® pipette.
[605] [605] 11. Apply one of the EP conditions in Table 9 above and follow steps 3 through 6 of Protocol No. 1 above.
[606] [606] Note: if iPSCs do not grow well, condition means are reported. Collect the used medium (without Puromycin) and filter it to remove cell debris. Mix in a 1: 1 ratio of used medium and fresh medium. The use of Matrigel (Cat. No. Corning 354277) and StemCell Technologies TeSR-E8 medium (Cat. No. 05940) is recommended for culturing human iPSCs in feeder-free conditions through the genetic editing process. The addition of Rock inhibitor (final concentration 10 µM) to the medium for 48 hours when plating cells after PE will help to preserve cell viability. Protocol No. 3 (Nucleofection using RNP):
[607] [607] 1. Lonza 4D Nucleofector, parameter setting: Lonza program, DS-150
[608] [608] 2. Prepare RNP (cas9 + RNA) and ssODN separately (bring the volume to a maximum of 10 µl), mix before use. See Table 10. (1) ssODN Advanced gRNA1 + CYP4V2 (2) gRNA2 + CYP4V2 Fortress ssODN (3) ssODN Reverse gRNA1 + CYP4V2 (4) gRNA2 + CYP4V2 Revise ssODN Table 10 Each group of 30µM gRNA ( µL) Cas9 20µM (µL) ssODN 30µM (µL) PBS (µL) reaction iPS cells P1 4 1 4 1 Case buffer / each Volume (µL) / sample 4 samples (µL) group Solution 16.4 65.6 Supplement 3 .6 14.4
[609] [609] Each expression plasmid construct containing CRISPR g1 or g2 (Item No. 1 or 2) and CRISPR RNP construct containing sgRNA1 or sgRNA2 (Item No. 4 or 5, Synthego, Silicon Valley, CA, USA) and SpCas9 (Item No. 9, Cat No.: A034a-a- 1000 from Feldan (Quebec, Canada) or Synthego (eg Cas9 2NLS, S. pyogenes), next to a CYP4V2 donor model (Item No. 7 or No. 8, ssODN, Ultramer DNA Oligonucleotides, Integrated DNA Technologies (IDT), Coralviile, Iowa, USA), is used to transfect patient iPSCs carrying the c mutation. 802-8_810del17insGC Evaluation of Gene Correction through Repair directed to Homology (HDR):
[610] [610] After transfection, the picked cells are collected for PCR followed by targeted amplicon sequencing to assess gene correction in the CYP4V2 region containing the c.802- 8_810del17insGC mutation. Deep sequencing of transfected cells shows that the readings contained precise correction of the mutation, with insertion of "TCATACAGGTCATCGCT" in 17 bp and deletion of "GC", resulting in correction of the mutation for the wild type sequence (SEQ ID NO: 47 ). Mutation correction is not seen in any non-transfected control iPSCs. The results also serve as an indication of HDR frequency among transfected cells. Obtaining iPS clones with no or minimal off-target editing
[611] [611] After HDR evaluation, transfection is performed again on patient iPSCs carrying the c.802- 8_810del17insGC mutation. Transfected cells undergo single cell cloning and expansion. Clonal cell lines with HDR on the confirmed target are then evaluated for off-target editing through sequencing. For clinical application, entire genome sequencing (60x coverage) is used to compare edited and untransfected cell lines from the same patient. A clonal iPS cell line edited without any off-target editing or minimal off-target editing without any material adverse consequences in the genome is selected. Differentiation of genetically corrected iPS in the desired type of cells
[612] [612] The selected iPS clonal cell line is then differentiated into iPS-RPE cells (see Examples here). The selected iPS clonal cell line can be differentiated into other cell types that are desired for use in cell therapy (for example, iPS-RPE cells, iPS-PRCs, iPS-CE cells, iPS-CECs or other cells iPS-eyepieces). Bioassay to confirm that genetically repaired or iPS derived iPS cells no longer have a phenotype
[613] [613] Genetically corrected (or genetically repaired) iPS-RPE cells are tested for biochemical function (see Examples here) and confirmed to have no more phenotype as seen in untreated patient iPS-RPE cells. CYP4V2 expression is detected in genetically repaired patient iPS-RPE cells.
[614] [614] Unlike a plasmid or other vector constructs (for example, AAV, lentivirus) that result in sustained expression of CRISPR components that it encodes (for example, RNA, the Cas nuclease and / or the donor model), a RNP CRISPR construct is quick to start activity and quick to stop activity. Components of an RNP construct are degraded relatively quickly in the transfected cells. In this way, the use of RNP constructs reduces the risk of off-target editing compared to plasmids and other constructs. This makes the RNA construct particularly suitable for transplantation, as well as for in vivo treatment (for example, injection of RNP constructs into an individual's eye for in vivo gene correction). In addition to BCD treatment, the CRISPR RNP constructs and methods provided here can be used to treat other diseases, including diseases associated with a mutated or defective gene shown in Table 4. Example 23: Use of Genetically Repaired Cells in Ocular Cell Therapy
[615] [615] iPS-RPE cells, iPS-PRCs, iPS-CECs, iPS-CE cells or other genetically repaired iPS-ocular cells can be transplanted into the patient's eye as an ocular cell therapy. For example, they can be used as autologous replacement cells for RPE cells, photoreceptors, or other ocular cells killed or degenerated in a patient with BCD. Genetically repaired cells can be transplanted either directly (for example, cell suspension) or in other forms, including, without limitation
[616] [616] If necessary, cells can be manufactured in a GMP facility for clinical use. GMP facilities for cell therapy products are commercially available from research institutes, contract manufacturing organizations (CMOs) and contract research organizations (CROs), for example, Cellular Therapy Integrated Services at Case Western Reserve University, Center for Cell and Gene Therapy at Baylor College of Medicine, CELLforCURE, New York Stem Cell Foundation and Lonza.
[617] [617] Autologous patient-specific administration can use the same administration methods as used in allogeneic ocular cell therapy (for example, embryonic stem cell-derived RPE transplantation (ES-RPE)) for retinal degeneration diseases, including those affected due to RPE degeneration, such as age-related macular degeneration (AMD). Such administration / surgical methods are known in the art. Example 24. Combination Treatment of Gene Therapy and Cell Therapy for Eye Diseases
[618] [618] The present descriptions were about compositions and methods for use in gene therapy and cell therapy for BCD. For eye diseases, gene therapy and cell therapy each have their own pros and cons. On the one hand, gene therapy works best in early to medium disease stage when the patient still has too many retinal (or ocular) cells left to receive and be rescued by gene therapy treatment. However, gene therapy does not work well or may not work at all for advanced patients who do not have the relevant eye cells remaining (for example, RPE or PRC). Cell therapy, on the other hand, provides replacement cells to replace dead or degenerate cells in the patient's eye and has its advantages over gene therapy particularly for patients in advanced stages and predominantly inherited diseases. However, cell therapy cannot rescue the “original” cells remaining in the patient's eye, whose survival not only preserves the patient's remaining vision, but also benefits from the replacement cells' integration.
[619] [619] To overcome the limitations of gene therapy and cell therapy and bring maximum benefits to patients, a combination treatment of gene therapy and cell therapy has been developed for BCD, which can also be used for other eye diseases. This method comprises: (a) applying gene therapy (for example, AAV.CYP4V2 gene therapy or CRISPR gene correction therapy) to the patient's eye in vivo; and (b) in vitro generation of genetically repaired patient-specific autologous iPS-ocular cells (eg, iPS-RPE cells, iPS-PRCs, iPS-CE cells, iPS-CECs or other types of eye cells that are affected by the disease) and transplantation of these cells into the patient's eye; where (a) and (b) can be applied sequentially (first (a), then (b) or first (b), then (a)) or simultaneously (for example, injection of gene therapy vectors and cells into a administration). Each of (a) and (b) can be applied one or more times to the same eye. Depending on the disease, stage of the disease and the individual situation of the patient, (a) and (b) may target the same or different types of eye cells. For example, in the case of BCD, gene therapy vectors driven by a ubiquitin promoter can result in CYP4V2 expression in RPE cells, photoreceptors and other retinal cells, while cell therapy can focus on the provision of RPE cells and / or regenerated photoreceptors. In this case, cell therapy can benefit from the provision of new cells (for example, RPE or photoreceptor cells), while gene therapy can improve the effect of cell therapy by rescuing the remaining RPE or photoreceptor cells and / or improve the conditions of choroid cells whose health affects the conditions of ocular cells. The combination of the "rescue" and "replacement" effect of gene therapy and cell therapy, respectively, makes combination treatment an improvement or either gene therapy or cell therapy. This combination treatment method can be applied to eye and other diseases caused by one or more genetic mutations, including, without limitation, diseases associated with a mutated or defective gene shown in Table 4.
[620] [620] It should be understood that, although the methods and compositions of importance have been described here in conjunction with several different aspects, the above description of the various aspects is intended to illustrate and not to limit the scope of the methods and compositions of importance. Other aspects, advantages and modifications are within the scope of the claims that follow.
[621] [621] Methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for or are products of the disclosed methods and compositions are disclosed. These and other materials are revealed here, and it is understood that combinations, subsets, interactions, groups, etc., of these methods and compositions are revealed. That is, although specific reference to each of the various individual and collective combinations and exchanges of these compositions and methods may not be explicitly revealed, each is specifically understood and described here. For example, if a particular composition of importance or a particular method is revealed and discussed and various compositions or methods are discussed, each and every combination and exchange of the compositions and methods is specifically understood unless specifically stated to the contrary. Likewise, any subset or combination of these is also specifically understood and revealed. SEQUENCES
SEQUENCE LIST * All reference numbers used in the strings are NCBI reference numbers unless otherwise mentioned.
Part I: Gene Therapy Sequences A. CYP4V2 cDNA and protein sequences SEQ ID NO: 1 - a cDNA sequence (1578 bp) encoding the human CYP4V2 protein (SEQ ID NO: 4), referred to herein as CYP4V2st. SEQ ID NO: 2 - a codon-optimized cDNA sequence (1578 bp) encoding the human CYP4V2 protein (SEQ ID NO: 4), referred to here as CYP4V2op. SEQ ID NO: 3 - a cDNA sequence (1578 bp) encoding a functional CYP4V2 protein (SEQ ID NO: 5), referred to herein as CYP4V2fv. SEQ ID NO: 4 - amino acid sequence (525 aa) of human CYP4V2 protein (NP_997235.3) SEQ ID NO: 5 - amino acid sequence (525 aa) of a functional variant of human CYP4V2 protein SEQ ID NO: 6 - fragment of human CYP4V2 protein without transmembrane domain (490 aa) SEQ ID NO: 7 - human CYP46A1 amino acid sequence
SEQ ID NO: 8 - human CYP4A11 amino acid sequence SEQ ID NO: 9 - human CYP4A22 amino acid sequence SEQ ID NO: 10 - human CYP4B1 amino acid sequence SEQ ID NO: 11 - human CYP4F2 amino acid sequence SEQ ID NO: 12 - human CYP4F3 amino acid sequence SEQ ID NO: 13 - human CYP4F8 amino acid sequence SEQ ID NO: 14 - human CYP4F11 amino acid sequence SEQ ID NO: 15 - human CYP4F12 amino acid sequence SEQ ID NO: 16 - human CYP4F22 amino acid sequence SEQ ID NO: 17 - human CYP4X1 amino acid sequence SEQ ID NO: 18 - human CYP4Z1 amino acid sequence SEQ ID NO: 19 - chimpanzee CYP4V2 amino acid sequence SEQ ID NO: 20 - RYESUS Monkey CYP4V2 amino acid sequence SEQ ID NO: 21 - dog CYP4V2 amino acid sequence SEQ ID NO: 22 - cattle CYP4V2 amino acid sequence SEQ ID NO: 23 - domestic mouse CYP4V2 amino acid sequence SEQ ID NO: 24 - Standard rate CYP4V2 amino acid sequence SEQ ID NO: 25 - chicken CYP4V2 amino acid sequence SEQ ID NO: 26 - tropical clawed toad CYP4V2 amino acid sequence SEQ ID NO: 27 - horse CYP4V2 amino acid sequence SEQ ID NO: 28 - rabbit CYP4V2 amino acid sequence SEQ ID NO: 29 - fruit fly CYP4V2 amino acid sequence SEQ ID NO: 30 - P450 signature element sequence SEQ ID NO: 31 - P450 signature element sequence SEQ B.
Exemplary regulatory sequences and ITR sequences
SEQ ID NO: 32 - CAG promoter sequence SEQ ID NO: 33 - WPRE enhancer sequence SEQ ID NO: 34 - polyA sequence bGH SEQ ID NO: 35 - EFS promoter sequence SEQ ID NO: 36 - small polyA sequence (SPA) SEQ ID NO: 37 - Kozak sequence SEQ ID NO: 38 - Kozak sequence SEQ ID NO: 39 - Late polyA sequence SV40 SEQ ID NO: 40 - CMV promoter sequence SEQ ID NO: 41 - EF promoter sequence -1 alpha SEQ ID NO: 42 - Left ITR sequence 5 'AAV2 SEQ ID NO: 43 - Right ITR sequence 3' AAV2 SEQ ID NO: 44 - mutant ITR sequence 5 'AAV2 used in scAAV SEQ ID NO: 45 - ITR 3 'AAV2 sequence used in scAAV
Part II.
Cell Therapy Sequences SEQ ID NO: 46 - human CYP4V2 gene region containing the c.802-8_810del17insGC mutation SEQ ID NO: 47 - wild-type human CYP4V2 gene region without the c.802-8_810del17insGC mutation SEQ ID NO: 48 - gRNA 1 SEQ ID NO: 49 - gRNA 2 SEQ ID NO: 50 - gRNA 3 SEQ ID NO: 51 - gRNA 4
SEQ ID NO: 52 - gRNA 5 SEQ ID NO: 53 - exemplary crRNA sequence SEQ ID NO: 54 - exemplary tracrRNA sequence SEQ ID NO: 55 - exemplary degRNA sequence SEQ ID NO: 56 - donor model sequence 1 SEQ ID NO: 57 - donor model sequence 2 SEQ ID NO: 58 - SpCas9 amino acid sequence SEQ ID NO: 59 - additional nucleotide inserted immediately after the U6 promoter sequence and before the protospacer element sequence in a construct plasmid and in an IVT sgRNA Part III: CYP4V2 Expression Cassette Sequences (including AAV ITRs and junction / ligand sequences).
SEQ ID NO: 60 - CYP4V2 expression cassette sequence in AAV2.CYP4V2op, AAV2tri (Y-F) .CYP4V2op and AAV5.CYP4V2op. SEQ ID NO: 61 - CYP4V2 expression cassette sequence in AAV5.CYP4V2st. AAV5.CYP4V2st has the same promoter (CAG), enhancer (WPRE) and polyA (bGH-polyA) sequences as AAV2.CYP4V2op, AAV2tri (YF) .CYP4V2op and AAV5.CYP4V2op (SEQ ID NO: 60) but cDNA different CYP4V2 in junction / ligand sequences. SEQ ID NO: 62 - CYP4V2 expression cassette sequence in AAV8.CYP4V2fv. AAV8.CYP4V2fv has the same promoter (CAG), enhancer (WORE) and polyA (bGH-polyA) and junction / ligand sequences as AAV5.CYP4V2st (SEQ ID NO: 61) and differ only in cDNA sequence of CYP4V2. SEQ ID NO: 63 - CYP4V2 expression cassette sequence in AAV5.CYP4V2op (new). AAV5.CYP4V2op (new) has the same promoter (CAG), enhancer (WPRE) and polyA (bGH-polyA) sequences and the same junction / ligand sequences as AAV5.CYP4V2st (SEQ ID NO: 61) and AAV8. CYP4V2fv (SEQ ID NO: 62) but different CYP4V2 cDNA sequences. SEQ ID NO: 64 - CYP4V2 expression cassette sequence in scAAV1.CYP4V2op, scAAV5.CYP4V2op and scAAV9.CYP4V2op.
SEQUENCES SEQ ID NO: 1 (cDNA CYP4V2st, 1578 bp) ATGGCGGGGCTCTGGCTGGGGCTCGTGTGGCAGAAGCTGCTGCTGTGGGGCGCGGCGAGTG- CCCTTTCCCTGGCCGGCGCCAGTCTGGTCCTGAGCCTGCTGCAGAGGGTGGCGAGCTACG- CGCGGAAATGGCAGCAGATGCGGCCCATCCCCACGGTGGCCCGCGCCTACCCACTGGTGGG- CCACGCGCTGCTGATGAAGCCGGACGGGCGAGAATTTTTTCAGCAGATCATTGAGTACACA- GAGGAATACCGCCACATGCCGCTGCTGAAGCTCTGGGTCGGGCCAGTGCCCATGGTGG- CCCTTTATAATGCAGAAAATGTGGAGGTAATTTTAACTAGTTCAAAGCAAATTGACAAA- TCCTCTATGTACAAGTTTTTAGAACCATGGCTTGGCCTAGGACTTCTTACAAGTACTGGA- AACAAATGGCGCTCCAGGAGAAAGATGTTAACACCCACTTTCCATTTTACCATTCTGGAA- GATTTCTTAGATATCATGAATGAACAAGCAAATATATTGGTTAAGAAACTTGAAAAACACA- TTAACCAAGAAGCATTTAACTGCTTTTTTTACATCACTCTTTGTGCCTTAGATATCATCTG- TGAAACAGCTATGGGGAAGAATATTGGTGCTCAAAGTAATGATGATTCCGAGTATGTCCG- TGCAGTTTATAGAATGAGTGAGATGATATTTCGAAGAATAAAGATGCCCTGGCTTTGG- CTTGATCTCTGGTACCTTATGTTTAAAGAAGGATGGGAACACAAAAAGAGCCTTCAGATCC- TACATACTTTTACCAACAGTGTCATCGCTGAACGGGCCAATGAAATGAACGCCAATGAAGA- CTGTAGAGGTGATGGCAGGGGCTCTGCCCCCTCCAAAAATAAACGCAGGGCCTTTCTTGAC- TTGCTTTTAAGTGTGACTGATGACGAAGGGAACAGGCTAAGTCATGAAGATATT- CGAGAAGAAGTTGACACCTTCATGTTTGAGGGGCACGATACAACTGCAGCTGCAATAAAC- TGGTCCTTATACCTGTTGGGTTCTAACCCAGAAGTCCAGAAAAAAGTGGATCATGAATTG- GATGACGTGTTTGGGAAGTCTGACCGTCCCGCTACAGTAGAAGACCTGAAGAAACTTCGG- TATCTGGAATGTGTTATTAAGGAGACCCTTCGCCTTTTTCCTTCTGTTCCTTTATTTG- CCCGTAGTGTTAGTGAAGATTGTGAAGTGGCAGGTTACAGAGTTCTAAAAGGCACTGAAG- CCGTCATCATTCCCTATGCATTGCACAGAGATCCGAGATACTTCCCCAACCCCGAGGAGTT- CCAGCCTGAGCGGTTCTTCCCCGAGAATGCACAAGGGCGCCATCCATATGCCTACGTG- CCCTTCTCTGCTGGCCCCAGGAACTGTATAGGTCAAAAGTTTGCTGTGATGGAAGAAAAGA- CCATTCTTTCGTGCATCCTGAGGCACTTTTGGATAGAATCCAACCAGAAAAGAGAAGAG- CTTGGTCTAGAAGGACAGTTGATTCTTCGTCCAAGTAATGGCATCTGGATCAAG-
TTGAAGAGGAGAAATGCAGATGAACGCTAA SEQ ID NO: 2 (CYP4V2op cDNA 1578 bp) ATGGCTGGACTGTGGCTGGGACTGGTGTGGCAGAAACTGCTGCTGTGGGGGGCCGCTTCCG- CACTGTCACTGGCTGGGGCTTCACTGGTGCTGAGCCTGCTGCAGAGGGTGGCCTCCTACG- CCAGAAAGTGGCAGCAGATGAGGCCCATCCCTACCGTGGCCAGAGCCTATCCACTGGTGG- GACACGCACTGCTGATGAAGCCTGACGGCAGAGAGTTCTTTCAGCAGATCATCGAGTACA- CAGAGGAGTATAGGCACATGCCACTGCTGAAGCTGTGGGTGGGACCCGTGCCTATGGTGG- CCCTGTACAACGCCGAGAATGTGGAAGTGATCCTGACCAGCAGCAAGCAGATCGATAAGTC- TAGCATGTATAAGTTCCTGGAGCCTTGGCTGGGCCTGGGCCTGCTGACCTCTACAGGCAA- CAAGTGGAGGAGCCGGAGAAAGATGCTGACCCCAACATTCCACTTTACAATCCTGGAGGAC- TTCCTGGACATCATGAACGAGCAGGCCAATATCCTGGTGAAGAAGCTGGAGAAGCACAT- CAACCAGGAGGCCTTTAATTGCTTCTTTTACATCACCCTGTGCGCCCTGGACATCATCTG- TGAGACAGCTATGGGCAAGAACATCGGCGCCCAGTCTAATGACGATAGCGAGTACGTG- CGGGCCGTGTATAGAATGAGCGAGATGATCTTTAGGCGCATCAAGATGCCCTGGCTGTGG- CTGGATCTGTGGTATCTGATGTTCAAGGAGGGCTGGGAGCACAAGAAGTCCCTGCAGA- TCCTGCACACCTTTACAAACTCTGTGATCGCCGAGAGAGCCAATGAGATGAACGCCAA- TGAGGACTGTAGGGGCGATGGAAGGGGCAGCGCCCCTTCCAAGAACAAGCGGAGAGCCTT- CCTGG ACCTGCTGCTGAGCGTGACCGACGATGAGGGCAATCGCCTGTCCCACGAGGACA- TCCGGGAGGAGGTGGATACATTCATGTTTGAGGGACACGACACCACAGCCGCCGCCAT- CAACTGGTCCCTGTACCTGCTGGGCTCTAATCCAGAGGTGCAGAAGAAGGTGGATCACGAG- CTGGACGACGTGTTCGGCAAGTCCGACAGGCCAGCAACCGTGGAGGATCTGAAGAAGCTGA- GATACCTGGAGTGCGTGATCAAGGAGACACTGCGCCTGTTCCCCTCTGTGCCTCTGTTTG- CCCGGTCCGTGTCTGAGGACTGTGAGGTGGCCGGCTATCGCGTGCTGAAGGGCACCGAGG- CCGTGATCATCCCTTACGCCCTGCACCGGGACCCCAGGTATTTCCCTAACCCAGAGGAG- TTTCAGCCAGAGAGATTCTTTCCCGAGAATGCCCAGGGCAGGCACCCTTACGCCTATGTG- CCATTCTCCGCCGGACCAAGGAACTGCATCGGACAGAAGTTTGCCGTGATGGAGGA- GAAAACCATCCTGTCTTGTATCCTGAGACACTTCTGGATCGAGAGCAATCAGAAGAGGGAG- GAGCTGGGCCTGGAGGGACAGCTGATCCTGCGGCCAAGCAACGGCATCTGGATCAAAC-
TGAAAAGAAGGAACGCTGACGAGAGGTAA SEQ ID NO: 3 (cDNA CYP4V2fv, 1578 bp) ATGGCGGGGCTCTGGCTGGGGCTCGTGTGGCAGAAGCTGCTGCTGTGGGGCGCGGCGAGTG- CCCTTTCCCTGGCCGGCGCCAGTCTGGTCCTGAGCCTGCTGCAGAGGGTGGCGAGCTACG- CGCGGAAATGGCAGCAGATGCGGCCCATCCCCACGGTGGCCCGCGCCTACCCACTGGTGGG- CCACGCGCTGCTGATGAAGCCGGACGGGCGAGAATTTTTTCAGCAGATCATTGAGTACACA- GAGGAATACCGCCACATGCCGCTGCTGAAGCTCTGGGTCGGGCCAGTGCCCATGGTGG- CCCTTTATAATGCAGAAAATGTGGAGGTAATTTTAACTAGTTCAAAGCAAATTGACAAA- TCCTCTATGTACAAGTTTTTAGAACCATGGCTTGGCCTAGGACTTCTTACAAGTACTGGA- AACAAATGGCGCTCCAGGAGAAAGATGTTAACACCCACTTTCCATTTTACCATTCTGGAA- GATTTCTTAGATATCATGAATGAACAAGCAAATATATTGGTTAAGAAACTTGAAAAACACA- TTAACCAAGAAGCATTTAACTGCTTTTTTTACATCACTCTTTGTGCCTTAGATATCATCTG- TGAAACAGCTATGGGGAAGAATATTGGTGCTCAAAGTAATGATGATTCCGAGTATGTCCG- TGCAGTTTATAGAATGAGTGAGATGATATTTCGAAGAATAAAGATGCCCTGGCTTTGG- CTTGATCTCTGGTACCTTATGTTTAAAGAAGGATGGGAACACAAAAAGAGCCTTAAGATCC- TACATACTTTTACCAACAGTGTCATCGCGGAACGGGCCAATGAAATGAACGCCAATGAAGA- CTGTAGAGGTGATGGCAGGGGCTCTGCCCCCTCCAAAAATAAACGCAGGGCCTTTCTTGAC- TTGCTTTTAAGTGTGACTGATGACGAAGGGAACAGGCTAAGTCATGAAGATATT- CGAGAAGAAGTTGACACCTTCATGTTTGAGGGGCACGATACAACTGCAGCTGCAATAAAC- TGGTCCTTATACCTGTTGGGTTCTAACCCAGAAGTCCAGAAAAAAGTGGATCATGAATTG- GATGACGTGTTTGGGAAGTCTGACCGTCCCGCTACAGTAGAAGACCTGAAGAAACTTCGG- TATCTGGAATGTGTTATTAAGGAGACCCTTCGCCTTTTTCCTTCTGTTCCTTTATTTG- CCCGTAGTGTTAGTGAAGATTGTGAAGTGGCAGGTTACAGAGTTCTAAAAGGCACTGAAG- CCGTCATCATTCCCTATGCATTGCACAGAGATCCGAGATACTTCCCCAACCCCGAGGAGTT- CCAGCCTGAGCGGTTCTTCCCCGAGAATGCACAAGGGCGCCATCCATATGCCTACGTG- CCCTTCTCTGCTGGCCCCAGGAACTGTATAGGTCAAAAGTTTGCTGTGATGGAAGAAAAGA- CCATTCTTTCGTG CATCCTGAGGCACTTTTGGATAGAATCCAACCAGAAAAGAGAAGAG- CTTGGTCTAGAAGGACAGTTGATTCTTCGTCCAAGTAATGGCATCTGGATCAAG-
TTGAAGAGGAGAAATGCAGATGAACGCTAA SEQ ID NO: 4 (human protein CYP4V2, NP_997235.3, 525 aa) MAGLWLGLVWQKLLLWGAASALSLAGASLVLSLLQRVASYARKWQQMRPIPTVA- RAYPLVGHALLMKPDGREFFQQIIEYTEEYRHMPLLKLWVGPVPMVALYNAENVE- VILTSSKQIDKSSMYKFLEPWLGLGLLTSTGNKWRSRRKMLTPTFHFTILEDFLDIMNEQA- NILVKKLEKHINQEAFNCFFYITLCALDIICETAMGKNIGAQSNDDSEYVRAVYRMSEMI- FRRIKMPWLWLDLWYLMFKEGWEHKKSLQILHTFTNSVIAERANEMNANEDCRGDGRGSAP- SKNKRRAFLDLLLSVTDDEGNRLSHEDIREEVDTFMFEGHDTTAAAINWSLYLLGSN- PEVQKKVDHELDDVFGKSDRPATVEDLKKLRYLECVIKETLRLFPSVPLFARSVSEDCE- VAGYRVLKGTEAVIIPYALHRDPRYFPNPEEFQPERFFPENAQGRHPYAYVPFSAGPRN-
CIGQKFAVMEEKTILSCILRHFWIESNQKREELGLEGQLILRPSNGIWIKLKRRNADER SEQ ID NO: 5 (functional variant of human protein CYP4V2; 525 aa) MAGLWLGLVWQKLLLWGAASALSLAGASLVLSLLQRVASYARKWQQMRPIPTVA- RAYPLVGHALLMKPDGREFFQQIIEYTEEYRHMPLLKLWVGPVPMVALYNAENVE- VILTSSKQIDKSSMYKFLEPWLGLGLLTSTGNKWRSRRKMLTPTFHFTILEDFLDIMNEQA- NILVKKLEKHINQEAFNCFFYITLCALDIICETAMGKNIGAQSNDDSEYVRAVYRMSEMI- FRRIKMPWLWLDLWYLMFKEGWEHKKSLKILHTFTNSVIAERANEMNANEDCRGDGRGSAP- SKNKRRAFLDLLLSVTDDEGNRLSHEDIREEVDTFMFEGHDTTAAAINWSLYLLGSN- PEVQKKVDHELDDVFGKSDRPATVEDLKKLRYLECVIKETLRLFPSVPLFARSVSEDCE- VAGYRVLKGTEAVIIPYALHRDPRYFPNPEEFQPERFFPENAQGRHPYAYVPFSAGPRN-
CIGQKFAVMEEKTILSCILRHFWIESNQKREELGLEGQLILRPSNGIWIKLKRRNADER SEQ ID NO: 6 (CYP4V2 functional fragment (without transmembrane domain, 490 aa) RVASYARKWQQMRPIPTVARAYPLVGHALLMKPDGREFFQQIIEYTEEYRHMPLLKLWVG- PVPMVALYNAENVEVILTSSKQIDKSSMYKFLEPWLGLGLLTSTGNKWRSRRKMLTPT- FHFTILEDFLDIMNEQANILVKKLEKHINQEAFNCFFYITLCALDIICETAMGKNI- GAQSNDDSEYVRAVYRMSEMIFRRIKMPWLWLDLWYLMFKEGWEHKKSLQILHTFTNSVI- AERANEMNANEDCRGDGRGSAPSKNKRRAFLDLLLSVTDDEGNRLSHEDIREEVDTFM- FEGHDTTAAAINWSLYLLGSNPEVQKKVDHELDDVFGKSDRPATVEDLKKLRYLECVI- KETLRLFPSVPLFARSVSEDCEVAGYRVLKGTEAVIIPYALHRDPRYFPNPEEFQPER- FFPENAQGRHPYAYVPFSAGPRNCIGQKFAVMEEKTILSCILRHFWIESNQKREELGLE- GQLILRPSNGIWIKLKRRNADER
SEQ ID NO: 7 (CYP46A1, NP_006659 500 aa) MSPGLLLLGSAVLLAFGLCCTFVHRARSRYEHIPGPPRPSFLLGHLPCFWKKDE- VGGRVLQDVFLDWAKKYGPVVRVNVFHKTSVIVTSPESVKKFLMSTKYNKDSKMYRAL- QTVFGERLFGQGLVSECNYERWHKQRRVIDLAFSRSSLVSLMETFNEKAEQLVEILEAKAD- GQTPVSMQDMLTYTAMDILAKAAFGMETSMLLGAQKPLSQAVKLMLEGITASRNTLAKFL- PGKRKQLREVRESIRFLRQVGRDWVQRRREALKRGEEVPADILTQILKAEEGAQDDE- GLLDNFVTFFIAGHETSANHLAFTVMELSRQPEIVARLQAEVDEVIGSKRYLDFEDLGR- LQYLSQVLKESLRLYPPAWGTFRLLEEETLIDGVRVPGNTPLLFSTYVMGRMDTYFEDPLT- FNPDRFGPGAPKPRFTYFPFSLGHRSCIGQQFAQMEVKVVMAKLLQRLEFRLVPGQRF-
GLQEQATLKPLDPVLCTLRPRGWQPAPPPPPC SEQ ID NO: 8 (CYP4A11, NP_000769, 519 aa) msvsvlspsr llgdvsgilq aasllillll likavqlylh rqwllkalqq fpcppshwlf ghiqelqqdq elqriqkwve tfpsacphwl wggkvrvqly dpdymkvilg rsdpkshgsy rflapwigyg llllngqtwf qhrrmltpaf hydilk- pyvg lmadsvrvml dkweellgqd splevfqhvs lmtldtimkc afshqgsiqv drnsqsyiqa isdlnnlvfs rvrnafhqnd tiysltsagr wthracqlah qhtd- qviqlr kaqlqkegel ekikrkrhld fldilllakm engsilsdkd lraevdtfmf eghdttasgi swilyalath pkhqercree ihsllgdgas itwnhldqmp yttmci- Keal rlyppvpgig relstpvtfp dgrslpkgim vllsiyglhh npkvwpnpev fdpfrfapgs aqhshaflpf sggsrncigk qfamnelkva taltllrfel lpd- ptripip iarlvlkskn gihlrlrrlp npcedkdql SEQ ID NO: 9 (CYP4A22, NP_001010969, 519 aa) msvsvlspsr rlggvsgilq vtsllillll likaaqlylh rqwllkalqq fpcppshwlf ghiqefqhdq elqriqervk tfpsacpywi wggkvrvqly dpdymkvilg rsdpkshgsy kflaprigyg llllngqtwf qhrrmltpaf hndilk- pyvg lmadsvrvml dkweellgqd splevfqhvs lmtldtimks afshqgsiqv drnsqsyiqa isdlnslvfc cmrnafhend tiysltsagr wthracqlah qhtd- qviqr kaqlqkegel ekikrkrhld fldilllakm engsilsdkd lraevdtfmf eghdttasgi swilyalath pkhqercree ihgllgdgas itwnhldqmp yttmci- Keal rlyppvpgig relstpvtfp dgrslpkgim vllsiyglhh npkvwpnlev fdpsrfapgs aqhshaflpf sggsrncigk qfamnqlkva raltllrfel lpd- ptripip marlvlkskn gihlrlrrlp npcedkdql SEQ ID NO: 10 (CYP4B1, NP_000770, 511 aa) mvpsflslsf sslglwasgl ilvlgflkli hlllrrqtla kamdkfpgpp thwl- fghale iqetgsldkv hplwfgqfig flniyepdya kavysrgdpk vswahqfpya apdvydfflq wigrgllvle gpkwlqhrkl ltpgfhydvl kpyvavftes trim- ldkwee karegksfdi fcdvghmaln tlmkctfgrg dtglghrdss yylavsdltl lmqqrlvsfq yhndfiywlt phgrrflrac qvahdhtdqv irerkaalqd ekvrk- kiqnr rhldfldill gardeddikl sdadlraevd tfmfeghdtt tsgiswflyc malypehqhr creevreilg dqdffqwddl gkmtyltmci kesfrlyppv pqvyrqlskp vtfvdgrslp agslismhiy alhrnsavwp dpevfdslrf stenaskrhp fafmpfsagp rncigqqfam semkvvtamc llrfefsldp SRL- pikmpql vlrskngfhl hlkplgpgsg k SEQ ID NO: 11 (CYP4F2, NP_001073, 520 aa) msqlslswlg lwpvaaspwl llllvgaswl lahvlawtya fydncrrlrc fpqp- prrnwf wghqgmvnpt eegmrvltql vatypqgfkv wmgpisplls lchpdiirsv inasaaiapk dkffysflep wlgdglllsa gdkwsrhrrm ltpafhfnil kpymkifnes vnimhakwql lasegsacld mfehislmtl dslqkcvfsf dshcqekpse yiaailelsa lvskrhheil lhidflyylt pdgqrfrrac rlvhdftdav iqerrrtlps qgvddflqak aksktldfid vlllskdedg kklsdedira eadtfmfegh dttasglswv lyhlakhpey qercrqevqe llkdrepkei ewddlahlpf ltmcmkeslr lhppvpvisr hvtqdivlpd grvip- kgiic lisvfgthhn pavwpdpevy dpfrfdpeni kersplafip fsagprncig qtfamaemkv vlaltllrfr vlpdhteprr kpelvlraeg glwlrvepls
SEQ ID NO: 12 (CYP4F3, NP_000887 520 aa) mpqlslsslg lwpmaaspwl llllvgaswl larilawtyt fydnccrlrc fpqppkrnwf lghlglihss eegllytqsl actfgdmccw wvgpwhaivr ifhptyikpv lfapaaivpk dkvfysflkp wlgdglllsa gekwsrhrrm ltpafhfnil kpymkifnes vnimhakwql lasegsarld mfehislmtl dslqkcvfsf dshcqekpse yiaailelsa lvtkrhqqil lyidflyylt pdgqrfrrac rlvhdftdav iqerrrtlps qgvddflqak aksktldfid vlllskdedg kklsdedira eadtfmfegh dttasglswv lyhlakhpey qercrqevqe llkdrepkei ewddlaqlpf ltmcikeslr lhppvpavsr cctqdi- vlpd grvipkgiic lisvfgthhn pavwpdpevy dpfrfdpkni kersplafip fsagprncig qafamaemkv vlgltllrfr vlpdhteprr kpell
SEQ ID NO: 13 (CYP4F8, NP_009184 520 aa) msllslswlg lrpvaaspwl lllvvgaswl larilawtya fyhngrrlrc fpqprkqnwf lghlglvtpt eeglrvltql vatypqgfvr wlgpitpiin lchpdivrsv intsdaitdk divfyktlkp wlgdglllsv gdkwrhhrrl ltpafhfnil kpyikifsks animhakwqr lamegstcld vfehislmtl dslqkcifsf dsncqekpse yitaimelsa lvvkrnnqff rykdflyflt pcgrrfhrac rlvhdftdav iqerrrtlts qgvddflqak aksktldfid vlllsedkng kelsdedira eadtfmfggh dttasglswv lynlarhpey qercrqevqe llkdrepkei ewddlaqlpf ltmclkeslr lhppiptfar gctqdvvlpd srvipkgnvc ninifaihhn psvwpdpevy dpfrfdpena qkrspmafip fsagprncig qkfamaemkv vlaltllrfr ilpdhreprr tpeivlraed glpe
SEQ ID NO: 14 (CYP4F11, NP_067010, 524 aa) mpqlslswlg lgpvaaspwl llllvggswl larvlawtyt fydncrrlqc fpqppkqnwf wghqglvtpt eegmktltql vttypqgfkl wlgptfplli lchpdiirpi tsasaavapk dmifygflkp wlgdglllsg gdkwsrhrrm ltpafhfnil kpymkifnks vnimhdkwqr lasegsarld mfehislmtl dslqkcvfsf esncqekpse yiaailelsa fvekrnqqil lhtdflyylt pdgqrfrrac hlvhdftdav iqerrctlpt qgiddflknk aksktldfid vlllskdedg kelsdedira eadtfmfegh dttasglswv lyhlakhpey qeqcrqevqe llkdrepiei ewddlaqlpf ltmcikeslr lhppvpvisr cctqdfvlpd grvipkgivc liniigihyn ptvwpdpevy dpfrfdqeni kersplafip fsagprncig qafamaemkv vlaltllhfr ilpthteprr kpelil- raeg glwlw
SEQ ID NO: 15 (CYP4F12, NP_076433, 524 aa) msllslpwlg lrpvatspwl llllvvgswl larilawtya fynncrrlqc fpqppkrnwf wghlglitpt eeglknstqm satysqgftv wlgpiipfiv lchpd- tirsi tnasaaiapk dnlfirflkp wlgegillsg gdkwsrhrrm ltpafhfnil ksyitifnks animldkwqh lasegssrld mfehislmtl dslqkcifsf dshcqerpse yiatilelsa lvekrsqhil qhmdflyyls hdgrrfhrac rlvhdftdav irerrrtlpt qgiddffkdk aksktldfid vlllskdedg kalsdedira eadtfmfggh dttasglswv lynlarhpey qercrqevqe llkdrd- pkei ewddlaqlpf ltmcvkeslr lhppapfisr cctqdivlpd grvipkgitc lidiigvhhn ptvwpdpevy dpfrfdpens kgrsplafip fsagprncig qafamaemkr vlalhllvllllhll
SEQ ID NO: 16 (CYP4F22, NP_775754, 531 aa) mlpitdrllh llglektafr iyavstlllf llfflfrlll rflrlcrsfy N- qpprrnwllg hlgmylpnea glqdekkvld crrlrcfp nmhhvllvwm gpvlpllvlv hpdyikpllg asaaiapkdd lfygflkpwl gdglllskgd kws- rhrrllt pafhfdilkp ymkifnqsad imhakwrhla egsavsldmf ehislmtlds lqkcvfsyns ncqekmsdyi saiielsals vrrqyrlhhy ldfiyyrsad grrfr- qacdm vhhftteviq errralrqqg aeawlkakqg ktldfidvll lardedgkel sdediraead tfmfeghdtt ssgiswmlfn lakypeyqek creeiqevmk greleelewd dltqlpfttm cikeslrqyp pvtlvsrqct ediklpdgri ip- kgiiclvs iygthhnptv wpdskvynp rfdrvrprrr
SEQ ID NO: 17 (CYP4X1, NP_828847, 509 aa) mefswletrw arpfylafvf clalgllqai klylrrqrll rdlrpfpapp thwflghqkf iqddnmekle eiiekypraf pfwigpfqaf fciydpdyak tllsrtdpks qylqkfsppl lgkglaaldg pkwfqhrrll tpgfhfnilk ayievmahsv kmmldkweki cstqdtsvev yehinsmsld iimkcafske tncqtnsthd pyakaifels kiifhrlysl lyhsdiifkl spqgyrfqkl srvlnqytdt iiqerkkslq agvkqdntpk rkyqdfldiv lsakdesgss fsdidvhsev stfllaghdt laasiswily clalnpehqe rcreevrgil gdgssitwdq lgemsyttmc iketcrlipa vpsisrdlsk pltfpdgctl pagitvvlsi wglhhnpavw knpkvfdplr fsqensdqrh pyaylpfsag srn- cigqefa mielkvtial illhfrvtpd ptrpltfpnh filkpkngmy lhlkklsec
SEQ ID NO: 18 (CYP4Z1, NP_835235, 505 aa) mepswlqelm ahpflllill cmslllfqvi rlyqrrrwmi ralhlfpapp ahwfy- ghkef ypvkefevyh klmekypcav plwvgpftmf fsvhdpdyak illkrqdpks avshkilesw vgrglvtldg skwkkhrqiv kpgfnisilk ifitmmsesv rmmlnk- weeh iaqnsrlelf qhvslmtlds imkcafshqg siqldstlds ylkavfnlsk isnqrmnnfl hhndlvfkfs sqgqifskfn qelhqftekv iqdrkeslkd klkqdttqkr rwdfldills aksentkdfs eadlqaevkt fmfaghdtts saiswilycl akypehqqrc rdeirellgd gssitwehls qmpyttmcik eclr- lyapvv nisrlldkpi tfpdgrslpa gitvfiniwa lhhnpyfwed pqvfnplrfs rensekihpy afipfsaglr ncigqhfaii eckvqtkhsr
SEQ ID NO: 19 (P50 isoform of the cytochrome X1 4V2 [chimpanzee Pan troglodytes)] XP_001165629.1, 525 aa) maglwlglvw qklllwgaas avslagaslv lsllqrvaty arkwqqmrpi ptva- rayplv ghallmkpdg reffqqiiey teeyrhmpll klwvgpvpmv alynaenvev iltsskqidk ssmykflepw lglglltstg nkwrsrrkml tptfhftile dfldim- neqa ntlvkklekh inqeafncff yitlcaldii cetamgknig aqsnddseyv ravyrmsemi frrikmpwlw ldlwylmfke gwehkkslki lhtftnsvia eranem- nane dcrgdgrgsa psknkrrafl dlllsvtdde gnrlshedir eevdtfmfeg hdttaaainw slyllgsnpe vqkkvdheld dvfgksdrpa tvedlkklry lecvi- ketlr lfpsvplfar svsedcevag yrvlkgteav iipyalhrdp ryfpnpeefq perffpknaq grhpyayvpf sagprncigq kfavmeekti lscilrhfwi esn- qkreelg legqlilrps ngiwiklkrr Nader
SEQ ID NO: 20 (4V2 P450 [Macaca mulatta (rhesus Ma- caque, Rhesus monkey)], NP_001180767.1, 525 aa) magiwlglvw qklllwgaas avslagaslv lsllqrvasy vrkwqqmrpi ptva- rayplv ghallmkrdg reffqqiiey teeyrhmpll klwvgpvpmv alynaenvev iltsskqidk ssmykflepw lglglltstg nkwrsrrkml tptfhftile dfldim-
neqa nilvkklekh vnqeafncfv yitlcaldii cetamgknig aqsnddseyv ravyrmsemi frrikmpwlw ldlwylmfke gwehkkslki lhaftnnvia eranem- nvde dcrgdgrdsa psknkrrafl dlllsvtdde gnrlshedir eevdtfmfeg hdttaaamnw slyllgsnpe vqkkvdheld dvfgrsdrpa tvedlkklry lecvi- ketlr lfpsvplfar svsedcevag yrvlkgteav iipyalhrdp ryfpnpeefr perffpenaq grhpyayvpf sagprncigq kfavmeekti lscilrhfwi esn- qkreelg legqlilrpt ngiwiklkrr nadep
SEQ ID NO: 21 (4V2 P450 [Canis lupus familiaris (ca- jet)] XP_013975571.1, 539 aa) mlkvkwkenv fregdkdsnm ldavqlpsik vesalsdaea ggspggrrpv ltvergrlaq gsmssllknp kdttrnslki kyflpeffqq vilyseesrh lpll- klwlgp ipivaiysae nveviltssr qidksyvykf lepwlglgll tstgnkwrsr rkmltptfhf tiledfldvm nehanilvnk lekhvnqeaf ncffyitlca ldiice- tamg knigaqnned seyvraiyrm sdtihrrmkm pwlwldflfl mfkegrehkr nleilhnftn nviterasel krdeehgsad kdcspsknkr rafldlllnv tddegn- klrh edvreevdtf mfeghdttaa ainwslyllg sypevqkqvd seledvfgks drpatledlk klkylecvik eslrlfpsvp lfarnlnedc vvagykvvkg sqaiiipyal hrdpryfpnp eefqperffp enlqgrhpya yipfsagprn cigqrfaime ektvlscvlr hfwvesnqkr eelglageli lrptngiwik des lkrrna-
SEQ ID NO: 22 (4V2 P450 [Bos taurus (cattle)], NP_001029545, 527 aa) mlapwllsvg pklllwsglc avslagatlt lnllkmvasy arkwrqmrpv ptigd- pyplv ghalmmkpda rdffqqiidf teecrhlpll klwlgpvplv alynaetvev ilssskhiek symykflepw lglglltstg nkwrsrrkml tptfhftile dfldvmneqa nilvtklekh vnqeafncff yvtlctldii cetamgknig aqrnddseyv ravyrmsdsi hqrmkmpwlw ldlifymfkn grehrrslki vhdftnnvit eranemkrhe egtsndkekd fpprktkcra fldlllnvtd dqgn- klshed ireevdtfmf eghdttaaai nwslyllgwy pevqqrvdte leevfgksdr pvtledlkkl kyldcvikes lrlfpsvpff arnltedcev aghkivqgcq viiv- pyalhr dpkyfpdpee fkperffpen lkgrhtyayv pfsagprnci gqkfaimeek tilscilrhf wvesnqkree lglagelilr psngiwiklk rrntdes
SEQ ID NO: 23 (Cyp4v3, 4V2 P450 [Mus musculus (domestic ca- Mundungus)], NP_598730.1, 525 aa) mlwlwlglsg qklllwgaas avslagatil isifpmlvsy arkwqqmrsi psva- rayplv ghalymkpnn aeffqqliyy teefrhlpii klwigpvplv alykaenvev iltsskqidk sflykflqpw lglglltstg skwrtrrkml tptfhftile nfldvmneqa nilvnklekh vnqeafncff yitlcaldii cetamgknig aqsnndseyv rtvyrmsdmi yrrmkmpwlw fdlwylvfke grdhkrglkc lht- ftnnvia ervkerkaee dwtgagrgpi psknkrkafl dlllsvtdee gnrlsqedir eevdtfmfeg hdttaaainw slyllgtnpe vqrkvdqeld evfgrshrpv tledlk- klky ldcviketlr vfpsvplfar slsedcevgg ykvtkgteai iipyalhrdp ryfpdpeefr perffpensq grhpyayvpf sagprncigq kfavmeekti lacilr- qfwv esnqkreelg lagdlilrpn ngiwiklkrr heddp
SEQ ID NO: 24 (Cyp4v3, 4V2 P450 [Rattus norvegicus (Norway rat)], NP_001129072, 525 aa) mlwlwlglsg qklllwgaas avsvagatvl lnilqmlvsy arkwqqmrpi psva- rayplv ghalfmkpnn teffqqiiqy teefrhlpii klwigpvplv alykaenvev iltsskqidk sfmykflqpw lglglltstg skwrarrkml tpsfhftile dfldvmneqa nilvnklekh vnqeafncff pitlcaldii cetamgknig aqsngdseyv rtvyrmsdmi yrrmkmpwfw fdlwylmfke grdhkkglks lht-
ftnnvia ervnarkaeq dcigagrgpl psktkrkafl dlllsvtdee gnklshedir eevdtfmfeg hdttaaainw slyllgsnpe vqrkvdkeld dvfgrshrpv tledlk- ldcviketlr vfpsvplfar slsedcevag klky ykiskgteav iipyalhrdp ryfpdpeefq perffpensq grhpyayvpf sagprncigq kfavmeekti lacil- refwi esnqkreelg lagdlilrpn ngiwiklkrr heddp
SEQ ID NO: 25 (4V2 P450 [Gallus gallus (chicken)], NP_001001879, 530 aa) mameitlgsm egtqllpwva gaitllltvv tvhflpslln ywwwwwvmkp ipgir- pcypf vgnalllern gegffkqlqq yadefrkmpm fklwlgplpv tvlfhpdsve vilssskhik ksflytflhp wlgtglltst gdkwrsrrkm itptfhfail ndflevmneq ggvlleklek hvdkepfnif tditlcaldi icetamgknl gaqdnk- dsey vravyrmsdl iqqrqkspwl whdlmyllfk egrehernlk ilhgftdtvi aekvaelent kltkhdtdvn teeesgskkr eafldmllna tddegkklsy kdiree- vdtf mfeghdttaa amnwvlyllg hhpeaqkkvh qeldevfgnt erpvtvddlk klrylecvvk ealrlfpsvp mfarslqedc yisgyklpkg tnvlvltyvl hrd- peifpep defrperffp enskgrhpya yvpfsagprn cigqrfaqme ektllalilr rfwvdcsqkp eelglsgeli lrpnngiwvq lkrrpktvte
SEQ ID NO: 26 (2 subfamily V member 4 cytochrome P450 family [Xenopus tropicalis (sapo-clutches with rain)], NP_001072667.1, 523 aa) melggevhll vwvaaavvll tllalsilpa lqdyvrkrri lkpipgpgpn yplig- dalfl knnggdfflq iceytesyrl qpllkvwigt ipfivvyhad tvepvlsssk hmdkaflykf lhpwlgkgll tstgekwrsr rkmitptfhf ailseflevm neqs- kilvek lqthvdgesf dcfmdvtlca ldiisetamg rkiqaqsnrd seyvqaiykm sdiiqrrqkm pwlwldflya hlrdgkehdk nlkilhsftd kaileraeel kkm- geqkkeh cdsdpesdkp kkrsafldml lmatddagnk msymdireev dtfmfeghdt taaalnwslf llgshpeaqr qvhkeldevf gksdrpvtmd dlkklrylea vikesl- riyp svplfgrtvt edcsirgfhv pkgvnvviip yalhrdpeyf pepeefrper ffpenasgrn pyayipfsag lrncigqrfa lmeekvvlss ilrnywveas qkreel- cllg elilrpqdgm wiklknreta pta
SEQ ID NO: 27 (4V2 P450 [Equus caballus (horse)] XP_014592182.1, 469 aa) mfvliefkik yslsdffqql iyyteenrhl pllklwlgpv pvvifynaen ve- viltssrq idksymykfl kpwlglgllt stgnkwrsrr kmltptfhft Nle dfldvmn eqanilvnkl ekhvnqeafn cflyitlcal diicetamgk nigaqrnnds eyvravyrms dmihrrmkmp wlwldifflm fkegrehrrl lived lkilhnftnn - semk kdeersrsdd ggsapsknkr rafldlllnv tddegnklsh edirqevdtf mfeghdttaa ainwslyllg cypevqkkvd seleevfgks drpatledlk klkylecvmk etlrlfpsvp lfarnlnedc evagykivkg sqaiivsyal hrds- ryfpnp eefkperffp ensqgrhpya yvpfsagprn cigqkfavme ekiilscilr hfwvesnqkr eelglageli lrpsngiwik lkrrntees
SEQ ID NO: 28 (4V2 P450 [Oryctolagus cuniculus (coe- him)] XP_002709379.1, 524 aa) mwlwlglvgq kllfwgaasa vslagaslfl nllqmvasya rkwqqmrpip ti- grpyplvg halymkpsgk effqqliqyt eeyrhlpllk lwlgplpiva lynaenve- seen smyqflepwl glglltstgy kwrsrrkmlt lnsskqinks ptfhftiled fldimneqan ilvhklekhv dqeafncffy itlcaldiic etamgkniga qsnedseyvr avyrmsdvif rrmkmpwlwl dlwylmfkeg wehkrclkil hrftnn- Viae rvsemktdee hrdadsncap stmkrkafld llltvtdeeg nklshedire evdtfmfegh dttaaainws lyllgshpev qrkvddelde vfgksdrpat sedlk- klkyl ecviketlrl fpsvplfars lsddcevagf rvvkgtqavi vpyalhrdpk yfpnpeefrp erffpenaqg rhpyayvpfs agprncigqk faimeektil scil- rklwve snqkmeelgl agelilrptn giwiklkrrn Adka SEQ ID: 29 (4c3 P450 [Drosophila melanogaster (fruit flies)], NP_524598, 535 aa) msskvitslm aesillskvg qvisgyspit vfllgsilif lvvynkrrsr lvkyiekipg paampflgna iemnvdhdel fnrvigmqkl wgtriginrv wqgta- prvll fepetvepil nsqkfvnksh dydylhpwlg eglltstdrk whsrrkiltp afhfkilddf idvfneqsav larklavevg tLC seafnlfpyv tldivce tamgr- snseseyvka vygigsivqs rqakiwlqsd riyaq fifsltaeyk lhqsyintlh gfsnmvirer kaelailqen nnnnnnnapd ayddvgkkkr lafldllida skeg- tvlsne direevdtfm feghdttsaa iswtlfllgc hpeyqervve eldsifgddk etpatmknlm dmryleccik dslrlfpsvp mmarmvgedv niggkivpag tqaiim- tyal hrnprvfpkp eqfnpdnflp encagrhpfa yipfsagprn cigqkfaile ekavistvlr kykieavdrr edltllgeli lrpkdglrvk itprd SEQ ID NO: 30 (P450 signature element "x" means any amino acid) FxxGxxxCxG SEQ ID nO: 31 (P450 signature element, "x" means any amino acid) ExxR SEQ ID nO: 32 (CAG promoter 1715 bp) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAG- CCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG- CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA- GGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTA- CATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG- CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG- TATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTG CTTCAC- TCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTA- TTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGG- CGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCG- CTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCG- CGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCG- CCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGG- CCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGG- CTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGG- CTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTG- CCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCG- CGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAA- CAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGG- TCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTT- CGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGG- CGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGA- GGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTG- CCTTTTATGGTAATCGTGCGAGAGG GCGCAGGGACTTCCTTTGTCCCAAATCTGGCGGAG- CCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCG- CCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTT- CTCCATCTCCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCA- GGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTT- CATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCAT- CATTTTGGCAAA
SEQ ID NO: 33 (WPRE enhancer, 589 bp) AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTG- CTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCG- TATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTG- TGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCAC- TGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCC- TATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTG- TTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCG- CCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAA- TCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCG-
CCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC SEQ ID NO: 34 (bGH polyA, 225 bp) CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC- CCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTG- TCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGA-
TTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG SEQ ID NO: 35 (EFS promoter, 235 bp) attggctccg g gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcg- cggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacggg- TTTG ccgccagaac acag SEQ ID NO: 36 (SPA, 54 bp)
GATCCAATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTG SEQ ID NO: 37 (Kozak sequence, 6 sc)
GCCACC SEQ ID NO: 38 (Kozak sequence, 5 bp)
CCACC SEQ ID NO: 39 (Late polyA SV40, 120 bp) TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAA-
TAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAT SEQ ID NO: 40 (CMV promoter, 576 bp) TAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC- TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAA- TGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTA- TTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCC- TATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG- GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG- TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAG- TCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT- CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG-
GAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAG SEQ ID NO: 41 (EF-1 alpha promoter 1184 bp) cgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaag- ttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgg- gaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtata-
taagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagg- taagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtg- ccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttgga- agtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgag- ttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctg- tctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacg- ctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcgg- tttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgagg- cggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctg- ctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctgg- cccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggag- ctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaagga- aaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccg- tccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttgggggga- ggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccag- cttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggtt- cattctcaagcctcagaca gtggttcaaagtttttttcttccatttcaggtgtcgtga SEQ ID NO: 42 (left ITR 5 'AAV 141 bp) cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccggg- TCGC gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc T SEQ ID NO: 43 (the right ITR 3' AAV 141 bp) g gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct CAC tgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcagg SEQ ID NO: 44 (5 'ITR scAAV AAV2 mutant construct, 117 bp) cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccggg- TCGC gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag SEQ ID ggagtgg NO: 45 3 'ITR in AAV2 scAAV construct, 141 bp) aggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcac- TGA ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct ca- gtgagcga gcgagcgcgc agctgcctgc agg SEQ ID NO: 46 (human CYP4V2 gene region containing mutation c.802 -8_810del17insGC)
CAAACAGAAGCATGTGATTATCATTCAAAGCGAACGGGCCAATGAAATGAACGCCAATGA SEQ ID NO: 47 (wild-type human CYP4V2 gene region without the c.802-8_810del17insGC mutation) CAAACAGAAGCATGTGATTATCATGACGA
ATGAACGCCAATGA SEQ ID NO: 48 (g1 protospace element, RNA sequence, 20 nt)
UGAUUAUCAUUCAAAGCGAA SEQ ID NO: 49 (g2 protospacer element, RNA sequence, 20 nt) GAUUAUCAUUCAAAGCGAAC
SEQ ID NO: 50 (g3 protospace element, RNA sequence, 20 nt)
GAUAAUCACAUGCUUCUGUU SEQ ID NO: 51 (g4 protospacer element, RNA sequence, 20 nt)
UUCAUUGGCGUUCAUUUCAU SEQ ID NO: 52 (g5 protospace element, RNA sequence, 20 nt)
CACAUGCUUCUGUUUGGACU SEQ ID NO: 53 (exemplary crRNA sequence (excluding 5 'protospacer element sequence, 16 nt)
GUUUUAGAGCUAUGCU SEQ ID NO: 54 (exemplary tracrRNA sequence, 67 nt) AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA-
GUCGGUGCUUU SEQ ID NO: 55 (exemplary sgRNA sequence, Excluding the 5 'proto-spacer element sequence and the optional “G” before the proto-spacer element. Sequence shown in DNA format as in a plasmid construct. For sequence in format of RNA, use “U” to replace “T”, 82 nt) gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaag- tggcaccgagtcggtgctttttt SEQ ID NO: 56 (model sequence 1 donor of CYP4V2, 200 bases) AGA AAA ATA AAT GAA AGA AAC TAG CAT ATT TTA TAA GAA AAT GTG TTA ACT AGG GTG CAT CCA AGT CCA AAC AGA AGC ATG TGA TTA TCA TTC AAA TCA TAC AGG TCA TCG CTG AAC GGG CCA ATG AAA TGA ACG CCA ATG AAG ACT GTA GAG GTG ATG GCA GGG GCT CTG CCC CCT CCA
AAA ATA AAC GCA GGG CCT TT SEQ ID NO: 57 (CYP4V2 donor model 2 sequence, the reverse complement of CYP4V2 donor model 1 sequence, 200 bases) AA AGG CCC TGC GTT TAT TTT TGG AGG GGG CAG AGC CCC TGC CAT CAC CTC TAC AGT CTT CAT TGG CGT TCA TTT CAT TGG CCC GTT CAG CGA TGA CCT GTA TGA TTT GAA TGA TAA TCA CAT GCT TCT GTT TGG ACT TGG ATG CAC CCT AGT TAA CAC ATT TTC TTA TAA AAT ATG CTA GTT
TAT ATT TTT TCT TTC TCT SEQ ID NO: 58 (amino acid sequence of exemplary SpCas9 (1368 aa)) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA- TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI- VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD- KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN- LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA- ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD- QSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI- HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEE- TITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY- VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNAS- LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDD- KVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF- KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA- RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVD- QELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR- QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT- KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK- KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEI- RKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSD- KLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN- PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVN- FLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKRVILADANLDKVL- SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI-
HQSITGLYETRIDLSQLGGD SEQ ID NO: 59 (additional nucleotide inserted immediately after the U6 promoter sequence and before the protospace element sequence in a plasmid construct and in an IVT sgR- NA, 1 nt) SEQ ID NO: 60 - Sequence of CYP4V2 expression cassette in AAV2.CYP4V2op, AAV2tri (YF) .CYP4V2op and AAV5.CYP4V2op .: Left ITR: 1-141 CAG promoter: 237-1951 cYNA CYP4V2op: 2002-3579 WPRE: 3736-24 1 right ITR -4574 4659-4799 CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG 51 CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC 101 GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TGCGGCCAAT 151 TCAGTCGATA ACTATAACGG TCCTAAGGTA GCGATTTAAA TACGCGCTCT 201 CTTAAGGTAG CCCCGGGACG CGTCAATTGA GATCTCGACA TTGATTATTG 251 ACTAGTTATT AATAGTAATC AATTACGGGG TCATTAGTTC ATAGCCCATA 301 TATGGAGTTC CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC 351 CGCCCAACGA IncorporaçõesCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA 401 GTAACGCCAA TAGGGACTTT CCATTGACGT CAATGGGTGG ACTATTT ACG 451 GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTACGC 501 CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG 551 TACATGACCT TATGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT 601 CATCGCTATT ACCATGGGTC GAGGTGAGCC CCACGTTCTG CTTCACTCTC 651 CCCATCTCCC CCCCCTCCCC ACCCCCAATT TTGTATTTAT TTATTTTTTA 701 ATTATTTTGT GCAGCGATGG GGGCGGGGGG GGGGGGGGCG CGCGCCAGGC 751 GGGGCGGGGC GGGGCGAGGG GCGGGGCGGG GCGAGGCGGA GAGGTGCGGC 801 GGCAGCCAAT CAGAGCGGCG CGCTCCGAAA GTTTCCTTTT ATGGCGAGGC 851 GGCGGCGGCG GCGGCCCTAT AAAAAGCGAA GCGCGCGGCG GGCGGGAGTC 901 GCTGCGTTGC CTTCGCCCCG TGCCCCGCTC CGCGCCGCCT CGCGCCGCCC 951 GCCCCGGCTC TGACTGACGCGGTG
1001 GCCCTTCTCC TCCGGGCTGT AATTAGCGCT TGGTTTAATG ACGGCTCGTT 1051 TCTTTTCTGT GGCTGCGTGA AAGCCTTAAA GGGCTCCGGG AGGGCCCTTT 1101 GTGCGGGGGG GAGCGGCTCG GGGGGTGCGT GCGTGTGTGT GTGCGTGGGG 1151 AGCGCCGCGT GCGGCCCGCG CTGCCCGGCG GCTGTGAGCG CTGCGGGCGC 1201 GGCGCGGGGC TTTGTGCGCT CCGCGTGTGC GCGAGGGGAG CGCGGCCGGG 1251 GGCGGTGCCC CGCGGTGCGG GGGGGCTGCG AGGGGAACAA AGGCTGCGTG 1301 CGGGGTGTGT GCGTGGGGGG GTGAGCAGGG GGTGTGGGCG CGGCGGTCGG 1351 GCTGTAACCC CCCCCTGCAC CCCCCTCCCC GAGTTGCTGA GCACGGCCCG 1401 GCTTCGGGTG CGGGGCTCCG TGCGGGGCGT GGCGCGGGGC TCGCCGTGCC 1451 GGGCGGGGGG TGGCGGCAGG TGGGGGTGCC GGGCGGGGCG GGGCCGCCTC 1501 GGGCCGGGGA GGGCTCGGGG GAGGGGCGCG GCGGCCCCGG AGCGCCGGCG 1551 GCTGTCGAGG CGCGGCGAGC CGCAGCCATT GCCTTTTATG GTAATCGTGC 1601 GAGAGGGCGC AGGGACTTCC TTTGTCCCAA ATCTGGCGGA GCCGAAATCT 1651 GGGAGGCGCC GCCGCACCCC CTCTAGCGGG CGCGGGCGAA GCGGTGCGGC 1701 GCCGGCAGGA AGGAAATGGG CGGGGAGGGC CTTCGTGCGT CGCCGCGCCG 1751 CCGTCCCCTT CTCCATCTCC AGCCTCGGGG CTGCCGCAGG GGGACGGCTG 1801 CCTTCGGGGG GGACGGGGCA GGGCGGGGTT CG GCTTCTGG CGTGTGACCG 1851 GCGGCTCTAG AGCCTCTGCT AACCATGTTC ATGCCTTCTT CTTTTTCCTA 1901 CAGCTCCTGG GCAACGTGCT GGTTATTGTG CTGTCTCATC ATTTTGGCAA 1951 AGAATTCTAA TACGACTCAC TATAGGGAGA CCCAAGCTGG CTAGAGCCAC 2001 CATGGCTGGA CTGTGGCTGG GACTGGTGTG GCAGAAACTG CTGCTGTGGG 2051 GGGCCGCTTC CGCACTGTCA CTGGCTGGGG CTTCACTGGT GCTGAGCCTG 2101 CTGCAGAGGG TGGCCTCCTA CGCCAGAAAG TGGCAGCAGA TGAGGCCCAT 2151 CCCTACCGTG GCCAGAGCCT ATCCACTGGT GGGACACGCA CTGCTGATGA 2201 AGCCTGACGG CAGAGAGTTC TTTCAGCAGA TCATCGAGTA CACAGAGGAG 2251 TATAGGCACA TGCCACTGCT GAAGCTGTGG GTGGGACCCG TGCCTATGGT 2301 GGCCCTGTAC AACGCCGAGA ATGTGGAAGT GATCCTGACC AGCAGCAAGC 2351 AGATCGATAA GTCTAGCATG TATAAGTTCC TGGAGCCTTG GCTGGGCCTG 2401 GGCCTGCTGA CCTCTACAGG CAACAAGTGG AGGAGCCGGA GAAAGATGCT 2451 GACCCCAACA TTCCACTTTA CAATCCTGGA GGACTTCCTG GACATCATGA 2501 ACGAGCAGGC CAATATCCTG GTGAAGAAGC TGGAGAAGCA CATCAACCAG 2551 GAGGCCTTTA ATTGCTTCTT TTACATCACC CTGTGCGCCC TGGACATCAT 2601 CTGTGAGACA GCTATGGGCA AGAACATCGG CGCCCAGTCT AATGACGATA 2651 GCGAGTACGT GCGG GCCGTG TATAGAATGA GCGAGATGAT CTTTAGGCGC 2701 ATCAAGATGC CCTGGCTGTG GCTGGATCTG TGGTATCTGA TGTTCAAGGA 2751 GGGCTGGGAG CACAAGAAGT CCCTGCAGAT CCTGCACACC TTTACAAACT 2801 CTGTGATCGC CGAGAGAGCC AATGAGATGA ACGCCAATGA GGACTGTAGG 2851 GGCGATGGAA GGGGCAGCGC CCCTTCCAAG AACAAGCGGA GAGCCTTCCT 2901 GGACCTGCTG CTGAGCGTGA CCGACGATGA GGGCAATCGC CTGTCCCACG 2951 AGGACATCCG GGAGGAGGTG GATACATTCA TGTTTGAGGG ACACGACACC 3001 ACAGCCGCCG CCATCAACTG GTCCCTGTAC CTGCTGGGCT CTAATCCAGA 3051 GGTGCAGAAG AAGGTGGATC ACGAGCTGGA CGACGTGTTC GGCAAGTCCG 3101 ACAGGCCAGC AACCGTGGAG GATCTGAAGA AGCTGAGATA CCTGGAGTGC 3151 GTGATCAAGG AGACACTGCG CCTGTTCCCC TCTGTGCCTC TGTTTGCCCG 3201 GTCCGTGTCT GAGGACTGTG AGGTGGCCGG CTATCGCGTG CTGAAGGGCA 3251 CCGAGGCCGT GATCATCCCT TACGCCCTGC ACCGGGACCC CAGGTATTTC 3301 CCTAACCCAG AGGAGTTTCA GCCAGAGAGA TTCTTTCCCG AGAATGCCCA 3351 GGGCAGGCAC CCTTACGCCT ATGTGCCATT CTCCGCCGGA CCAAGGAACT 3401 GCATCGGACA GAAGTTTGCC GTGATGGAGG AGAAAACCAT CCTGTCTTGT 3451 ATCCTGAGAC ACTTCTGGAT CGAGAGCAAT CAGAAGAGGG AGGAGCTGGG3501 CCTGGAGGGA CAGCTGATCC TGCGGCCAAG CAACGGCATC TGGATCAAAC 3551 TGAAAAGAAG GAACGCTGAC GAGAGGTAAA AGCTTGGTAC CGATATCGCG 3601 GCCGCCCTAG GGAGCTCCTC GAGGCGGCCC GCTCGAGTCT AGAGGGCCCT 3651 TCGAAGGTAA GCCTATCCCT AACCCTCTCC TCGGTCTCGA TTCTACGCGT 3701 ACCGGTCATC ATCACCATCA CCATTGAGTT TCGATAATCA ACCTCTGGAT
3751 TACAAAATTT GTGAAAGATT GACTGGTATT CTTAACTATG TTGCTCCTTT 3801 TACGCTATGT GGATACGCTG CTTTAATGCC TTTGTATCAT GCTATTGCTT 3851 CCCGTATGGC TTTCATTTTC TCCTCCTTGT ATAAATCCTG GTTGCTGTCT 3901 CTTTATGAGG AGTTGTGGCC CGTTGTCAGG CAACGTGGCG TGGTGTGCAC 3951 TGTGTTTGCT GACGCAACCC CCACTGGTTG GGGCATTGCC ACCACCTGTC 4001 AGCTCCTTTC CGGGACTTTC GCTTTCCCCC TCCCTATTGC CACGGCGGAA 4051 CTCATCGCCG CCTGCCTTGC CCGCTGCTGG ACAGGGGCTC GGCTGTTGGG 4101 CACTGACAAT TCCGTGGTGT TGTCGGGGAA ATCATCGTCC TTTCCTTGGC 4151 TGCTCGCCTG TGTTGCCACC TGGATTCTGC GCGGGACGTC CTTCTGCTAC 4201 GTCCCTTCGG CCCTCAATCC AGCGGACCTT CCTTCCCGCG GCCTGCTGCC 4251 GGCTCTGCGG CCTCTTCCGC GTCTTCGCCT TCGCCCTCAG ACGAGTCGGA 4301 TCTCCCTTTG GGCCGCCTCC CCGCATCGAA ACCCGCTGAT CAGCCTCGAC 4351 TGTGCCTTCT AGTTGCCAGC CATCTGTTGT TTGCCCCTCC CCCGTGCCTT 4401 CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA ATAAAATGAG 4451 GAAATTGCAT CGCATTGTCT GAGTAGGTGT CATTCTATTC TGGGGGGTGG 4501 GGTGGGGCAG GACAGCAAGG GGGAGGATTG GGAAGACAAT AGCAGGCATG 4551 CTGGGGATGC GGTGGGCTCT ATGGCTTCTG AG GCGGAAAG AACCAGATCC 4601 TCTCTTAAGG TAGCATCGAG ATTTAAATTA GGGATAACAG GGTAATGGCG 4651 CGGGCCGCAG GAACCCCTAG TGATGGAGTT GGCCACTCCC TCTCTGCGCG 4701 CTCGCTCGCT CACTGAGGCC GGGCGACCAA AGGTCGCCCG ACGCCCGGGC 4751 TTTGCCCGGG CGGCCTCAGT GAGCGAGCGA GCGCGCAGCT GCCTGCAGG
SEQ ID NO: 61 - CYP4V2 expression cassette sequence in AAV5.CYP4V2st.
AAV5.CYP4V2st has the same promoter (CAG), enhancer (WPRE) and polyA (polyA bGH) sequences as AAV2.CYP4V2op, AAV2tri (YF) .CYP4V2op and AAV5.CYP4V2op (SEQ ID NO: 60) but different from CYP4V1 cDNA and junction / linker: Left ITR: 1-141 CAG promoter: 166-1880 CYP4V2st cDNA: 1938-3515 WPRE enhancer: 3551-4139 polyA bGH: 4163-4387 ITR Right: 4399-4539
1 CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG 51 CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC 101 GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TGCGGCCTAA 151 GGCAATTGAG ATCTCGACAT TGATTATTGA CTAGTTATTA ATAGTAATCA 201 ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA 251 ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT 301 TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC 351 CATTGACGTC AATGGGTGGA CTATTTACGG TAAACTGCCC ACTTGGCAGT 401 ACATCAAGTG TATCATATGC CAAGTACGCC CCCTATTGAC GTCAATGACG 451 GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT ATGGGACTTT 501 CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGGTCG 551 AGGTGAGCCC CACGTTCTGC TTCACTCTCC CCATCTCCCC CCCCTCCCCA 601 CCCCCAATTT TGTATTTATT TATTTTTTAA TTATTTTGTG CAGCGATGGG 651 GGCGGGGGGG GGGGGGGCGC GCGCCAGGCG GGGCGGGGCG GGGCGAGGGG 701 CGGGGCGGGG CGAGGCGGAG AGGTGCGGCG GCAGCCAATC AGAGCGGCGC 751 GCTCCGAAAG TTTCCTTTTA TGGCGAGGCG GCGGCGGCGG CGGCCCTATA 801 AAAAGCGAAG CGCGCGGCGG GCGGGAGTCG CTGCGTTGCC TTCGCCCCGT 851 GCCCCGCTCC GCGCCGCCTC GCGCCGCCCG CCCCGGCTCT GACTGACCGC 901 GTTACTCCCA CAGGTGAGCG GGCGGGACGG CCCTTCTCCT CCGGGCTGTA 951 ATTAGCGCTT GGTTTAATGA CGGCTCGTTT CTTTTCTGTG GCTGCGTGAA 1001 AGCCTTAAAG GGCTCCGGGA GGGCCCTTTG TGCGGGGGGG AGCGGCTCGG
1051 GGGGTGCGTG CGTGTGTGTG TGCGTGGGGA GCGCCGCGTG CGGCCCGCGC 1101 TGCCCGGCGG CTGTGAGCGC TGCGGGCGCG GCGCGGGGCT TTGTGCGCTC 1151 CGCGTGTGCG CGAGGGGAGC GCGGCCGGGG GCGGTGCCCC GCGGTGCGGG 1201 GGGGCTGCGA GGGGAACAAA GGCTGCGTGC GGGGTGTGTG CGTGGGGGGG 1251 TGAGCAGGGG GTGTGGGCGC GGCGGTCGGG CTGTAACCCC CCCCTGCACC 1301 CCCCTCCCCG AGTTGCTGAG CACGGCCCGG CTTCGGGTGC GGGGCTCCGT 1351 GCGGGGCGTG GCGCGGGGCT CGCCGTGCCG GGCGGGGGGT GGCGGCAGGT 1401 GGGGGTGCCG GGCGGGGCGG GGCCGCCTCG GGCCGGGGAG GGCTCGGGGG 1451 AGGGGCGCGG CGGCCCCGGA GCGCCGGCGG CTGTCGAGGC GCGGCGAGCC 1501 GCAGCCATTG CCTTTTATGG TAATCGTGCG AGAGGGCGCA GGGACTTCCT 1551 TTGTCCCAAA TCTGGCGGAG CCGAAATCTG GGAGGCGCCG CCGCACCCCC 1601 TCTAGCGGGC GCGGGCGAAG CGGTGCGGCG CCGGCAGGAA GGAAATGGGC 1651 GGGGAGGGCC TTCGTGCGTC GCCGCGCCGC CGTCCCCTTC TCCATCTCCA 1701 GCCTCGGGGC TGCCGCAGGG GGACGGCTGC CTTCGGGGGG GACGGGGCAG 1751 GGCGGGGTTC GGCTTCTGGC GTGTGACCGG CGGCTCTAGA GCCTCTGCTA 1801 ACCATGTTCA TGCCTTCTTC TTTTTCCTAC AGCTCCTGGG CAACGTGCTG 1851 GTTATTGTGC TGTCTCATCA TTTTGGCAAA GA ATTCTAAT ACGACTCACT 1901 ATAGGGAGAC CCAAGCTGGC TAGCCAAAGC TTCCACCATG GCGGGGCTCT 1951 GGCTGGGGCT CGTGTGGCAG AAGCTGCTGC TGTGGGGCGC GGCGAGTGCC 2001 CTTTCCCTGG CCGGCGCCAG TCTGGTCCTG AGCCTGCTGC AGAGGGTGGC 2051 GAGCTACGCG CGGAAATGGC AGCAGATGCG GCCCATCCCC ACGGTGGCCC 2101 GCGCCTACCC ACTGGTGGGC CACGCGCTGC TGATGAAGCC GGACGGGCGA 2151 GAATTTTTTC AGCAGATCAT TGAGTACACA GAGGAATACC GCCACATGCC 2201 GCTGCTGAAG CTCTGGGTCG GGCCAGTGCC CATGGTGGCC CTTTATAATG 2251 CAGAAAATGT GGAGGTAATT TTAACTAGTT CAAAGCAAAT TGACAAATCC 2301 TCTATGTACA AGTTTTTAGA ACCATGGCTT GGCCTAGGAC TTCTTACAAG 2351 TACTGGAAAC AAATGGCGCT CCAGGAGAAA GATGTTAACA CCCACTTTCC 2401 ATTTTACCAT TCTGGAAGAT TTCTTAGATA TCATGAATGA ACAAGCAAAT 2451 ATATTGGTTA AGAAACTTGA AAAACACATT AACCAAGAAG CATTTAACTG 2501 CTTTTTTTAC ATCACTCTTT GTGCCTTAGA TATCATCTGT GAAACAGCTA 2551 TGGGGAAGAA TATTGGTGCT CAAAGTAATG ATGATTCCGA GTATGTCCGT 2601 GCAGTTTATA GAATGAGTGA GATGATATTT CGAAGAATAA AGATGCCCTG 2651 GCTTTGGCTT GATCTCTGGT ACCTTATGTT TAAAGAAGGA TGGGAACACA 2701 AAAAGAGCCT TCAG ATCCTA CATACTTTTA CCAACAGTGT CATCGCTGAA 2751 CGGGCCAATG AAATGAACGC CAATGAAGAC TGTAGAGGTG ATGGCAGGGG 2801 CTCTGCCCCC TCCAAAAATA AACGCAGGGC CTTTCTTGAC TTGCTTTTAA 2851 GTGTGACTGA TGACGAAGGG AACAGGCTAA GTCATGAAGA TATTCGAGAA 2901 GAAGTTGACA CCTTCATGTT TGAGGGGCAC GATACAACTG CAGCTGCAAT 2951 AAACTGGTCC TTATACCTGT TGGGTTCTAA CCCAGAAGTC CAGAAAAAAG 3001 TGGATCATGA ATTGGATGAC GTGTTTGGGA AGTCTGACCG TCCCGCTACA 3051 GTAGAAGACC TGAAGAAACT TCGGTATCTG GAATGTGTTA TTAAGGAGAC 3101 CCTTCGCCTT TTTCCTTCTG TTCCTTTATT TGCCCGTAGT GTTAGTGAAG 3151 ATTGTGAAGT GGCAGGTTAC AGAGTTCTAA AAGGCACTGA AGCCGTCATC 3201 ATTCCCTATG CATTGCACAG AGATCCGAGA TACTTCCCCA ACCCCGAGGA 3251 GTTCCAGCCT GAGCGGTTCT TCCCCGAGAA TGCACAAGGG CGCCATCCAT 3301 ATGCCTACGT GCCCTTCTCT GCTGGCCCCA GGAACTGTAT AGGTCAAAAG 3351 TTTGCTGTGA TGGAAGAAAA GACCATTCTT TCGTGCATCC TGAGGCACTT 3401 TTGGATAGAA TCCAACCAGA AAAGAGAAGA GCTTGGTCTA GAAGGACAGT 3451 TGATTCTTCG TCCAAGTAAT GGCATCTGGA TCAAGTTGAA GAGGAGAAAT 3501 GCAGATGAAC GCTAAGCGGC CGCAACTCGA GACTCTAGAG GTTAATCGAT3551 AATCAACCTC TGGATTACAA AATTTGTGAA AGATTGACTG GTATTCTTAA 3601 CTATGTTGCT CCTTTTACGC TATGTGGATA CGCTGCTTTA ATGCCTTTGT 3651 ATCATGCTAT TGCTTCCCGT ATGGCTTTCA TTTTCTCCTC CTTGTATAAA 3701 TCCTGGTTGC TGTCTCTTTA TGAGGAGTTG TGGCCCGTTG TCAGGCAACG 3751 TGGCGTGGTG TGCACTGTGT TTGCTGACGC AACCCCCACT GGTTGGGGCA
3801 TTGCCACCAC CTGTCAGCTC CTTTCCGGGA CTTTCGCTTT CCCCCTCCCT 3851 ATTGCCACGG CGGAACTCAT CGCCGCCTGC CTTGCCCGCT GCTGGACAGG 3901 GGCTCGGCTG TTGGGCACTG ACAATTCCGT GGTGTTGTCG GGGAAATCAT 3951 CGTCCTTTCC TTGGCTGCTC GCCTGTGTTG CCACCTGGAT TCTGCGCGGG 4001 ACGTCCTTCT GCTACGTCCC TTCGGCCCTC AATCCAGCGG ACCTTCCTTC 4051 CCGCGGCCTG CTGCCGGCTC TGCGGCCTCT TCCGCGTCTT CGCCTTCGCC 4101 CTCAGACGAG TCGGATCTCC CTTTGGGCCG CCTCCCCGCA TCGAAACCCG 4151 CTGACTAGAC GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC 4201 TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC 4251 CTAATAAAAT GAGGAAATTG CATCGCATTG TCTGAGTAGG TGTCATTCTA 4301 TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA TTGGGAAGAC 4351 AATAGCAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGCCG CGGGCCGCAG 4401 GAACCCCTAG TGATGGAGTT GGCCACTCCC TCTCTGCGCG CTCGCTCGCT 4451 CACTGAGGCC GGGCGACCAA AGGTCGCCCG ACGCCCGGGC TTTGCCCGGG 4501 CGGCCTCAGT GAGCGAGCGA GCGCGCAGCT GCCTGCAGG
SEQ ID NO: 62 - CYP4V1 expression cassette sequence in AAV8.CYP4V2fv.
AAV8.CYP4V2fv has the same promoter (CAG), enhancer (WPRE) and polyA (polyA bGH) and junction / ligand sequences as AAV5.CYP4V2st (SEQ ID NO: 61) and differs only in cDNA sequence of CYP4V2:
Left ITR: 1-141 CAG promoter: 166-1880 CYP4V2fv cDNA: 1938-3515 WPRE enhancer: 3551-4139 PoliA bGH: 4163-4387 Right ITR: 4399-4539
1 CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG 51 CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC 101 GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TGCGGCCTAA 151 GGCAATTGAG ATCTCGACAT TGATTATTGA CTAGTTATTA ATAGTAATCA 201 ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA 251 ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT 301 TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC 351 CATTGACGTC AATGGGTGGA CTATTTACGG TAAACTGCCC ACTTGGCAGT 401 ACATCAAGTG TATCATATGC CAAGTACGCC CCCTATTGAC GTCAATGACG 451 GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT ATGGGACTTT 501 CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGGTCG 551 AGGTGAGCCC CACGTTCTGC TTCACTCTCC CCATCTCCCC CCCCTCCCCA 601 CCCCCAATTT TGTATTTATT TATTTTTTAA TTATTTTGTG CAGCGATGGG 651 GGCGGGGGGG GGGGGGGCGC GCGCCAGGCG GGGCGGGGCG GGGCGAGGGG 701 CGGGGCGGGG CGAGGCGGAG AGGTGCGGCG GCAGCCAATC AGAGCGGCGC 751 GCTCCGAAAG TTTCCTTTTA TGGCGAGGCG GCGGCGGCGG CGGCCCTATA 801 AAAAGCGAAG CGCGCGGCGG GCGGGAGTCG CTGCGTTGCC TTCGCCCCGT 851 GCCCCGCTCC GCGCCGCCTC GCGCCGCCCG CCCCGGCTCT GACTGACCGC 901 GTTACTCCCA CAGGTGAGCG GGCGGGACGG CCCTTCTCCT CCGGGCTGTA 951 ATTAGCGCTT GGTTTAATGA CGGCTCGTTT CTTTTCTGTG GCTGCGTGAA 1001 AGCCTTAAAG GGCTCCGGGA GGGCCCTTTG TGCGGGGGGG AGCGGCTCGG 1051 GGGGTGCGTG CGTGTGTGTG TGCGTGGGGA GCGCCGCGTG CGGCCCGCGC 1101 TGCCCGGCGG CTGTGAGCGC TGCGGGCGCG GCGCGGGGCT TTGTGCGCTC 1151 CGCGTGTGCG CGAGGGGAGC GCGGCCGGGG GCGGTGCCCC GCGGTGCGGG
1201 GGGGCTGCGA GGGGAACAAA GGCTGCGTGC GGGGTGTGTG CGTGGGGGGG 1251 TGAGCAGGGG GTGTGGGCGC GGCGGTCGGG CTGTAACCCC CCCCTGCACC 1301 CCCCTCCCCG AGTTGCTGAG CACGGCCCGG CTTCGGGTGC GGGGCTCCGT 1351 GCGGGGCGTG GCGCGGGGCT CGCCGTGCCG GGCGGGGGGT GGCGGCAGGT 1401 GGGGGTGCCG GGCGGGGCGG GGCCGCCTCG GGCCGGGGAG GGCTCGGGGG 1451 AGGGGCGCGG CGGCCCCGGA GCGCCGGCGG CTGTCGAGGC GCGGCGAGCC 1501 GCAGCCATTG CCTTTTATGG TAATCGTGCG AGAGGGCGCA GGGACTTCCT 1551 TTGTCCCAAA TCTGGCGGAG CCGAAATCTG GGAGGCGCCG CCGCACCCCC 1601 TCTAGCGGGC GCGGGCGAAG CGGTGCGGCG CCGGCAGGAA GGAAATGGGC 1651 GGGGAGGGCC TTCGTGCGTC GCCGCGCCGC CGTCCCCTTC TCCATCTCCA 1701 GCCTCGGGGC TGCCGCAGGG GGACGGCTGC CTTCGGGGGG GACGGGGCAG 1751 GGCGGGGTTC GGCTTCTGGC GTGTGACCGG CGGCTCTAGA GCCTCTGCTA 1801 ACCATGTTCA TGCCTTCTTC TTTTTCCTAC AGCTCCTGGG CAACGTGCTG 1851 GTTATTGTGC TGTCTCATCA TTTTGGCAAA GAATTCTAAT ACGACTCACT 1901 ATAGGGAGAC CCAAGCTGGC TAGCCAAAGC TTCCACCATG GCGGGGCTCT 1951 GGCTGGGGCT CGTGTGGCAG AAGCTGCTGC TGTGGGGCGC GGCGAGTGCC 2001 CTTTCCCTGG CCGGCGCCAG TCTGGTCCTG AG CCTGCTGC AGAGGGTGGC 2051 GAGCTACGCG CGGAAATGGC AGCAGATGCG GCCCATCCCC ACGGTGGCCC 2101 GCGCCTACCC ACTGGTGGGC CACGCGCTGC TGATGAAGCC GGACGGGCGA 2151 GAATTTTTTC AGCAGATCAT TGAGTACACA GAGGAATACC GCCACATGCC 2201 GCTGCTGAAG CTCTGGGTCG GGCCAGTGCC CATGGTGGCC CTTTATAATG 2251 CAGAAAATGT GGAGGTAATT TTAACTAGTT CAAAGCAAAT TGACAAATCC 2301 TCTATGTACA AGTTTTTAGA ACCATGGCTT GGCCTAGGAC TTCTTACAAG 2351 TACTGGAAAC AAATGGCGCT CCAGGAGAAA GATGTTAACA CCCACTTTCC 2401 ATTTTACCAT TCTGGAAGAT TTCTTAGATA TCATGAATGA ACAAGCAAAT 2451 ATATTGGTTA AGAAACTTGA AAAACACATT AACCAAGAAG CATTTAACTG 2501 CTTTTTTTAC ATCACTCTTT GTGCCTTAGA TATCATCTGT GAAACAGCTA 2551 TGGGGAAGAA TATTGGTGCT CAAAGTAATG ATGATTCCGA GTATGTCCGT 2601 GCAGTTTATA GAATGAGTGA GATGATATTT CGAAGAATAA AGATGCCCTG 2651 GCTTTGGCTT GATCTCTGGT ACCTTATGTT TAAAGAAGGA TGGGAACACA 2701 AAAAGAGCCT TAAGATCCTA CATACTTTTA CCAACAGTGT CATCGCGGAA 2751 CGGGCCAATG AAATGAACGC CAATGAAGAC TGTAGAGGTG ATGGCAGGGG 2801 CTCTGCCCCC TCCAAAAATA AACGCAGGGC CTTTCTTGAC TTGCTTTTAA 2851 GTGTGACTGA TGAC GAAGGG AACAGGCTAA GTCATGAAGA TATTCGAGAA 2901 GAAGTTGACA CCTTCATGTT TGAGGGGCAC GATACAACTG CAGCTGCAAT 2951 AAACTGGTCC TTATACCTGT TGGGTTCTAA CCCAGAAGTC CAGAAAAAAG 3001 TGGATCATGA ATTGGATGAC GTGTTTGGGA AGTCTGACCG TCCCGCTACA 3051 GTAGAAGACC TGAAGAAACT TCGGTATCTG GAATGTGTTA TTAAGGAGAC 3101 CCTTCGCCTT TTTCCTTCTG TTCCTTTATT TGCCCGTAGT GTTAGTGAAG 3151 ATTGTGAAGT GGCAGGTTAC AGAGTTCTAA AAGGCACTGA AGCCGTCATC 3201 ATTCCCTATG CATTGCACAG AGATCCGAGA TACTTCCCCA ACCCCGAGGA 3251 GTTCCAGCCT GAGCGGTTCT TCCCCGAGAA TGCACAAGGG CGCCATCCAT 3301 ATGCCTACGT GCCCTTCTCT GCTGGCCCCA GGAACTGTAT AGGTCAAAAG 3351 TTTGCTGTGA TGGAAGAAAA GACCATTCTT TCGTGCATCC TGAGGCACTT 3401 TTGGATAGAA TCCAACCAGA AAAGAGAAGA GCTTGGTCTA GAAGGACAGT 3451 TGATTCTTCG TCCAAGTAAT GGCATCTGGA TCAAGTTGAA GAGGAGAAAT 3501 GCAGATGAAC GCTAAGCGGC CGCAACTCGA GACTCTAGAG GTTAATCGAT 3551 AATCAACCTC TGGATTACAA AATTTGTGAA AGATTGACTG GTATTCTTAA 3601 CTATGTTGCT CCTTTTACGC TATGTGGATA CGCTGCTTTA ATGCCTTTGT 3651 ATCATGCTAT TGCTTCCCGT ATGGCTTTCA TTTTCTCCTC CTTGTATAAA3701 TCCTGGTTGC TGTCTCTTTA TGAGGAGTTG TGGCCCGTTG TCAGGCAACG 3751 TGGCGTGGTG TGCACTGTGT TTGCTGACGC AACCCCCACT GGTTGGGGCA 3801 TTGCCACCAC CTGTCAGCTC CTTTCCGGGA CTTTCGCTTT CCCCCTCCCT 3851 ATTGCCACGG CGGAACTCAT CGCCGCCTGC CTTGCCCGCT GCTGGACAGG 3901 GGCTCGGCTG TTGGGCACTG ACAATTCCGT GGTGTTGTCG GGGAAATCAT
3951 CGTCCTTTCC TTGGCTGCTC GCCTGTGTTG CCACCTGGAT TCTGCGCGGG 4001 ACGTCCTTCT GCTACGTCCC TTCGGCCCTC AATCCAGCGG ACCTTCCTTC 4051 CCGCGGCCTG CTGCCGGCTC TGCGGCCTCT TCCGCGTCTT CGCCTTCGCC 4101 CTCAGACGAG TCGGATCTCC CTTTGGGCCG CCTCCCCGCA TCGAAACCCG 4151 CTGACTAGAC GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC 4201 TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC 4251 CTAATAAAAT GAGGAAATTG CATCGCATTG TCTGAGTAGG TGTCATTCTA 4301 TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA TTGGGAAGAC 4351 AATAGCAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGCCG CGGGCCGCAG 4401 GAACCCCTAG TGATGGAGTT GGCCACTCCC TCTCTGCGCG CTCGCTCGCT 4451 CACTGAGGCC GGGCGACCAA AGGTCGCCCG ACGCCCGGGC TTTGCCCGGG 4501 CGGCCTCAGT GAGCGAGCGA GCGCGCAGCT GCCTGCAGG SEQ ID NO: 63 - CYP4V2 expression cassette sequence in AAV5.CYP4V2op (new). AAV5.CYP4V2op (new) has the same promoter (CAG), enhancer (WPRE) and polyA (polyA bGH) sequences and the same junction / ligand sequences as AAV5.CYP4V2st (SEQ ID NO: 61) and AAV8.CYP4V2fv (SEQ ID NO: 62) but different CYP4V2 cDNA sequences: left ITR: 1-141 CAG promoter: 166-1880 CYP4V2op cDNA: 1938-3515 WPRE enhancer: 3551-4139 polyA bGH: 4163-4387 right ITR: 4399- 4539 CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TGCGGCCTAA GGCAATTGAG ATCTCGACAT TGATTATTGA CTAGTTATTA ATAGTAATCA ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC IncorporaçõesGCCCAT TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA CTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTACGCC CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT ATGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGGTCG AGGTGAGCCC CACGTTCTGC TTCACTCTCC CCATCTCCCC→TCCCCA CccaCATTT TGTATTTATT TATTTTTTAA TTATTTTGTG GGCGGGGGGG GGGGGGGCGC GCGCCAGGCG GGGCGGGGCG GGGCGAGGGG CGGGGCGGGG CGAGGCGGAG AGGTGCGGCG GCAGCCAATC AGAGCGGCGC GCTCCGAAAG TTTCCTTTTA TGGCGAGGCG GCGGCGGCGG CGGCCCTATA AAAAGCGAAG CGCGCGGCGG GCGGGAGTCG CTGCGTTGCC TTCGCCCCGT GCCCCGCTCC GCGCCGCCTC GCGCCGCCCG IncorporaçõesGGCTCT GACTGACCGC GTTACTCCCA CAGGTGAGCG GGCGGGACGG CCCTTCTCCT CCGGGCTGTA ATTAGCGCTT GGTTTAATGA CGGCTCGTTT CTTTTCTGTG GCTGCGTGAA AGCCTTAAAG GGCTCCGGGA GGGCCCTTTG TGCGGGGGGG AGCGGCTCGG GGGGTGCGTG CGTGTGTGTG TGCGTGGGGA GCGCCGCGTG CGGCCCGCGC TGCCCGGCGG CTGTGAGCGC TGCGGGCGCG GCGCGGGGCT TTGTGCGCTC CGCGTGTGCG CGAGGGGAGC GCGGCCGGGG GCGGTGCCCC GCGGTGCGGG GGGGCTGCGA GGGGAACAAA GGCTGCGTGC GGGGTGTGTG CGTGGGGGGG TGAGCAGGGG GTGTGGGCGC GGCGGTCGGG CTGTAACCCC SAMSTGCACC →TCCCCG AGTTGCTGAG CACGGCCCGG CTTCGGGTGC GGGGCTCCGT GCGGGGCGTG GCGCGGGGCT CGCCGTGCCG GGCGGGGGGT GGCGGCAGGT GGGGGTGCCG GGCGGGGCGG GGCCGCCTCG GGCCGGGGAG GGCTCGGGGG AGGGGCGCGG CGGCCCCGGA GCGCCGGCGG CTGTCGAGGC GCGGCGAGCC GCAGCCATTG CCTTTTATGG TAATCGTGCG AGAGGGCGCA GGGACTTCCT TTGTCCCAAA TCTGGCGGAG CCGAAATCTG GGAGGCGCCG CCGCACCCCC TCTAGCGGGC GCGGGCGAAG CGGTGCGGCG CCGGCAGGAA GGAAATGGGC GGGGAGGGCC TTCGTGCGTC GCCGCGCCGC CGTCCCCTTC TCCATCTCCA GCCTCGGGGC TGCCGCAGGG GGACGGCTGC CTTCGGGGGG GACGGGGCAG GGCGGGGTTC GGCTTCTGGC GTGTGACCGG CGGCTCTAGA GCCTCTGCTA ACCATGTTCA TGCCTTCTTC TTTTTCCTAC AGCTCCTGGG CAACGTGCTG GTTATTGTGC TGTCTCATCA TTTTGGCAAA GAATTCTAAT ACGACTCACT ATAGGGAGAC CCAAGCTGGC TAGCCAAAGC TTCCACC ATGGCTGGACTGTGGCTGGGACTGGTGTGGCAGAAACTGCTGCTGTGGGGGGCCGCTTCCG- CACTGTCACTGGCTGGGGCTTCACTGGTGCTGAGCCTGCTGCAGAGGGTGGCCTCCTACG- CCAGAAAGTGGCAGCAGATGAGGCCCATCCCTACCGTGGCCAGAGCCTATCCACTGGTGG- GACACGCACTGCTGATGAAGCCTGACGGCAGAGAGTTCTTTCAGCAGATCATCGAGTACA- CAGAGGAGTATAGGCACATGCCACTGCTGAAGCTGTGGGTGGGACCCGTGCCTATGGTGG- CCCTGTACAACGCCGAGAATGTGGAAGTGATCCTGACCAGCAGCAAGCAGATCGATAAGTC- TAGCATGTATAAGTTCCTGGAGCCTTGGCTGGGCCTGGGCCTGCTGACCTCTACAGGCAA- CAAGTGGAGGAGCCGGAGAAAGATGCTGACCCCAACATTCCACTTTACAATCCTGGAGGAC- TTCCTGGACATCATGAACGAGCAGGCCAATATCCTGGTGAAGAAGCTGGAGAAGCACAT- CAACCAGGAGGCCTTTAATTGCTTCTTTTACATCACCCTGTGCGCCCTGGACATCATCTG- TGAGACAGCTATGGGCAAGAACATCGGCGCCCAGTCTAATGACGATAGCGAGTACGTG- CGGGCCGTGTATAGAATGAGCGAGATGATCTTTAGGCGCATCAAGATGCCCTGGCTGTGG- CTGGATCTGTGGTATCTGATGTTCAAGGAGGGCTGGGAGCACAAGAAGTCCCTGCAGA- TCCTGCACACCTTTACAAACTCTGTGATCGCCGAGAGAGCCAATGAGATGAACGCCAA- TGAGGACTGTAGGGGCGATGGAAGGGGCAGCGCCCCTTCCAAGAACAAGCGGAGAGCCTT- CCTGGACCTGCTGCTGAGCGTGACCGACGATGA GGGCAATCGCCTGTCCCACGAGGACA- TCCGGGAGGAGGTGGATACATTCATGTTTGAGGGACACGACACCACAGCCGCCGCCAT- CAACTGGTCCCTGTACCTGCTGGGCTCTAATCCAGAGGTGCAGAAGAAGGTGGATCACGAG- CTGGACGACGTGTTCGGCAAGTCCGACAGGCCAGCAACCGTGGAGGATCTGAAGAAGCTGA- GATACCTGGAGTGCGTGATCAAGGAGACACTGCGCCTGTTCCCCTCTGTGCCTCTGTTTG- CCCGGTCCGTGTCTGAGGACTGTGAGGTGGCCGGCTATCGCGTGCTGAAGGGCACCGAGG- CCGTGATCATCCCTTACGCCCTGCACCGGGACCCCAGGTATTTCCCTAACCCAGAGGAG- TTTCAGCCAGAGAGATTCTTTCCCGAGAATGCCCAGGGCAGGCACCCTTACGCCTATGTG- CCATTCTCCGCCGGACCAAGGAACTGCATCGGACAGAAGTTTGCCGTGATGGAGGA- GAAAACCATCCTGTCTTGTATCCTGAGACACTTCTGGATCGAGAGCAATCAGAAGAGGGAG- GAGCTGGGCCTGGAGGGACAGCTGATCCTGCGGCCAAGCAACGGCATCTGGATCAAAC- TGAAAAGAAGGAACGCTGACGAGAGGTAAGCGGC CGCAACTCGA GACTCTAGAG GTTA- ATCGAT AATCAACCTC TGGATTACAA AATTTGTGAA AGATTGACTG GTATTCTTAA CTATGTTGCT CCTTTTACGC TATGTGGATA CGCTGCTTTA ATGCCTTTGT ATCATGCTAT TGCTTCCCGT ATGGCTTTCA TTTTCTCCTC CTTGTATAAA TCCTGGTTGC TGTCTCTTTA TGAGGAGTTG TGGCCCGTTG TCAGGCAACG TGGCGTGGTG TGCACTGTGT TTGCTGACGC AACCCCCACT GGTTGGGGCA TTGCCACCAC CTGTCAGCTC CTTTCCGGGA CTTTCGCTTT ACCTCTCT ATTGCCACGG CGGAACTCAT CGCCGCCTGC CTTGCCCGCT GCTGGACAGG GGCTCGGCTG TTGGGCACTG ACAATTCCGT GGTGTTGTCG GGGAAATCAT CGTCCTTTCC TTGGCTGCTC GCCTGTGTTG CCACCTGGAT TCTGCGCGGG ACGTCCTTCT GCTACGTCCC TTCGGCCCTC AATCCAGCGG ACCTTCCTTC CCGCGGCCTG CTGCCGGCTC TGCGGCCTCT TCCGCGTCTT CGCCTTCGCC CTCAGACGAG TCGGATCTCC CTTTGGGCCG CCTCCCCGCA TCGAAACCCG CTGACTAGAC GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC CTAATAAAAT GAGGAAATTG CATCGCATTG TCTGAGTAGG TGTCATTCTA TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA TTGGGAAGAC AATAGCAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGCCG CGGGCCGCAG GAACCCCTAG TGATGGAGTT GGCCACTCCC TCTCTGCGCG CTCGCTCGCT CACTGAGGCC GGGCGACCAA AGGTCGCCCG ACGCCCGGGC TTTGCCCGGG
CGGCCTCAGT GAGCGAGCGA GCGCGCAGCT GCCTGCAGG SEQ ID NO: 64 - CYP4V2 expression cassette sequence in scAAV1.CYP4V2op, scAAV5.CYP4V2op and scAAV9.CYP4V2op. Left ITR (truncated): 1-117 Promoter EFS: 130-364 cDNA CYP4V2op: 520-2097 SPA: 2116-2169 Right ITR: 2263-2403 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgggggggggggggggggggggggggggggggggggggggggggg 121 cgtaggcctg attggctccg gtgcccgtca gtgggcagag cgcaca- TCGC 181 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt 241 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 301 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacggg- TTTG ccgccagaac 361 acaggtgtcg tgacgcgacc aggtatgcat ctgcagctct aaggtaa- taaaattttt 421 minutes aagtgtataa tgtgttaaac tactgattct aattgtttct ctcttt- taga ttccaacctt 481 tggaactgac tgcagggatc caagctttct agagccacca tggctg- GACT gtggctggga 541 ctggtgtggc agaaactgct gctgtggggg gccgcttccg cactgt- CACT 601 ggctggggct tcactggtgc tgagcctgct gcagagggtg gcctcctacg CCA gaaagtg gcagcagatg 661 aggcccatcc ctaccgtggc cagagcctat ccactggtgg gacacg- CACT 721 gctgatgaag cctgacggca tc gagagttctt agcagatc atcgagtaca cagagga- gta 781 taggcacatg ccactgctga agctgtgggt gggacccgtg cctatggtgg ccctgta- CAA cgccgagaat 841 gtggaagtga tcctgaccag cagcaagcag atcgataagt ctag- catgta taagttcctg 901 gagccttggc tgggcctggg cctgctgacc tctacaggca acaagtg- gag gagccggaga 961 aagatgctga ccccaacatt ccactttaca atcctggagg actt- cctgga catcatgaac 1021 gagcaggcca atatcctggt gaagaagctg gagaagcaca tcaacca- gga ggcctttaat 1081 tgcttctttt acatcaccct gtgcgccctg gacatcatct gtgaga- CAGC 1141 tatgggcaag aacatcggcg cccagtctaa tgacgatagc gagtacgtgc gggccg- tgta tagaatgagc 1201 gagatgatct ttaggcgcat caagatgccc tggctgtggc tgga- tctgtg gtatctgatg 1261 ttcaaggagg gctgggagca caagaagtcc ctgcagatcc tgcacac- ctt tacaaactct
1321 gtgatcgccg agagagccaa tgagatgaac gccaatgagg actg- tagggg cgatggaagg 1381 ggcagcgccc cttccaagaa caagcggaga gccttcctgg acctg- ctgct gagcgtgacc 1441 gacgatgagg gcaatcgcct gtcccacgag gacatccggg aggagg- tgga tacattcatg 1501 tttgagggac acgacaccac agccgccgcc atcaactggt ccctg- tacct gctgggctct 1561 aatccagagg tgcagaagaa ggtggatcac gagctggacg acgtgtt- caagtccgac CGG 1621 aggccagcaa ccgtggagga tctgaagaag ctgagatacc tggagtg- cgt gatcaaggag 1681 acactgcgcc tgttcccctc tgtgcctctg tttgcccggt ccgtg- tctga ggactgtgag 1741 gtggccggct atcgcgtgct gaagggcacc gaggccgtga tcatccctta cgccctgcac 1801 cgggacccca ggtatttccc taacccagag gagtttcagc cagaga- gatt ctttcccgag 1861 aatgcccagg gcaggcaccc ttacgcctat gtgccattct ccgccg- GACC aaggaactgc 1921 atcggacaga agtttgccgt gatggaggag aaaaccatcc tgtcttg- tat cctgagacac 1981 ttctggatcg agagcaatca gaagagggag gagctgggcc tggagg- gaca gctgatcctg 2041 cggccaagca acggcatctg gatcaaactg aaaagaagga acgctga- cga gaggtaaaag 2101 cttgaattcc tcgaggatcc aataaaagat ctttattttcta attt ga- tctg tgtgttggtt 2161 ttttgtgtgt ctagttgcca gccatctgtt gtttgcccct cccccg- CCGT ttccttgacc 2221 ctggaaggtg ccactcccag tttaaactta attaagggcc gcagga- ctagtgatgg ACCC 2281 agttggccac tccctctctg cgcgctcgct cgctcactga ggccggg- cga ccaaaggtcg 2341 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcg- agg cgc 2401 agctgcctgc

权利要求:
Claims (334)
[1]
1. Composition for the treatment or prevention of an eye disease in a human individual characterized by the fact that it comprises a vector, the vector comprising an expression cassette, the expression cassette comprising a nucleic acid molecule or a non-variant pathogenic of the same encoding a functional or non-mutant CYP4V2 protein operably linked to one or more regulatory sequence, in which the disease is associated with dysfunction, dystrophy, disorder, degeneration, atrophy and / or death of ocular cells.
[2]
2. Composition for preventing, stopping, decreasing the progression of, treating or improving the dysfunction, dystrophy, disorder, degeneration, atrophy and / or death of an eye cell characterized by the fact that it comprises a vector, the vector comprising a cassette of expression, the expression cassette comprising a nucleic acid molecule or a non-pathogenic variant thereof encoding a functional or non-mutant CYP4V2 protein operably linked to one or more regulatory sequence.
[3]
3. Composition according to claim 1 or 2, characterized by the fact that eye disease or ocular cell degeneration is an inherited retinal degeneration (IRD) or pigmented retinitis (RP) with biallelic mutation in the CYP4V2 gene .
[4]
4. Composition according to claim 1, 2 or 3, characterized by the fact that the disease is degeneration of the ocular cell in association with Bietti's Crystalline Dystrophy (also known as Bietti's Corneoretinal Crystalline Dystrophy; BCD) .
[5]
5. Composition according to any one of claims 1 to 4, characterized by the fact that the vector is a viral vector, a plasmid or a non-viral vector.
[6]
6. Composition according to claim 5, characterized
by the fact that the viral vector is selected from the group consisting of an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, a herpes simplex virus vector, a bacterovirus vector, a Sendai virus vector and a retrovirus vector.
[7]
7. Composition according to claim 5 or 6, characterized by the fact that the vector is a recombinant AAV vector (rAAV).
[8]
8. Composition according to claim 5, 6 or 7, characterized by the fact that the AAV genome or the AAV capsid protein in rAAV is any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or another serotype or isolate or Glade of AAV or any derivative, variant or hybrid thereof.
[9]
9. Composition according to any one of claims 5 to 8, characterized by the fact that the rAAV is a pseudo-dotipified AAV (for example, AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 9, AAV2 / 6, AAV2 / 4, AAV2 / 6, AAV5 / 2, AAV8 / 1, AAV8 / 2, AAV2 / 7, AAV2 / 12 andA- AV2 / 10) or a hybrid AAV (for example, AAV-DJ, AAV -DJ / 8 or AAV- DJ / 9).
[10]
10. Composition according to any one of claims 5 to 9, characterized in that the rAAV comprises one or more capsid mutations (for example, YF, KR, TA, SA and / or TV mutations (for example , AAV2 with one or more capid mutations within Y444F, Y500F, Y730F, Y252F, Y272F, Y700F, Y704F and T491V, or the corresponding mutation for a different AAV serotype (for example, AAV2 (Y444F + Y500F + Y730F), AAV2 (quadY-F + TV) or AAV2 / 8 (Y733F)))).
[11]
11. Composition according to any one of claims 5 to 10, characterized by the fact that the rAAV serotype is selected or derived from the group consisting of AAV1, AAV2, AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Anc80, rh10 and / or ShH10.
[12]
12. Composition according to any one of claims 5 to 11, characterized by the fact that rAAV is selected or derived from the group consisting of AAV2 / 5, AAV2 / 8, AAV2 / 2, AAV2 (Y444F + Y500F + Y730F), AAV2 / 1, AAV2 / 9, AAV2 / 8 (Y733F), AAV2 / 6, AAV2 / 4, AAV2 / 7, AAV5, AAV2, AAV8, AAV1, AAV9, AAV6, AAV10, AAV4, AAV7, AAV12, Anc80, AAV 7m8, AAV-DJ, ShH10, AAV-PHP.B or a hybrid, a derivative or variant thereof.
[13]
13. Composition according to any one of claims 5 to 12, characterized by the fact that the rAAV vector is a single-stranded AAV vector or a self-complementing AAV vector (scAAV).
[14]
14. Composition according to claim 5, characterized by the fact that the vector is a plasmid, a naked nucleic acid, liposomes (for example, cationic or anionic liposomes), denimers, nanoparticles, polymers (for example , polyplexes), lipid-polymer system, solid lipid nanoparticle or protamine / DNA lipid-containing lipoplex (LPD).
[15]
15. Composition according to any one of the preceding claims, characterized by the fact that the functional or non-mutant CYP4V2 protein encoded by the nucleic acid sequence comprises a polypeptide having at least 80% amino acid sequence identity ( e.g. at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) with any of the sequences selected from the group consisting of SEQ ID NOs: 4-6.
[16]
16. Composition according to any of the preceding claims, characterized by the fact that the acid molecule
of the nucleic has at least 75% sequence identity with any of the sequences in SEQ ID NO: 1, 2 or 3.
[17]
17. Composition according to any one of the preceding claims, characterized by the fact that the nucleic acid molecule has at least 80% sequence identity with any of the sequences in SEQ ID NO: 1, 2 or 3.
[18]
18. Composition according to any one of the preceding claims, characterized by the fact that the nucleic acid molecule comprises a sequence shown in SEQ ID NO: 1, 2 or 3.
[19]
19. Composition according to any one of the preceding claims, characterized by the fact that the nucleic acid molecule comprises an codon-optimized sequence encoding a protein comprising an amino acid sequence selected from SEQ ID NO: 4 , 5 or 6.
[20]
20. Composition, according to any one of the preceding claims, characterized by the fact that the regulatory sequence comprises a promoter.
[21]
21. Composition according to claim 20, characterized by the fact that the promoter is a specific promoter of RPE cell, a specific promoter of retinal cell, a specific promoter of corneal cell, a promoter specific eye cell or constitutive promoter.
[22]
22. Composition according to claim 20, characterized by the fact that the promoter is a beta-actin promoter or a viral promoter or a hybrid thereof.
[23]
23. Composition, according to claim 20, characterized by the fact that the promoter is selected from the group consisting of a CAG promoter (early CMV hybrid / beta-actin enhancer promoter from Galinha, also known as promoter HERE-
GGS, CB promoter or CBA promoter), a beta-actin promoter of the gamma, a small CBA promoter (smCBA), a CBSB promoter, or a CBh promoter, another beta-actin promoter such as the human beta-actin promoter , a short alpha 1 elongation factor (EFS) promoter, a 1 alpha elongation factor (EF-1 alpha) promoter, a CMV promoter, a PGK promoter, a UBC promoter, a GUSB promoter, a UCOE promoter, a promoter VMD2 (macular dystrophy, lifelike 2; also known as BEST1), an RPE65 promoter or a hybrid, a variant or a derivative thereof.
[24]
24. Composition according to any of the preceding claims, characterized by the fact that the regulatory sequence is a polyA signal selected from the group consisting of bovine growth hormone polyadenylation signal (polyA bGH), a signal small polyA (SPA), a human growth hormone polyadenylation signal (poly hGH), a SV40 polyA signal, late SV40 polyA signal or a derivative, a hybrid or a variant thereof.
[25]
25. Composition according to any one of the preceding claims, characterized by the fact that the regulatory sequence comprises a Kozak sequence.
[26]
26. Composition according to any one of the preceding claims, characterized by the fact that the regulatory sequence comprises an enhancer.
[27]
27. Composition according to claim 26, characterized by the fact that the enhancer is a viral enhancer, including, without limitation, a WPRE enhancer, an HPRE enhancer, a CTE enhancer or a derivative or hybrid or variant the same.
[28]
28. Composition for treating or preventing BCD or for producing a composition for treating or preventing BCD characterized by the fact that it comprises a nucleic acid molecule sharing at least 80% sequence identity with any of the cassette sequence of CYP4V2 expression in SEQ ID NOs: 60 to 64.
[29]
29. Composition according to any one of the preceding claims, characterized by the fact that the composition is formulated with a pharmaceutically acceptable carrier and additional components suitable for the specific route of administration.
[30]
30. Method of treatment or prevention of an eye disease in a human individual, characterized by the fact that it comprises administering a vector to the individual, in which the vector comprises an expression cassette, the expression cassette comprising a molecule of nucleic acid or a non-pathogenic variant thereof encoding a functional or non-mutant CYP4V2 protein operably linked to one or more regulatory sequence, in which the disease is associated with dysfunction, dystrophy, disorder, degeneration, atrophy and / or death of ocular cells.
[31]
31. Method of prevention, arrest, reduction of progression, treatment or improvement of dysfunction, dystrophy, disorder, degeneration, atrophy and / or death of an ocular cell characterized by the fact that it comprises the administration of a vector to the cell ocular, in which vector comprises an expression cassette, the expression cassette comprising a nucleic acid molecule or a non-pathogenic variant thereof encoding a functional or non-mutant CYP4V2 protein operably linked to one or more regulatory sequences .
[32]
32. Method according to claim 30 or 31, characterized by the fact that the ocular cells are retinal cells, corneal cells, choroidal cells, retinal pigment epithelium (RPE) cells, photoreceptor cells and / or choroidal epithelial cells.
[33]
33. The method of claim 30 or 31, characterized by
characterized by the fact that eye disease or ocular cell degeneration is inherited retinal degeneration (IRD) or pigmented retinitis (RP) with biallelic mutation in the CYP4V2 gene.
[34]
34. Method, according to claim 30 or 31, characterized by the fact that the disease is the ocular cell degeneration associated with Bietti's Crystalline Dystrophy (also known as Bietti's Corneoretinal Crystalline Dystrophy; BCD).
[35]
35. Method according to any of claims 30 to 34, characterized by the fact that the vector is a viral vector, a plasmid or a non-viral vector.
[36]
36. Method according to claim 35, characterized by the fact that the viral vector is selected from the group consisting of an adeno-associated virus vector (AAV), an adenovirus vector, a lentivirus vector, a virus vector herpes simplex, a baculovirus vector, a Sendai virus vector and a retrovirus vector.
[37]
37. Method, according to claim 36, characterized by the fact that the vector is a recombinant AAV vector (rAAV).
[38]
38. Method according to claim 37, characterized by the fact that the rAAV comprises an AAV genome or a derivative thereof and / or an AAV capsid protein or a derivative or variant thereof.
[39]
39. Method according to claim 37 or 38, characterized by the fact that the rAAV is a chimeric AAV, a shuffled AAV or a modified capsid AAV.
[40]
40. Method according to claim 36, 27, 38 or 39, characterized in that the AAV genome or the AAV capsid protein in rAAV is any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 , AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or another serotype or isolate or Glade of AAV or any derivative, variant or hybrid thereof.
[41]
41. Method according to any of claims 37 to 40, characterized by the fact that the rAAV is a pseudo-dotipified AAV (for example, AAV2 / 5, AAV2 / 8, AAV2 / 1, AAV2 / 9, AAV2 / 6, AAV2 / 4, AAV2 / 6, AAV5 / 2, AAV8 / 1, AAV8 / 2, AAV2 / 7, AAV2 / 12 andA- AV2 / 10) or a hybrid AAV (for example, AAV-DJ, AAV -DJ / 8 or AAV- DJ / 9).
[42]
42. Method according to any one of claims 37 to 41, characterized in that the rAAV comprises one or more capsid mutations (for example, YF, KR, TA, SA and / or TV mutations (for example , AAV2 with one or more capid mutations within Y444F, Y500F, Y730F, Y252F, Y272F, Y700F, Y704F and T491V, or the corresponding mutation for a different AAV serotype (for example, AAV2 (Y444F + Y500F + Y730F), AAV2 (quadY-F + TV) or AAV2 / 8 (Y733F)))).
[43]
43. Method according to any of claims 37 to 42, characterized by the fact that the rAAV serotype is selected or derived from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Anc80, rh10 and / or ShH10.
[44]
44. Method according to any of claims 37 to 42, characterized by the fact that rAAV is selected or derived from the group consisting of AAV2 / 5, AAV2 / 8, AAV2 / 2, AAV2 (Y444F + Y500F + Y730F), AAV2 / 1, AAV2 / 9, AAV2 / 8 (Y733F), AAV2 / 6, AAV2 / 4, AAV2 / 7, AAV5, AAV2, AAV8, AAV1, AAV9, AAV6, AAV10, AAV4, AAV7, AAV12, Anc80, AAV 7m8, AAV-DJ, ShH10, AAV-PHP.B or a hybrid, a derivative or variant thereof.
[45]
45. Method according to any of claims 37 to 44, characterized by the fact that the rAAV vector is a single-stranded AAV vector or a self-complementing AAV vector (scAAV).
[46]
46. Method according to any of claims 30 to 34, characterized in that the vector is a plasmid, a naked nucleic acid, liposomes (for example, cationic or anionic liposomes), dendrimer, nanoparticle, polymers ( for example, polyplexes), lipid-polymer system, solid lipid nanoparticle or liposome protamine / DNA liposome (LPD).
[47]
47. Method according to any of claims 30 to 46, characterized in that the functional or non-mutant CYP4V2 protein encoded by the nucleic acid sequence comprises a polypeptide having at least 80% sequence identity. amino acid (eg at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% sequence identity) with any of the selected sequences from the group consisting of SEQ ID NOs: 4-6.
[48]
48. Method according to any one of claims 30 to 47, characterized in that the nucleic acid molecule has at least 75% sequence identity with any of the sequences in SEQ ID NO: 1, 2 or 3.
[49]
49. Method according to any one of claims 30 to 48, characterized in that the nucleic acid molecule has at least 80% sequence identity with any of the sequences in SEQ ID NO: 1, 2 or 3.
[50]
50. Method according to any one of claims 30 to 49, characterized in that the nucleic acid molecule comprises a sequence shown in SEQ ID NO: 1, 2 or 3.
[51]
51. Method according to any one of claims 30 to 50, characterized in that the nucleic acid molecule comprises a codon-optimized sequence encoding a protein comprising a selected amino acid sequence of SEQ ID NO : 4, 5 or 6.
[52]
52. Method according to any one of claims 30 to 51, characterized in that the regulatory sequence comprises a promoter.
[53]
53. Method according to claim 52, characterized by the fact that the promoter is a specific cell promoter of RPE, a specific promoter of retinal cell, a specific promoter of corneal cell, a specific promoter of ocular cell or a constitutive promoter.
[54]
54. Method according to claim 52, characterized by the fact that the promoter is a beta-actin promoter or a viral promoter or a hybrid thereof.
[55]
55. Method, according to claim 52, characterized by the fact that the promoter is selected from the group consisting of a chicken CAG promoter (CMV hybrid / beta-actin early enhancer promoter, also known as CAGGS promoter, promoter). CB tor or CBA promoter), a chicken beta-actin promoter, a small CBA (smCBA) promoter, a CBSB promoter, or a CBh promoter, another beta-actin promoter such as the human beta-actin promoter, a short alpha 1 elongation factor (EFS) promoter, 1 alpha elongation factor (EF-1 alpha) promoter, CMV promoter, PGK promoter, UBC promoter, GUSB promoter, UCOE promoter, VMD2 promoter (dystrophy vitelliform macular 2; also known as BEST1), an RPE65 promoter or a hybrid, variant or derivative thereof.
[56]
56. Method according to any of claims 30 to 55, characterized in that the regulatory sequence comprises a polyadenylation signal (polyA).
[57]
57. Method according to claim 56, characterized
by the fact that the polyA signal is a sign of bovine growth hormone polyadenylation (polyA bGH), a small polyA signal (SPA), a sign of human growth hormone polyadenylation (poly hGH), a SV40 polyA signal, late SV40 polyA signal or a derivative, hybrid or variant thereof.
[58]
58. Method according to any of claims 30 to 57, characterized in that the regulatory sequence comprises a Kozak sequence.
[59]
59. Method according to any one of claims 30 to 57, characterized by the fact that the regulatory sequence comprises an enhancer.
[60]
60. Method according to claim 59, characterized by the fact that the enhancer is a viral enhancer, including, without limitation, a WPRE enhancer, an HPRE enhancer, a CTE enhancer or a derivative or hybrid or variant the same.
[61]
61. Method for treating or preventing BCD characterized by the fact that it comprises administering a vector comprising a nucleic acid molecule sharing at least 80% sequence identity with any of the CYP4V2 expression cassette sequence in SEQ ID NOs: 60 to 64.
[62]
62. Method according to any one of claims 30 to 61, characterized in that the composition is formulated with a pharmaceutically acceptable carrier and additional components suitable for the specific route of administration.
[63]
63. Composition for use in the production of a vector for the treatment or prevention of BCD, characterized by the fact that the composition comprises a nucleic acid molecule comprising at least 80% sequence identity with any of the expression cassette sequence of CYP4V2 in SEQ ID NOs: 60 to 64.
[64]
64. Method according to any one of claims 30 to 63, characterized in that for in vitro treatment, the target cell is infected at a dose (MOI) of about 1 x 10 ^ 3 GC at about 1 x 10 ^ 6 GC per cell (GC: genomic copies, measuring genome containing AAV particles).
[65]
65. Method according to any one of claims 30 to 64, characterized by the fact that for injectable administration to an individual's eye, a single administration may be of the order of about 1 x 10 ^ 6 to 2 x 10 ^ 13 GC (for example, a high dose range of about 1 x 10 ^ 11 GC to about 1 x 10 ^ 12 GC, an average dose range of about 1 x 10 ^ 10 GC at about 1 x 10 ^ 11 GC, a low dose range of about 1 x 10 ^ 9 GC at about 1 x 10 ^ 10 GC, a very low dose range of about 1 x 10 ^ 6 GC at about 1 x 10 ^ 9 GC and a very high dose range of about 1 x 10 ^ 12 GC at about 2 x 10 ^ 13 GC) or any dose within those ranges that is sufficient to provide the effect wanted.
[66]
66. Method according to any one of claims 30 to 65, characterized by the fact that the administration step occurs before the onset of symptoms of the disease or after the onset of symptoms of the disease.
[67]
67. Method according to any one of claims 30 to 66, characterized by the fact that the targeted administration and / or administration is to the eye and / or an ocular cell.
[68]
68. Method according to any one of claims 30 to 67, characterized by the fact that the administration is through sub-retinal injection, intravitreal injection or through intravitreal implant of a device encapsulating the vector.
[69]
69. Method according to any one of claims 30 to 68, characterized by the fact that administration is by any other method of administration that is effectively administered.
between the vectors to the sub-retinal site, the posterior segment of the eye, the cornea, the retina, the choroid, the RPE cells, the photoreceptors or the corneal epithelial cells, to the individual's CE cells.
[70]
70. Method according to claim 67, characterized by the fact that administration to the eye is obtained through administration through the bloodstream.
[71]
71. Method according to any of the preceding claims, characterized by the fact that the ocular cells are selected from the group consisting of retinal pigment epithelial cells (RPE), photoreceptor cells (PRCs), epithelial cells of the retina cornea (SCCs), choroidal endothelial cells (CE), retinal cells, corneal cells, lens cells, ganglion cells, optic nerve cells and / or choroidal cells, as well as said cell types derived from a cell -stem (including, without limitation, an iPSC, an ES cell, an MSC, an adult stem cell and / or a tissue-specific stem cell).
[72]
72. Method according to any one of claims 30 to 71, characterized in that the vector is formulated with a pharmaceutically acceptable carrier and additional components suitable for the specific route of administration.
[73]
73. Method according to any one of claims 30 to 72, characterized by the fact that it further comprises identification of an individual having BCD or at risk of developing BCD or having a biallelic CYP4V2 mutation.
[74]
74. Method of treatment or prevention of Bietti's Crystal Dystrophy (BCD) in a human individual, characterized by the fact that it comprises administering to the individual's eye cells a vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 2 encoding a human CYP4V2 protein or a nucleic acid sequence
sharing at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 2, operably linked to one or more regulatory sequence.
[75]
75. Method, according to claim 74, characterized by the fact that the vector is an adenoassociated virus (AAV) vector.
[76]
76. Method according to claims 74 and 75, characterized by the fact that the regulatory sequence is a promoter.
[77]
77. Nucleic acid molecule characterized by the fact that it comprises the nucleic acid sequence of SEQ ID NO: 2 encoding a human CYP4V2 protein or a nucleic acid sequence sharing at least 90% sequence identity with the acid sequence nucleic of SEQ ID NO: 2.
[78]
78. Expression cassette characterized by the fact that it comprises the nucleic acid molecule as defined in claim 77 and one or more regulatory sequence operably linked to such a nucleic acid sequence.
[79]
79. Vector characterized by the fact that it comprises the nucleic acid molecule as defined in claim 77 or the expression case as defined in claim 78.
[80]
80. Vector, according to claim 79, characterized by the fact that the vector is a viral vector or is selected from the group consisting of a recombinant adenoassociated virus (rA-AV) vector, a recombinant adenovirus vector, a vector of recombinant lentivirus, a recombinant herpes simplex virus vector, a recombinant baculovirus vector, a recombinant Sendai virus vector and a recombinant retrovirus vector.
[81]
81. Vector, according to claim 79, characterized by the fact that the vector is a plasmid vector or a non-viral vector.
[82]
82. Vector according to claim 81, characterized by the fact that the non-viral vector is selected from a naked nucleic acid, liposomes (for example, cationic or anionic liposomes), dendrimers, nanoparticles, polymers (for example, polyplexes) , lipid-polymer system, solid lipid nanoparticle or liposome protamine / DNA liposome (LPD).
[83]
83. Host cell characterized by the fact that it comprises the nucleic acid molecule as defined in claim 77 and / or the vector as defined in any of claims 79 to 82.
[84]
84. Method to reduce immune responses to viral vectors, preserve transduction efficiency, decrease dose of viral and / or immunosuppressive vector and / or maximize therapeutic effect for different patients of the same genetic disease, in gene therapy mediated by viral vector, characterized by the fact that it comprises: (a) establishment of a group of more than one recombinant viral vector (for example, rAAVs) with sufficient transduction efficiency in the type of target cell for gene therapy by creating variants with mutations antigenic region or other mutations or variants in the capsids of said viral vectors after such mutations or variations are confirmed with sufficient transduction efficiency in the target cells relevant to the disease (for example, in iPS-RPE or RPE cell lines for gene therapy with CYP4V2 for BCD); (b) detection of pre-existing neutralizing antiviral vector antibodies (NAbs) against different viral vector serotypes and / or capsid mutations or variants in the individual in need of gene therapy, and / or testing and comparing different viral vectors in patient-specific cells (for example, iPS-RPE cells) derived from such an individual; (c) selection of a viral vector from said group of viral vectors with (i) sufficient transduction efficiency in the diseased target cells and (ii) low cross-reactivity with the pre-existing NAbs in the individual and / or (iii) good phenotype rescue result in individual patient-specific patient target cells (eg, iPS-RPE or RPE cell lines for CYP4V2 gene therapy for BCD), where such a viral vector group comprises serotype viral vectors different and / or modified capsid (for example, including, without limitation, mutant capsid AAVs and / or capsid protein variant AAVs). (d) use of the viral vector selected from (c) for administration to the individual; and (e) repetition of (b) to (d) (only the part related to pre-existing NAbs) before each time the individual requires administration of gene therapy, including, without limitation, a follow-up administration for the organ (for example, a contralateral eye or eye) or another organ.
[85]
85. Method of using AAV vector-mediated gene therapy to treat or prevent human retinal diseases characterized by the fact that the AAV vector comprises a nucleic acid molecule comprising an EFS promoter (SEQ ID NO: 35) and / or a small polyA (SEQ ID NO: 36) operably linked to the therapeutic transgene or a nucleic acid molecule shares at least 90% sequence identity with SEQ ID NO: 35 or at least 85% sequence identity with SEQ ID NO: 36.
[86]
86. Method, according to claim 85, characterized by the fact that the AAV vector is a single-stranded AAV.
[87]
87. Method, according to claim 85, characterized by the fact that the AAV vector is a self-complementary AAV (scAAV).
[88]
88. Cellular disease model characterized by the fact that it comprises a cell line composition comprising
either (a) a stem cell provided from an individual or reprogrammed from a cell provided from an individual or (2) a cell derived from a stem cell provided from an individual or reprogrammed from a cell provided from an individual comprising one or more mutations in a target gene.
[89]
89. Composition according to claim 88, characterized by the fact that the stem cell is an induced pluripotent stem cell (iPS).
[90]
90. Composition according to claim 88, characterized by the fact that the stem cell is an embryonic stem cell (ES), somatic (or adult) stem cell, tissue-specific stem cell or mesenchymal stem cell (MSC).
[91]
91. Composition according to claim 88, characterized by the fact that the cell provided from an individual is a somatic cell.
[92]
92. Composition according to claim 88, characterized by the fact that the cell provided from an individual is a skin cell, a fibroblast or a blood cell.
[93]
93. Composition according to claim 88 or 92, characterized in that the cell provided from an individual is a skin fibroblast or a peripheral blood mononuclear cell (PBMC).
[94]
94. Composition according to claim 88, characterized by the fact that the cell provided from an individual is a urinary cell, a renal epithelial cell, a hair follicle or a dermal papillary cell.
[95]
95. Composition according to claim 88, characterized in that the cell derived from a stem cell is an ocular cell.
[96]
96. Composition according to claim 95, characterized by
characterized by the fact that the eye cell is a retinal pigment epithelium (RPE) cell, photoreceptor cell (PRC, including rod cell, cone cell and photoreceptor progenitor cell), retinal cell, corneal cell, corneal epithelial cell (CPB), optic nerve cell, lens cell, choroidal endothelial cell (CE), optic nerve cell or choroidal cell.
[97]
97. Composition according to claim 88, characterized by the fact that the cell derived from a stem cell is a neuron cell.
[98]
98. Composition, according to claim 88, characterized by the fact that the mutation is endogenous to the individual.
[99]
99. Composition according to claim 88 or 89, characterized by the fact that the mutation is introduced artificially through genetic editing or genetic manipulation.
[100]
100. Composition, according to claim 88, characterized by the fact that the cell line comprises a plurality of mutations that are endogenous and / or exogenous to the individual.
[101]
101. Composition according to claim 88, characterized by the fact that the individual is a mammal.
[102]
102. Composition, according to claim 88, characterized by the fact that the individual is a human being.
[103]
103. Composition according to claim 88, characterized by the fact that the target gene comprises a set of genes shown in Table 4.
[104]
104. Composition according to claim 88, characterized in that the target gene comprises a CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH , C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3,
OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PARK2 PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4X1, CYP4X1, CYP4X1, CYP4X1, CYP4 , PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, TEG2 , WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A11 , CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or -CYP46A mutated or defective that encodes a protein having defective or partial function or activity.
[105]
105. Composition according to claim 88, characterized by the fact that the target gene is CYP4V2.
[106]
106. Composition according to claim 105, characterized by the fact that the cell line comprises an iPS cell.
[107]
107. Composition according to claim 105, characterized by the fact that the cell line comprises an iPS-RPE cell.
[108]
108. Composition according to claim 105, characterized by the fact that the cell line comprises an iPS-photoreceptor cell (iPS-PRC), an iPS-epithelial corneal cell (iPS-CEC), a cell iPS-choroidal endothelial (CE), an iPS-corneal cell, an iPS-choroidal cell, an iPS-optic nerve cell, an iPS-ocular cell or an iPS-neuron cell.
[109]
109. Composition, according to any of claims 88 or 105 to 108, characterized by the fact that the CYP4V2 mutation in the cell line is endogenous to the individual.
[110]
110. Composition according to claim 88 or 109, characterized by the fact that the individual has a pathological mutation in the CYP4V2 gene or in an orthologist of the CYP4V2 gene.
[111]
111. Composition according to claim 88 or 109, characterized by the fact that the individual has at least one homozygous mutation or two compound heterozygous mutations shown in Table 1.
[112]
112. Composition according to claim 88, characterized by the fact that the individual has inherited retinal degeneration (IRD) or retinitis pigmentosa (RP).
[113]
113. Composition according to claim 88, characterized by the fact that the individual has Bietti's Crystalline Dystrophy (BCD, Bietti's Corneoretinal Crystalline Dystrophy, Bietti's Crystal Retinopathy, Bietti's Retinal Dystrophy) or is under risk of developing BCD.
[114]
114. Composition according to claim 88 or 105, characterized in that the cell line comprises at least one mutation of CYP4V2 that is exogenous to the individual and is introduced artificially through genetic editing or genetic manipulation.
[115]
115. Composition according to claim 88 or 114, characterized in that the cell line comprises an iPS cell, an ES cell, MSC, tissue-specific stem cell or adult stem cell, or a cell RPE, photoreceptor cell, corneal epithelial cell, choroidal endothelial cell (CE) or choroidal cell derived from an iPS cell, ES cell, MSC, tissue-specific stem cell or adult stem cell.
[116]
116. BCD human cell model composition or CYP4V2 dysfunction cell model composition, characterized by the fact that it comprises an iPS cell or iPS cell line or an iPS-RPE cell or derived iPS-RPE cell line from a cell or cell line from a patient with BCD or derived from a cell or cell line with an artificially created biallelic CYP4V2 mutation.
[117]
117. Composition according to any one of claims 88 or 105 to 115, characterized by the fact that the cell line has an abnormal biochemical profile in one or more compounds in the following groups of compounds: (i) fatty acids, (ii) ceramides, (iii) sphingomyelin, (iv) sphingosine, (v) sphinganine, (vi) hydroxy fatty acids, (vii) corticosteroid or (viii) proteins (except CYP4V2) or abnormal RPE function or level of greater cell atrophy, degeneration or death compared to a corresponding cell line for healthy control.
[118]
118. Composition, according to any of claims 88 or 105 to 117, characterized by the fact that the cell line has an abnormal biochemical profile in one or more compounds shown in Table 2 compared to the corresponding cell line of healthy control.
[119]
119. Abnormality or phenotype detection method in a disease cell model, characterized by the fact that it comprises assessment and comparison of cell viability levels of a patient's cell line (or a genetically edited or manipulated cell line comprising an exogenous mutation in the gene causing such a disease) and healthy control.
[120]
120. Abnormality or phenotype detection method in a cell model of disease, characterized by the fact that it comprises assessment and comparison of the levels of RPE function (for example, phagocytic activity, transepithelial resistance) of a patient's cell line ( or a genetically edited or manipulated cell line comprising an exogenous mutation in the gene causing such a disease) and a healthy control, where the cell line is an RPE cell line (including RPE cell line derived from a stem cell ).
[121]
121. Method according to claim 119 or 120, characterized by the fact that the comparison between cell lines is made without exposure to light.
[122]
122. Method according to claim 119 or 120, characterized by the fact that comparisons between cell lines are made after exposure to light.
[123]
123. Method according to claim 119, 120 or 122, characterized by the fact that comparisons between cell lines are made after exposure to blue light.
[124]
124. Method according to claim 119, 121, 122 or 123, characterized by the fact that cell viability is measured by the ratio of dead / living cell, ratio of sick / healthy cell or similar ratios or percentage of dead cells / totals or live / total cells.
[125]
125. Abnormality or phenotype detection method in a disease cell model characterized by the fact that it comprises assessment and comparison of the levels of one or more compounds between a patient's cell line (or a genetically edited cell line or manipulated comprising an exogenous mutation in the gene causing such a disease) and a healthy control, in which the one or more compound is selected from the groups that follow: (i) fatty acids, (ii) ceramides, (iii) sphingomyelin, (iv) sphingosine , (v) sphinganine, (vi) hydroxy fatty acids, (vii) corticosteroids and / or (viii) proteins.
[126]
126. Method according to claim 125 or 126, characterized by the fact that one or more of the compounds evaluated is shown in Table 2.
[127]
127. Method according to claim 125 or 126, characterized in that the identification and / or evaluation of compound levels is carried out using LC-MS, LC-MS / MS, GC-MS, GC-MS / MS and / or FIA-MS / MS.
[128]
128. Method according to claim 119, 120 or 125, characterized in that the cell model of disease comprises a mutated or defective gene shown in Table 4.
[129]
129. Method according to claim 119, 120 or 125, characterized in that the disease cell model comprises a mutated or defective gene among the CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15 gene , CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, TEG, SPG7, POLG, SPG7 , ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A2, CYP4A22, CYP4A22 , CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A.
[130]
130. Method of evaluating a test agent for therapeutic efficacy against BCD characterized by the fact that it comprises: contacting the cells of an iPS-RPE cell line derived from a patient with BCD or an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene as a result of editing or artificial genetic manipulation with a test agent; and evaluating cells for normalization at levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid sequence in cells; an increase in the amount of CYP4V2 polypeptides in cells; and / or improved cell structure, morphology or function, or improved cell viability, compared to before contact by such a test agent; where normalization to levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid sequence in cells; an increase in the amount of CYP4V2 polypeptides in cells; and / or improved cell structure, morphology, function or viability, compared to before treatment by such a test agent, is indicative of a test agent that exhibits therapeutic efficacy against BCD; in which the comparison between cell lines can be made after exposure to light and / or without exposure to light.
[131]
131. Method according to claim 130, characterized in that the test agents are selected from the group consisting of nucleic acids or analogues thereof, vectors containing nucleic acid sequence or encoding polypeptides, polypeptides or analogs thereof , antibodies, chemical agents, small molecules and / or any combination thereof.
[132]
132. Method, according to claim 130, characterized by the fact that cells are evaluated using PCR techniques, immunoassays, sequencing, biochemical assay, function assay, cell viability assay, microscopy or combination thereof.
[133]
133. Method of evaluating the effectiveness or efficiency of a formulation, vector or construct, characterized by the fact that it comprises a test agent for BCD characterized by the fact that it comprises:
contacting multiple cell samples from an iPS-RPE cell line derived from a patient with BCD or an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene as a result of artificial genetic manipulation or editing with a formulated testing agent or packaged in various formulations, vectors or constructs; and evaluate cell samples for normalization at levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid sequence in cells; an increase in the amount of CYP4V2 polypeptides in cells; improved cell structure, morphology, function or viability; and / or tolerance or cell death, compared to before treatment by such test agent and / or cell samples treated by the same test agent, but formulated or packaged in a different formulation, vector or construct, to determine and compare the efficiency or effectiveness of such formulation, vector or construct; in which cells are evaluated using PCR techniques, immunoassays, sequencing, biochemical assay, cell viability assay, microscopy or combination thereof; in which the comparison between cell lines can be made after exposure to light and / or without exposure to light.
[134]
134. Method of evaluating the effective and safe dosage range of a test agent for BCD characterized by the fact that it comprises: contacting multiple cell samples from an iPS-RPE cell line derived from a patient with BCD or a line iPS-RPE cell comprising a mutated or defective CYP4V2 gene as a result of editing or artificial genetic manipulation with a testing agent at a different dose for each cell sample;
evaluate cell samples for normalization at levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid sequence in cells; an increase in the amount of CYP4V2 polypeptides in cells; improved cell structure, morphology, viability or function; and / or tolerance or cell death, as compared before treatment by such test agent and / or cell samples treated by the same test agent, but with a different dose, to determine and compare the effectiveness and safety of different doses of this test agent. way by determining an appropriate dosage range; in which cells are evaluated using PCR techniques, immunoassays, sequencing, biochemical assay, function assay, cell viability assay, microscopy or combination thereof; in which the comparison between cell lines can be made after exposure to light and / or without exposure to light.
[135]
135. Screening method or evaluation of the efficacy or efficiency of a device or method of administration for administering a therapeutic agent to the retina or retinal cells, characterized by the fact that it comprises: (i) contact of a cell sample from a lineage iPS-RPE cell derived from a patient with BCD or an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene as a result of artificial genetic manipulation or editing with a test agent without use of the device or method of administration; (ii) contacting another cell sample from an iPS-RPE cell line derived from a patient with BCD or an iPS-RPE cell line comprising a mutated or defective CYP4V2 gene as a result of artificial genetic editing or manipulation with the agent testing the same dosage as in (i), using the device or method of administration; (iii) evaluation and comparison of cell samples from (i) and (ii) for normalization to levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid sequence in cells; an increase in the amount of CYP4V2 polypeptides in cells; improved cell structure, morphology or function; tolerance or cell death; and / or the levels of the test agent in the cells, compared to before treatment by such test agent and / or treatment by the same test agent of the same dose but without the use of the device or methods of administration, to determine the effectiveness or efficiency such a device or administration technique; in which cells are evaluated using PCR techniques, immunoassays, sequencing, biochemical assay, function assay, cell viability, microscopy or combination thereof; in which the comparison between cell lines can be made after exposure to light and / or without exposure to light.
[136]
136. Method according to claim 135, characterized by the fact that the retinal cells are RPE cells.
[137]
137. Method of generating an isogenic control characterized by the fact that it comprises genetically correcting the mutation in a patient's cell line through any of the claims in gene editing therapy or present RNP claims.
[138]
138. Method of using patient-specific iPS-ocular cells to evaluate or suggest effective therapeutic dosage for in vivo treatment characterized by the fact that it comprises multiplying the optimal dose level (for example, indicated as MOI for gene therapy in vivo) in vitro) determined in a patient-specific iPS-ocular cell model in vitro by the estimated number of ocular cells (for example, RPE cells, photoreceptor cells or RPE cells and photoreceptor cells) targeted for in vivo treatment to reach the dose level of gene therapy vectors for in vivo use (eg GC or gp) and such a vector dose level is adjusted by a multiplier (eg 1 to 10) (eg 1 to 5 for sub-retinal injection or 5 to 10 for intravitreal injection); the other factors affecting the multiplier to be applied include the size of the area targeted, and the individual being treated (eg, age, weight, stage of disease development and condition of the individual to be treated and potential immune reactions (ie, pre-existing NAbs); the location and density of eye cells targeted for treatment) or suggest the range of effective therapeutic dose for in vivo treatment, which can be confirmed or further refined by clinical tests.
[139]
139. Method according to claim 138, characterized by the fact that the method is used to assess or suggest an optimal personalized dose used in in vivo treatment for an individual patient.
[140]
140. Method according to claim 138 or 139, characterized in that the disease is an inherited retinal disorder (IRD) or retinitis pigmentosa (RP).
[141]
141. Method according to claim 138 or 139, characterized in that the disease is BCD and the iPS-ocular cells are iPS-RPE cells and the eye cells targeted for in vivo treatment are RPE cells.
[142]
142. Composition characterized by the fact that it comprises: (a) a CRISPR guide RNA targeting a nucleic acid sequence (the “target sequence”) of or within 100 bps for the CYP4V2 gene and (b) a protein associated with functional CRISPR (Cas).
[143]
143. Composition according to claim 142, characterized in that it further comprises (c) a donor nucleic acid sequence comprising all or a portion of a wild-type sequence or a functional sequence of the CYP4V2 gene for correction, disruption or replacement of the CYP4V2 gene or a portion of it.
[144]
144. Composition according to claim 142 or 143, characterized in that one or more components of the same are provided in the form of a DNA molecule encoding such a component, an mRNA molecule encoding such a component, an RNA molecule, a polypeptide and / or a ribonucleoprotein (RNP) or protein-RNA complex.
[145]
145. Composition according to claim 142 or 143, characterized in that two or more components of the same are in a separate molecule or combined in a molecule or in a complex, are in separate vectors or combined in one vector, they are in one or more nucleic acid complexes, they are in one or more RNP complexes.
[146]
146. Composition according to claim 143, characterized by the fact that the donor nucleic acid sequence is provided in a single-stranded donor oligonucleotide (ssODN) or a vector.
[147]
147. Composition according to any one of claims 142 to 146, characterized by the fact that the vector is a plasmid, a recombinant AAV vector, a recombinant lentivirus vector and / or a combination thereof .
[148]
148. Composition characterized by the fact that it comprises a cell with a pathological CYP4V2 mutation comprising a composition of any one of claims 142 to
147.
[149]
149. Composition according to any one of claims 142 to 148, characterized by the fact that (a) the CRISPR guide RNA comprises (i) a CRISP RNA (crRNA) that comprises a protospace element sequence that is complementary to the target sequence of or within 100 bps to a target gene (the “target gene”) and a sequence that corresponds to a complementary region of the transactivation crRNA (tracrRNA) and (ii) a tracrRNA that comprises it contains a region that is complementary to the corresponding region of the crRNA and a sequence that interacts with a protein associated with CRISPR 9 (Cas9) and (b) the protein associated with functional CRISPR comprises Cas9.
[150]
150. Composition according to claim 149, characterized by the fact that the protospacer element is about 20 bases, about 19 bases, about 21 bases, about 19-21 bases, about 18 -22 bases or about 16-24 bases.
[151]
151. Composition according to claim 149 or 150, characterized in that the crRNA and tracrRNA are in separate molecules.
[152]
152. Composition according to claim 149 or 150, characterized by the fact that crRNA and tracrRNA are combined into a single guide RNA (sgRNA).
[153]
153. Composition according to claim 152, characterized by the fact that the sgRNA is about 88-150 pbs.
[154]
154. Composition, according to any one of claims 149 to 153, characterized by the fact that Cas9 comprises a Cas9 or Cas9 mutant orthologist selected from: Streptococcus pyogenes (SpCas9), SpCas9 nickase (Cas9n D10A ), SpCas9 (D1135E), eSpCas9, SpCas9-HF1, SpCas9 VRER, SpCas9 VQR, SpCas9EQR, Staphylococcus aureus (SaCas9), Neisseria Meningitis, Streptococcus thermophilus, Streptococcus pneumnoniae,
Campylobacter coli, Campylobacter jejuni, Streptococcus mutans, Pasteurella multocida, Bifidobacterium longum, Ba-cillus smithii, Treponema denticola, mycoplasma canis and enterococcus fae-calis.
[155]
155. Composition according to any one of claims 142 to 148, characterized by the fact that (a) the CRISPR guide RNA comprises a crRNA that comprises a protospacer element sequence that is complementary to the target sequence of or out of 100 pbs for a target gene and (b) the protein associated with functional CRISPR comprises Cpf1.
[156]
156. Composition according to any of claims 142 to 155, characterized by the fact that the protein associated with CRISPR, Cas9, or Cpf1, further comprises one, two, three or more nuclear localization sequences (NLS ) at the N and / or C terminal, and / or a selection marker, including, without limitation, GFP or EGFP.
[157]
157. Composition according to claim 142, characterized by the fact that the sequence of the protospacer element is selected from the group consisting of SEQ ID NOs: 48 to 52 or shares at least 85% sequence identity with any one of SEQ ID NO: 48 to 52 for use with a Cas protein that has NGG as an adjacent protospacer motif (PAM) to target the c.802-8_810del17insGC mutation of the CYP4V2 gene.
[158]
158. Composition according to claim 142, 143 or 157, characterized in that the donor nucleic acid sequence is selected from SEQ ID NOs: 56 and 57 or shares at least 90% sequence identity with one of SEQ ID NO: 56 or 57, or a sequence that is complementary to it, for use to correct, disrupt or replace the c.802-8_810del17insGC mutation of the CYP4V2 gene.
[159]
159. Method of treatment or prevention of BCD in an individual or a cell with a mutated CYP4V2 gene characterized by the fact that it comprises: (i) identification of the pathological mutation in the individual or in the cell through sequencing; (ii) finding of PAM sites related to Cas within the region extending from about 100 bps upstream of the first nucleotide involved in the mutation to about 100 bps downstream of the last nucleotide involved in the mutation; (iii) identification of several proto-spaether sequences targeting the relevant CYP4V2 sequence for each MAP site identified in (ii); (iv) evaluation of the activity level of each CRISPR guide RNA comprising a sequence of protospacer element identified in (iii) and an out-of-target editing profile based on the sequence of protospacer element and MAP; (v) selection of one or more CRISPR guide RNA projects based on (iv); (vi) design of one or more donor nucleic acid sequences based on homology-directed repair (HDR) to correct, disrupt or replace the targeted CYP4V2 mutation; (vii) constructing the CRISPR, Cas or donor guide RNA nucleic acid sequence as provided in composition claims 1 to 18; (viii) optionally validation and additional selection of the components of (vii) in an isolated cell of the individual; either an iPS cell derived from the individual or a cell differentiated from a stem cell derived from the individual or the genomic DNA isolated from the individual to assess the level of activity and / or editing profile outside the target; and (ix) administration of the components in (viii) to the individual or to the cell through a delivery system selected from the group consisting of a ribonucleoprotein or protein-RNA complex, a vector, a protein, a nucleic acid molecule, a nanoparticle, a liposome, a micelle, a virosome, a nucleic acid complex, and / or a combination thereof, where administration is carried out by electroporation or by lipid-mediated transfection or nucleofection or viral transduction or injection or a combination thereof.
[160]
160. Genetic editing composition to correct or replace the c.802-8_810del17insGC mutation in a CYP4V2 gene in an in vivo individual or in an in vitro cell characterized by the fact that it comprises: (i) a CRISPR guide RNA comprising a protospace element sequence selected from one of SEQ ID NOs: 48 to 52 or sharing at least 80% sequence identity with one of the sequences in SEQ ID NOs: 48 to 52; (ii) a donor nucleic acid sequence selected from one of SEQ ID NOs: 56 and 57 or shares at least 90% sequence identity with one of SEQ ID NOs: 56 and 57, or a sequence that is complementary to it; and (iii) a Cas9 protein (exemplary sequence shown in SEQ ID NO: 58), optionally containing 1, 2, 3 or more NLS, and / or a selection marker including, without limitation, GFP or EGFP.
[161]
161. Composition according to claim 160, characterized by the fact that an optional G nucleotide (SEQ ID NO: 59) is added before the protospace element sequence.
[162]
162. Composition according to claim 160 or 161, characterized in that the CRISPR guide RNA comprises a crRNA (exemplary sequence (excluding the sequence of the 5 'protospacer element) shown in SEQ ID NO: 53) and a tracrRNA
(exemplary sequence shown in SEQ ID NO: 54); and the proto-space element sequence is contained in the crRNA.
[163]
163. Composition according to any one of claims 160 to 162, characterized by the fact that the CRISPR guide RNA comprises a single guide RNA (sgRNA) comprising the protospacer element sequence (example sgRNA sequence) - plar (excluding the 5 'protospace element sequence) shown in SEQ ID NO: 55).
[164]
164. Composition according to any one of claims 160 to 163, characterized by the fact that one or more components of (i), (ii) and (iii) are provided in the form of a DNA molecule encoding such a component, an mRNA molecule encoding such a component, a nucleic acid molecule, a vector, an RNA molecule, a polypeptide, a ribonucleoprotein (RNP) or a protein-RNA complex and / or combinations thereof.
[165]
165. Method of treatment or prevention of an eye disease in an individual, in which the disease is associated with a pathological genetic or epigenetic change in the CYP4V2 gene, characterized by the fact that it comprises administering a cell composition to the individual, in which the cell composition comprises: retinal pigment epithelium (RPE) cells, photoreceptor or photoreceptor progenitor cells (PRCs), corneal epithelial cells (SCCs), choroidal endothelial cells (SC) and / or other ocular or other cells cells derived from a stem cell.
[166]
166. Method according to claim 165, characterized by the fact that the stem cell is an embryonic stem cell (ES), an iPC cell, an MSC, an adult stem cell or a cell. specific tissue trunk.
[167]
167. Method according to claim 165, characterized by the fact that the stem cell is from or derived from one or more individuals without BCD or without a pathological CYP4V2 gene.
[168]
168. Method according to claim 165, characterized by the fact that the stem cell is from or derived from one or more individuals with pathological mutations in the CYP4V2 gene.
[169]
169. Method according to any of claims 165 to 168, characterized by the fact that the individual is a human individual.
[170]
170. Cell composition characterized by the fact that it comprises (a) a stem cell reprogrammed from a cell isolated from or a stem cell isolated from an individual affected by BCD or having pathological mutations in the CYP4V2 gene or (2) a cell differentiated from a stem cell isolated from an individual or reprogrammed from a cell isolated from an individual affected by BCD or having pathological mutations in the CYP4V2 gene.
[171]
171. Composition according to claim 170, characterized by the fact that the stem cell reprogrammed from an isolated cell of the individual is an iPC cell.
[172]
172. Composition according to claim 170, characterized by the fact that the iPS cell is reprogrammed from an individual's somatic cell.
[173]
173. Composition according to claim 170, characterized by the fact that the iPS cell is reprogrammed from a skin cell, a blood cell, a urinary cell, a hair cell, a fibroblast, a mononuclear cell of peripheral blood (PBMC), a renal epithelial cell, a hair follicle or a dermal papillary cell.
[174]
174. Composition according to claim 170, characterized by the fact that the individual's isolated stem cell is an MSC, an adult stem cell or a specific tissue stem cell.
[175]
175. Composition according to claim 170, characterized by the fact that the cell differentiated from a stem cell is an ocular cell.
[176]
176. Composition according to claim 170 or 175, characterized in that the differentiated cell of a stem cell is an RPE cell, a PRC, a retinal cell, a corneal cell, a cell choroidal, a CEC or a CE cell.
[177]
177. Composition according to claim 170, characterized by the fact that the differentiated cell of a stem cell is an iPS-RPE, iPS-PRC, iPS-CEC or iPS-CE cell.
[178]
178. Composition according to claim 170, characterized by the fact that (i) the cell isolated from an individual affected by BCD or having pathogenic mutations in the CYP4V2 gene for use to reprogram in an iPSC, (ii) the stem cell isolated from an individual or iPS cell reprogrammed from a cell isolated from an individual affected by BCD or having pathological mutations in the CYP4V2 gene or (iii) the cell differentiated from a stem cell isolated from an individual or a cell iPS reprogrammed from a cell isolated from an individual affected by BCD or having pathological mutations in the CYP4V2 gene is genetically repaired to improve the effect of the mutated CYP4V2 gene.
[179]
179. Composition according to claim 178, characterized by the fact that genetic repair is through gene transfer therapy.
[180]
180. Composition according to claim 178, characterized by the fact that genetic repair is through gene transfer therapy using any composition or method of any of the gene therapy claims.
[181]
181. Composition, according to any of the preceding claims, characterized by the fact that genetic repair
co is through gene editing therapy.
[182]
182. Composition according to claim 181, characterized by the fact that genetic repair is through gene editing therapy through the use of any composition or method of any of the CRISPR gene therapy claims.
[183]
183. Method of treatment or prevention of an eye disease in an individual affected by BCD or having pathological genetic or epigenetic changes in the CYP4V2 gene, characterized by the fact that it comprises administering a composition of any one of the claims related to autologous cellular compositions - CYP4V2 gas to the individual, in which the cellular composition comprises: genetically repaired cells comprising retinal pigment epithelium (RPE) cells, photoreceptors or photoreceptor progenitors (PRCs), corneal epithelial cells (CECs), en cells - choroidal dothelial (EC) and / or other eye cells or other cells derived from an individual's stem cell.
[184]
184. Method according to claim 183, characterized by the fact that the stem cell is an iPC cell, an MSC, an adult stem cell or a tissue-specific stem cell.
[185]
185. Method according to claim 184, characterized by the fact that the iPS cell is reprogrammed using one or more of the OCT4, SOX2, KLF4 and c-MYC transcription factors.
[186]
186. Method, according to any of the preceding claims, characterized by the fact that the genetically repaired cells demonstrate one or more of the following: normalization to levels of one or more compounds shown in Table 2; an increase in non-defective CYP4V2 nucleic acid sequence in cells; an increase in the amount of functional CYP4V2 polypeptides in cells; and / or improved cell structure, morphology, viability, or function, compared to before genetic repair was performed
used.
[187]
187. Method, according to any of the preceding claims, characterized by the fact that the amount of cells administered is about 1,000 to about 10 million cells in a single administration.
[188]
188. Method, according to any of the preceding claims, characterized by the fact that the administration is by injection, sub-retinal injection or intravitreal injection.
[189]
189. Method according to any one of the preceding claims, characterized by the fact that the administration is through another method of administration that effectively delivers the cells to the sub-retinal site, the posterior segment or the cornea of the individual's eye .
[190]
190. Method, according to any of the preceding claims, characterized by the fact that the cells are administered through injection of cell suspension.
[191]
191. Method according to any one of the preceding claims, characterized by the fact that the cells are administered as part of a 3D sheet, matrix, base, tissue or retinal structure.
[192]
192. Method according to any one of the preceding claims, characterized in that the RPE cells are administered using natural and / or synthetic bases to generate a functional RPE monolayer.
[193]
193. Method, according to any of the preceding claims, characterized by the fact that the individual is a human individual.
[194]
194. Cell composition characterized by the fact that it comprises (a) a stem cell reprogrammed from an isolated cell from or an isolated stem cell from an individual affected by a disease caused by a mutated or defective gene in a gene encoding a protein having defective or partial function or activity or (2) a cell differentiated from a stem cell isolated from an individual or reprogrammed from a cell isolated from an individual affected by a disease caused by a mutated gene or defective or gene encoding a protein having defective or partial function or activity.
[195]
195. Composition according to claim 194, characterized by the fact that the stem cell reprogrammed from an isolated cell of the individual is an iPS cell.
[196]
196. Composition according to claim 195, characterized by the fact that the iPS cell is reprogrammed from an individual's somatic cell.
[197]
197. Composition according to claim 195 or 196, characterized in that the iPS cell is reprogrammed from a skin cell, a blood cell, a urinary cell, a hair cell, a fibroblast, a cell peripheral blood mononuclear (PBMC), a renal epithelial cell, a hair follicle or a dermal papillary cell.
[198]
198. Composition according to claim 194, characterized by the fact that the individual's isolated stem cell is an MSC, an adult stem cell or a specific tissue stem cell.
[199]
199. Composition according to claim 194, characterized by the fact that the gene is involved in ocular development or function and / or mutation which causes or is a risk factor for causing an eye disease.
[200]
200. Composition according to claim 194, characterized by the fact that the gene is involved in neuronal development or function and / or mutation which causes or is a risk factor for causing a neurodegenerative disease.
[201]
201. Composition according to claim 194, characterized by the fact that the gene is a cytochrome P450 gene.
[202]
202. Composition according to claim 194, characterized by the fact that the gene is one shown in Table 4.
[203]
203. Composition according to claim 194, characterized by the fact that the gene comprises a CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C1265 gene , CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1, RPEE, , RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F11, CYP4F8, , CYP4Z1 or CYP46A gene, or a CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOX, LT, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A mutated or defective.
[204]
204. Composition, according to any of the preceding claims, characterized by the fact that the cell differentiated from a stem cell is any type of cell.
[205]
205. Composition, according to any of the preceding claims, characterized by the fact that the cell differs
stem cell is an eye cell.
[206]
206. Composition, according to any of the preceding claims, characterized by the fact that the cell differentiated from a stem cell is an RPE cell, a PRC, a retinal cell, a corneal cell, a choroidal cell, a CPB, a CE cell or an optic nerve cell.
[207]
207. Composition, according to any of the preceding claims, characterized by the fact that the cell differentiated from a stem cell is an iPS-RPE, iPS-PRC, iPS-CEC or iPS-CE cell .
[208]
208. Composition, according to any of the preceding claims, characterized by the fact that the cell differentiated from a stem cell is a neuron.
[209]
209. Composition, according to any of the preceding claims, characterized by the fact that (i) the isolated cell of an individual affected by a disease caused by a mutated or defective gene or a gene encoding a protein having a defective or partial function or activity for use to reprogram in an iPSC, (ii) the stem cell isolated from an individual or iPS cell reprogrammed from a cell isolated from an individual affected by a disease caused by a mutated gene, or defective or a gene encoding a protein having defective or partial function or activity, or (iii) the cell differentiated from an individual stem cell isolated or an iPS cell reprogrammed from an isolated cell from an individual affected by a disease caused by a mutated or defective gene or a gene encoding a protein having defective or partial function or activity is genetically repaired to improve the effect of the mutated or defective gene.
[210]
210. Composition, according to any of the preceding claims, characterized by the fact that genetic repair
co is through gene transfer therapy.
[211]
211. Composition, according to any of the preceding claims, characterized by the fact that genetic repair is through gene transfer therapy using any composition or method of any of the claims related to gene therapy.
[212]
212. Composition, according to any of the preceding claims, characterized by the fact that genetic repair is through gene editing therapy.
[213]
213. Composition, according to any of the preceding claims, characterized by the fact that genetic repair is through gene editing therapy using any composition or method of any of the claims related to CRISPR gene editing therapy.
[214]
214. Method of treating or preventing a disease in an individual affected by a disease caused by a mutated or defective gene or a gene encoding a protein having a defective or partial function or activity shown in Table 4 characterized by the fact that it comprises administering a cell composition of any of the claims related to an autologous cell composition to the individual, wherein the cell composition comprises: genetically repaired cells comprising retinal pigment epithelium (RPE) cells, photoreceptors or photoreceptor progenitors (PRCs), corneal epithelial cells (SCCs), neurons, choroidal endothelial cells (CE) and / or other eye cells or other cells derived from an individual's stem cell, and in which the mutated gene or defective in the cellular composition has been genetically repaired.
[215]
215. Method of autologically treating an individual characterized by the fact that he understands:
(h) providing cells from an individual having an eye disease; (ii) induce pluripotency in the individual's cells to produce iPSCs; (iii) genetically repairing one or more mutations in a mutated or defective gene shown in Table 4 in the iPSCs derived from the individual through gene addition therapy; (iv) differentiate iPSCs in ocular cells or other cells as needed for replacement transplantation; (v) alternative to step (iii), to genetically repair cells derived from iPS through gene transfer therapy; and (vi) introducing the iPS-derived cells into the individual, thereby autologously treating the individual having the disease associated with a mutated or defective gene (the "target gene").
[216]
216. Method according to claim 214 or 215, characterized in that the stem cell is an iPC cell, an MSC, an adult stem cell or a specific tissue stem cell.
[217]
217. Method according to claim 216, characterized by the fact that the iPS cell is reprogrammed using one or more of the OCT4, SOX2, KLF4 and c-MYC transcription factors.
[218]
218. Method according to claim 214 or 215, characterized in that the genetically repaired cells demonstrate one or more of the following: an increase in non-defective target gene nucleic acid sequence in the cells; an increase in the amount of functional polypeptides encoded by the target gene in cells; improved structure, morphology, viability or function and / or improved or normalized biochemical function in cells, compared to before genetic repair is performed.
[219]
219. Method, according to any of the preceding claims, characterized by the fact that the amount of cells
administered squid is about 1,000 to about 10 million cells in a single administration.
[220]
220. Method, according to any of the preceding claims, characterized by the fact that the administration is through injection, sub-retinal injection or intravitreal injection.
[221]
221. Method according to any of the preceding claims, characterized by the fact that the administration is by any other method of administration that effectively delivers the cells to the sub-retinal site, the posterior segment or the cornea of the eye. individual or tissue or organ in need of replacement cells.
[222]
222. Method, according to any of the preceding claims, characterized by the fact that the cells are administered through injection of cell suspension.
[223]
223. Method, according to any of the preceding claims, characterized by the fact that the cells are administered as part of a sheet, a matrix, a base, a tissue or a 3D retinal structure.
[224]
224. Method according to any of the preceding claims, characterized by the fact that RPE cells are administered using natural and / or synthetic bases to generate a functional RPE monolayer.
[225]
225. Method, according to any of the preceding claims, characterized by the fact that the individual is a human individual.
[226]
226. Method, according to any of the preceding claims, characterized by the fact that the disease is associated with a genetic or epigenetic alteration or risk factor in the individual in a gene shown in Table 4.
[227]
227. Method according to any one of the claims
previous conditions, characterized by the fact that the disease is photoreceptor degeneration, retinal pigment epithelium cell degeneration, retinal degeneration, corneal degeneration and / or choroidal disorders.
[228]
228. Method, according to any of the preceding claims, characterized by the fact that the disease is an inherited retinal degeneration (IRD).
[229]
229. Method, according to any of the preceding claims, characterized by the fact that the disease is retinitis pigmentosa (RP).
[230]
230. Method, according to any of the preceding claims, characterized by the fact that the disease is Bietti's Crystalline Dystrophy (also known as Bietti's Coronororetinal Crystalline Dystrophy; BCD).
[231]
231. Method, according to any of the preceding claims, characterized by the fact that the disease is related to neurological degeneration.
[232]
232. Method, according to any of the preceding claims, characterized by the fact that the disease is corneal dystrophy.
[233]
233. Method, according to any of the preceding claims, characterized by the fact that the individual has BCD or is at risk of developing BCD.
[234]
234. Composition, according to any of the preceding claims, characterized by the fact that the cells are fibroblasts, blood cells or eye cells.
[235]
235. Composition, according to any of the preceding claims, characterized by the fact that the cells are obtained from urine or hair or hair follicles.
[236]
236. Composition, according to any of the claims
preceding indications, characterized by the fact that the ocular cells are retinal pigment epithelial cells (RPE), corneal epithelial cells (SCCs), choroidal endothelial cells (SC) or phororreceptor cells (PRCs).
[237]
237. Composition, according to any of the preceding claims, characterized by the fact that the genetic or epigenetic alteration is selected from the group consisting of a mutation, an insertion, a single nucleotide polymorphism, improper methylation, improper demethylation and combinations thereof.
[238]
238. Composition, according to any of the preceding claims, characterized by the fact that the genetic or epigenetic alteration is a mutation.
[239]
239. Composition, according to any of the preceding claims, characterized by the fact that the genetic or epigenetic alteration in the individual's iPS-ocular cells has been genetically repaired using gene editing therapy.
[240]
240. Composition, according to any of the preceding claims, characterized by the fact that the genetic editing technique method uses a zinc-finger nuclease, TALEN technology or CRISPR technology.
[241]
241. Composition, according to any of the preceding claims, characterized by the fact that the genetic or epigenetic alteration in the individual's iPSC-ocular cells was genetically repaired using gene transfer therapy.
[242]
242. Composition, according to any of the preceding claims, characterized by the fact that the gene transfer therapy method uses a recombinant AAV vector or another viral or non-viral vector to deliver a healthy copy of target gene (eg cDNA) to the cells to be transplanted.
[243]
243. Method, according to any of the preceding claims, characterized by the fact that the administration stage takes place before the onset of symptoms of the disease or after the onset of symptoms of the disease.
[244]
244. Method according to any of the preceding claims, characterized by the fact that the administration is to the eye or another organ or tissue comprising neurons.
[245]
245. Method, according to any of the preceding claims, characterized by the fact that the administration is by injection, sub-retinal or intravitreal injection, direct retinal injection, or through intravitreal implant of an encapsulating device. of the vector.
[246]
246. Method according to any one of the preceding claims, characterized by the fact that administration is by any other method of administration that effectively delivers cells to the sub-retinal site, the posterior segment or the cornea of the eye individual or tissue or organ in need of replacement cells.
[247]
247. Method, according to any of the preceding claims, characterized by the fact that it also comprises, before administration or transplantation, genotypic analysis in cells to identify the presence or absence of the genetic or epigenetic alteration in a or more genes shown in Table 4.
[248]
248. Method, according to any of the preceding claims, characterized by the fact that the genetic or epigenetic alteration is a mutation.
[249]
249. Method, according to any of the preceding claims, characterized by the fact that the mutation is in the CYP4V2 nucleic acid molecule.
[250]
250. Method according to any of the claims
preceding instructions, characterized by the fact that it comprises before the administration, evaluation of the individual's eye to identify the area (s) and extent of photoreceptors, retinal cells or damaged or retained corneal cells.
[251]
251. Method, according to any of the preceding claims, characterized by the fact that it also includes, following administration, monitoring of the individual.
[252]
252. Method, according to any one of the preceding claims, characterized by the fact that monitoring comprises performing non-invasive retinal images, corneal tests, dark adaptation, contrast sensitivity, perimetry, ERG, OCT, visual acuity and / or functional studies.
[253]
253. Method, according to any of the preceding claims, characterized by the fact that monitoring comprises an individual's assessment of an immune response.
[254]
254. Method, according to any of the preceding claims, characterized by the fact that it also comprises, following administration, evaluation of the individual's eye to identify the area (s) and extent of photoreceptors, retinal cells or cells cornea damaged or retained.
[255]
255. Composition characterized by the fact that it comprises: (a) a CRISPR guide RNA targeting a nucleic acid sequence (the “target sequence”) of or within 100 bps for a target gene (the “ target gene ”) and (b) a functional CRISPR-associated protein, in a ribonucleoprotein (RNP) or protein-RNA complex.
[256]
256. Composition according to claim 255, characterized in that it comprises (c) a donor nucleic acid sequence comprising all or a portion of a wild-type sequence or a functional sequence of the target gene for correction or replacement of such target gene or a portion thereof.
[257]
257. Composition according to claim 255 or 256, characterized by the fact that the target gene is involved in ocular development or function and / or mutation which causes or is a risk factor for causing an eye disease.
[258]
258. Composition according to claim 255 or 256, characterized by the fact that the target gene is involved in neuronal development or function and / or mutation which causes or is a risk factor for causing a neurodegenerative disease.
[259]
259. Composition according to claim 255 or 256, characterized by the fact that the target gene is cytochrome P450 gene.
[260]
260. Composition according to claim 255 or 256, characterized in that the target gene comprises a gene shown in Table 4 that is mutated or defective or encodes a protein having defective or partial function or activity.
[261]
261. Composition according to claim 256, characterized by the fact that the nucleic acid sequence is provided in a single-stranded donor oligonucleotide (ssODN) or a vector.
[262]
262. Composition, according to any one of claims 255 to 261, characterized by the fact that (a) the CRISPR guide RNA comprising (i) a CRISPR RNA (crRNA) comprising an element sequence protospacer that is complementary to the target sequence of or within 100 bps for a target gene and a sequence that corresponds to a complementary region of the transactivation crRNA (tracrRNA) and (ii) a tracrRNA that comprises a region that is complementary to corresponding regions of the crRNA and a sequence that interacts with a protein 9 associated with
CRISPR (Cas9) and (b) the protein associated with functional CRISPR comprises Cas9.
[263]
263. Composition according to claim 261 or 262, characterized in that the crRNA and tracrRNA are in different nucleic acid molecules.
[264]
264. Composition according to claim 261 or 262, characterized by the fact that crRNA and tracrRNA are combined into a single guide RNA (sgRNA).
[265]
265. Composition, according to any of claims 261-264, characterized by the fact that Cas9 comprises a Cas9 orthologist or a mutant Cas9 selected from: Streptoococcus pyogenes (SpCas9), SpCas9 nickase (Cas9n D10A) , SpCas9 (D1135E), eS-pCas9, SpCas9-HF1, SpCas9 VRER, SpCas9 VQR, SpCas9EQR, Sta-phylococcus aureus (SaCas9), Neisseria Meningitidys, Streptococcus thermophilus, Streptococcus pneumoci Pasteurella multocida, Bifidobacterium longum, Bacillus smithii, Treponema denticola, mycoplasma canis and enterococcus faecalis.
[266]
266. Composition according to any one of claims 255 to 265, characterized by the fact that (a) the CRISPR guide RNA comprises a crRNA that comprises a sequence of protospacer element that is complementary to the target sequence of or within 100 bps for a target gene and (b) the functional CRISPR-associated protein comprises Cpf1.
[267]
267. Composition, according to any one of claims 255 to 266, characterized by the fact that the protein associated with CRISPR, Cas9 or Cpf1 further comprises one, two, three or more nuclear localization sequences (NLS) in the terminal N and / or terminal C and / or a selection marker, including, without limitation, GFP or EGFP.
[268]
268. Composition according to any one of claims 255 to 267, characterized by the fact that the proto-spaether element is 100% complementary to the target sequence or contains 1, 2, 3, 4 or 5 nucleotide incompatibilities with the target string.
[269]
269. Composition according to claim 255 or 256, characterized in that the crRNA sequence further comprises a G nucleotide optionally added to the crRNA sequence immediately before the protospace element.
[270]
270. Composition according to any one of claims 255 to 269, characterized by the fact that the CRISPR guide RNA, crR-NA and / or tracrRNA, or sgRNA, is chemically modified.
[271]
271. Composition according to any one of claims 255 to 270, characterized by the fact that the wild-type version of the target gene encoded is an enzyme.
[272]
272. Composition according to claim 255 or 256, characterized in that the target gene comprises a gene CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH , C12orf65, CISD2, FOXC1, FOXF2, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1, ABPE , PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F4, CYP4F3, CYP4F3 , CYP4X1, CYP4Z1 or CYP46A gene or a CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, CXOR, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1,
RPE65, CEP290, PDE6B, RPGR, MERTK, MT-ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4F3, CYP4F4, CYP4 CYP4F12, CYP4F22, CYP4X1, CYP4Z1 or CYP46A mutated or defective that encodes a protein having defective or partial function or activity.
[273]
273. Composition according to any one of claims 255 to 272, characterized by the fact that any one or more components thereof including the CRISPR guide RNA, CRISPR-associated protein, and / or the sequence of donor nucleic acid, is provided separately and / or additionally in a vector, a DNA and / or an mRNA that can transcribe and / or translate into such a component.
[274]
274. Pharmaceutically acceptable formulation characterized by the fact that it comprises the composition as defined in claim 255 or 273.
[275]
275. Method of treating an individual's disease caused by a mutated or defective gene, or a gene encoding a protein having defective or partial function or activity, characterized by the fact that it comprises administering to the individual a composition as defined in any one of claims 255 to
274.
[276]
276. Method of treatment of an eye disease or improvement of a risk factor associated with that of an individual caused by a mutated or defective gene, or a gene encoding a protein having defective or partial function or activity, characterized by the fact that it comprises administering to the individual a composition as defined in any one of claims 255 to 157.
[277]
277. Method of treatment of a neurodegenerative disease or improvement of a risk factor related to it of an individual
caused by a mutated or defective gene, or a gene encoding a protein having defective or partial function or activity, characterized in that it comprises administering to the individual a composition as defined in any one of claims 255, 256 or 258-276 .
[278]
278. Method of treating a disease or improving an individual's related risk factor caused by a mutated or defective cytochrome P450 gene, or a cytochrome P450 gene encoding a protein having a defective or partial function or activity , characterized by the fact that it comprises administering to the individual a composition as defined in any one of claims 255 to 277.
[279]
279. The method of claim 278, characterized by the fact that the mutated or defective gene, or gene encoding a protein having defective or partial, broken, corrected or replaced function or activity, is a mutated version or defective gene shown in Table 4 or a version of a gene shown in Table 4 that encodes a protein having defective or partial function or activity.
[280]
280. Method according to claim 278, characterized by the fact that the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is present in fibroblasts, blood cells, RPE, photoreceptors, retinal, corneal, choroidal, ocular, optic nerve, neuron or trunk or any type of cells derived from a stem cell.
[281]
281. Method according to claim 278 or 279, characterized in that the composition is administered to fibroblasts, blood cells, RPE, photoreceptors, retinal, corneal, choroidal, ocular, nerve optical, neuron or stem cells or any type of cells derived from a stem cell.
[282]
282. Method according to claim 281, characterized by the fact that administration is carried out by electroporation or by lipid-mediated transfection or nucleofection or viral transduction or injection or a combination thereof.
[283]
283. Method according to any of claims 278 to 282, characterized in that any one or more components thereof including the CRISPR guide RNA, CRISPR-associated protein and / or the donor nucleic acid sequence it is administered to the individual or cells via a delivery system selected from the group consisting of a ribonucleoprotein or protein-RNA complex, a nanoparticle, a liposome, a micelle, a virosome, a nucleic acid complex and / or a combination thereof.
[284]
284. Method according to any of claims 278 to 283, characterized by the fact that the treatment is carried out for an individual in vivo.
[285]
285. Method according to any of claims 278-283, characterized in that the treatment is carried out in vitro on fibroblasts, blood cells, RPE, photoreceptor, retinal, corneal, choroidal, ocular, nerve optical, neuron or stem cells or any type of cells derived from a stem cell.
[286]
286. Method according to claim 285, characterized by the fact that the treated cells are transplanted to an individual in vivo, or if the treated cell is a stem cell, such a stem cell is differentiated in the desired type of cells for transplantation and then the differentiated cells are transplanted to an individual in vivo.
[287]
287. Method according to any of claims 278 to 280, characterized by the fact that the mutated or de-
feitous, or gene encoding a protein having defective or partial function or activity, is replaced.
[288]
288. Method according to any of claims 278 to 280, characterized in that the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, has one or more mutations corrected or replaced.
[289]
289. Method according to any one of claims 278 to 280, characterized in that the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is disrupted.
[290]
290. Method according to any one of claims 278 to 280, characterized in that the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, has 1-20, 21- 40, 41-60, 61-80, 81-100, 101- 1000, 1001-10000 base pairs of broken, corrected or replaced nucleotides or mutations.
[291]
291. Method according to any of claims 278 to 280, characterized in that a region of the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is disrupted, corrected or replaced.
[292]
292. Method according to any one of claims 278 to 280, characterized in that a region of less than about 10, 8, 6, 4, 2 or 1 kb of the mutated or defective gene, or gene encoding a protein having a defective or partial function or activity, it is disrupted, corrected or replaced.
[293]
293. Method according to any one of claims 278 to 280, characterized in that the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is disrupted, corrected or replaced by nucleotide insertion and / or deletion.
[294]
294. Method according to any one of claims 278 to 280, characterized in that the mutated or defective gene, or gene encoding a protein having defective or partial function or activity, is disrupted, corrected or replaced in one allele or both alleles.
[295]
295. Method according to any of claims 278 to 280, characterized by the fact that two or more CRISPR guide RNAs, proteins associated with CRISPR and / or donor nucleic acid sequences are used to disrupt, correct or replace one or more mutations or defects in the mutated or defective gene or gene encoding a protein having defective or partial function.
[296]
296. Method according to claim 278, characterized by the fact that the individual is a mammal.
[297]
297. Method, according to claim 278, characterized by the fact that the individual is a human.
[298]
298. Method according to claim 278, characterized by the fact that the method improves ocular development or function or prevents eye, retinal or corneal degeneration.
[299]
299. Method according to claim 278, characterized by the fact that the method improves neurological development or function or prevents neural degeneration.
[300]
300. Method according to claim 278, characterized by the fact that the method improves expression or function of a P450 enzyme.
[301]
301. Composition according to any one of claims 255, 257, 258 or 259, characterized by the fact that it further comprises (c) a donor nucleic acid sequence comprising all or a portion of a gene target shown in Table 4 with a mutation or alteration to generate a mutated or altered gene or portion thereof.
[302]
302. Composition characterized by the fact that it comprises a cell with a mutated or defective gene shown in Table 4.
[303]
303. Composition characterized by the fact that it comprises a cell with a CYP4V2, CYP1B1, MYO7A, DFNB31, USH1C, USH1G, CDH23, PCDH15, CLRN1, ACO2, AFG3L2, ATXN2, AUH, C12orf65, CISD2, FOX, FO1, LTBP2, MTPAP, MYOC, NDUFS1, NR2F1, OPA1, OPA3, OPTN, PAX6, PDGF, PITX2, POLG, SPG7, TEK, TXNRD2, WFS1, ABCA4, REP-1, RPE65, CEP290, PDE6B, RPGR, MERTK, RPGR, MERTK, ND4, FAM47E, GBA, GCH1, HTRA2, LRRK2, PARK2, PINK1, SNCA, SYNJ1, NPC1, NPC2, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F1, CYP4F1, CYP4F1, CYP4F4, - tuoso comprising a composition of any of the present claims.
[304]
304. Composition according to claim 303, characterized by the fact that the vector is an AAV vector.
[305]
305. Composition, according to any of the preceding claims, characterized by the fact that the sequence of the protospace element is selected from the group consisting of SEQ ID NOs: 48 to 52, or shares at least 80% of the identity of the sequence with one of SEQ ID NOs: 48 to 52, for use with a Cas protein that has NGG as a motif adjacent to the protospacer (PAM) to target the C.802-8_810del17insGC mutation of the CYP4V2 gene.
[306]
306. Composition, according to any of the preceding claims, characterized by the fact that the donor nucleic acid sequence is selected from SEQ ID NOs: 56 and 57, or shares at least 90% sequence identity with one of
SEQ ID NO: 56 and 57, or a sequence that is complementary to the same, for use to correct, disrupt or replace the C.802- 8_810del17insGC mutation of the CYP4V2 gene.
[307]
307. Method of treating an individual's disease caused by a mutated or defective gene, or a gene encoding a protein having defective or partial function or activity, characterized by the fact that it comprises administering to the individual a combination of (i) a composition targeting diseased cells in the subject comprising a gene editing therapy composition or a gene transfer therapy composition and (ii) a cell composition comprising replacement cells.
[308]
308. Method, according to claim 307, characterized by the fact that the individual is a human being.
[309]
309. Method, according to claim 307, characterized by the fact that the disease causes degeneration in the individual's cells.
[310]
310. Method according to claims 307 and 308, characterized by the fact that degeneration occurs in an RPE cell, an RPC, an ECC, a CE cell, a retinal cell, a corneal cell, a choroidal cell, an optic nerve cell, an eye cell, a neuron, a neuronal cell, a brain cell, a liver cell, a lung cell, and a cardiac cell.
[311]
311. Method according to claim 307, characterized by the fact that the disease is caused by a gene shown in Table 4.
[312]
312. Method according to claim 307, characterized by the fact that the gene editing therapy composition is a composition as defined in any of the present gene editing therapy claims.
[313]
313. The method of claim 307, characterized by the fact that the gene transfer therapy composition is a composition as defined in any of the present gene transfer therapy claims.
[314]
314. Method according to claim 307, characterized in that the cell composition comprises a composition as defined in any of the cell therapy claims or the present autologous cell therapy claims.
[315]
315. Method according to claim 307, characterized by the fact that the cellular composition is allogeneic for the individual being treated.
[316]
316. Method, according to claim 307, characterized by the fact that the cell composition is autologous for the individual being treated.
[317]
317. Method according to claim 307 or 314, characterized in that the cell composition is genetically repaired prior to administration to the individual.
[318]
318. Method according to claim 307 or 317, characterized by the fact that the genetic repair is through gene editing therapy.
[319]
319. Method according to claim 307 or 318, characterized by the fact that gene editing therapy is through any of the claims in the present gene editing therapy claims.
[320]
320. Method according to claim 307, 318 or 319, characterized by the fact that gene editing therapy is through any of the present claims of RNP CRISPR.
[321]
321. Method according to claim 307 or 317, characterized by the fact that genetic repair is through gene transfer therapy.
[322]
322. Method according to claim 307 or 321, characterized by the fact that gene transfer therapy is through any of the present claims for gene transfer therapy or gene therapy.
[323]
323. Method according to claim 307, characterized in that the compositions of (i) and (ii) are administered in a single administration.
[324]
324. Method according to claim 307, characterized in that the compositions of (i) and (ii) are administered separately or in separate administrations.
[325]
325. Method according to claim 307, characterized by the fact that the replacement cells are derived from a stem cell.
[326]
326. Method according to claims 307 and 325, characterized in that the replacement cells are derived from an iPS cell.
[327]
327. Method according to claim 307 or 326, characterized in that the iPS cell is derived from the same individual being treated.
[328]
328. Method according to claim 307 or 326, characterized in that the replacement cell is an iPS-RPE, iPS-PRC, iPS-CEC or iPS-CE cell.
[329]
329. Method according to claim 307 or 326, characterized in that the replacement cell is an iPS-ocular cell.
[330]
330. Method according to claim 307 or 326, characterized in that the replacement cell is an iPS-neuron cell.
[331]
331. Method according to claim 307, characterized
due to the fact that the method improves ocular development or function or prevents eye, retinal or corneal degeneration in the individual.
[332]
332. Method, according to claim 307, characterized by the fact that the method improves neurological development or function or prevents neural degeneration in the individual.
[333]
333. Method, according to claim 307, characterized by the fact that the method improves expression or function of a P450 enzyme in the individual.
[334]
334. Method according to any of claims 307 to 332, characterized by the fact that the individual suffers from ocular, retinal, corneal, choroidal or neuronal degenerations.

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CN111733174B|2020-08-07|2021-02-09|北京大学第三医院（北京大学第三临床医学院）|Isolated nucleic acid molecule and application thereof|

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CN113015804A|2020-10-13|2021-06-22|北京中因科技有限公司|Nucleic acid molecules for the treatment of crystalline retinal degeneration and uses thereof|

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RU2749459C1|2020-11-10|2021-06-11|Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека |UNIVERSAL INTEGRATION VECTOR pVEAL AND RECOMBINANT PLASMID pVEAL-15742, PROVIDING SYNTHESIS AND SECRETION OF scFv-Fc ANTIBODIES AGAINST EBOLA VIRUS ADI-15742 IN MAMMALIAN CELLS AND OBTAINED USING pVEAL VECTOR|

法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762539473P| true| 2017-07-31|2017-07-31|

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US62/539,473|2017-07-31|

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PCT/IB2018/055755|WO2019025984A1|2017-07-31|2018-07-31|Cellular models of and therapies for ocular diseases|

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