adeno-associated virus variant capsid protein, isolated nucleic acid, infectious raav virus and resp

专利摘要:
Variant adenoassociated virus (AAV) capsid proteins are provided here having one or more modifications in the amino acid sequence relative to a parental AAV capsid protein which, when present in an AAV virus, confers an increased infectivity of one or more retinal cell types compared to the infectivity of retinal cells by an AAV virus comprising the unmodified parental AAV capsid protein. Recombinant AAV virions and pharmaceutical compositions thereof are also provided comprising a variant AAV capsid protein as described herein, methods for producing these proteins and rAAV capsid virions and methods for using these rAAV capsid proteins and virions in research and in clinical practice, for example, in applying nucleic acid sequences to one or more cells of the retina for the treatment of retinal disorders and diseases.
公开号:BR112018072849B1
申请号:R112018072849-7
申请日:2017-05-12
公开日:2020-12-22
发明作者:David H. Kirn；Melissa KOTTERMAN；David Schaffer
申请人:4D Molecular Therapeutics Inc.；
IPC主号:

专利说明:

DESCRIPTIVE REPORT
[001] According to 35 U.S.C. §119 (e), this Order claims priority for the filing date of the United States provisional patent application with serial number 62 / 336,441 filed on May 13, 2016; United States provisional patent application with serial number 62 / 378,106, filed on August 22, 2016; United States provisional patent application with Serial No. 62 / 384,590, filed on September 7, 2016; United States provisional patent application with serial number 62 / 454,612, filed on February 3, 2017; the disclosures of which are hereby incorporated by reference. INCORPORATION BY SEQUENCE LISTING REFERENCE PROVIDED AS TEXT FILE
[002] A Sequence Listing is provided here as a text file, “4D_ST25.txt” created on May 12, 2017 and with a size of 198 KB. The content of the text file is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION
[003] The invention disclosed herein refers in general to the field of adeno-associated virus viruses (AAV) comprising variant capsid proteins and the generation of such variant capsids with the use of directed evolution techniques. BACKGROUND OF THE INVENTION
[004] Hereditary retinal diseases comprise a large group of heterogeneous genetic diseases that affect approximately 1 in 3,000 people (greater than 2 million people worldwide) and are an important source of severe vision loss or blindness. Complex multifactorial retinal diseases such as wet age-related macular degeneration (wAMD) and diabetic retinopathy (DR) impact even more individuals, with 1.7 million Americans currently living with wAMD-associated severe central vision loss and nearly a third of adults over 40 with diabetes being visually impaired. These diseases are typically associated with the dysfunction or death of one or more types of retinal cells, in some cases due to the absence of expression or function of a key protein, for example, RPE65 in LCA2, in other cases due to genetic mutations that they create toxic gene products, for example, dominant mutations that affect the rhodopsin protein flexion, or in other cases, due to changes in the physiology of the retina induced by the ectopic expression of a protein, for example, VEGF in wAMD.
[005] One approach to address this major non-medical need is gene-mediated adeno-associated virus (AAV) therapy, in which a recombinant adeno-associated virus (rAAV) is used to release a gene to one or more types of cells in the retina, for example, to replace a missing gene, to correct a defective dominant gene or to provide a model for continuous protein therapy. Although AAV-based clinical gene therapy has been increasingly successful, it is still fraught with deficiencies in the properties of the viral vector, including, for example, targeting the desired retinal cells with high efficiency. For example, multiple AAV serotypes from homologous primates and numerous serotypes of non-human primates have been identified and characterized, with AAV2 being the best characterized among AAV serotypes and the first to be adapted as a vehicle for gene release in the eye. However, these AAVs (including AAV2) have not been reported to be effective in transducing the deeper retinal cell types when released via intravitreal administration. Accordingly, there is a need in the art for new AAV variants with superior transduction capabilities that provide a more effective gene-based release to retinal cells for the treatment of eye disease. There is a need in the art for such AAV variants that exhibit an improved retinal transduction profile. In some cases, in general terms, in other cases preferably for certain types of retinal cells as compared to wild-type AAVs and AAV variants as known in the art.
[006] The naturally occurring AAV is a single-stranded DNA virus that contains three open reading frames, rep, cap and aap. The first gene, rep, encodes four proteins necessary for genome replication (Rep78, Rep68, Rep52 and Rep40), the second, cap, expresses three structural proteins (VP1-3) that come together to form the viral capsid, and the third expresses the assembly activation protein (AAP) which is essential for the assembly of the capsid. AAV depends on the presence of an auxiliary virus, such as an adenovirus or herpesvirus, for active replication. In the absence of a helper virus, AAV establishes a latent state in which its genome is maintained episomally or integrated with the host chromosome at the AAVS1 site.
[007] In vitro and in vivo directed evolution techniques can be used to select AAV variants that provide an improvement over current AAV-based gene application vectors. Such directed evolution techniques are known in the art and described, for example, in the publication PCT WO 2014/194132 and Kotterman & Schaffer (Nature Review Genetics, AOP, published online on May 20, 2014; doi: 10.1038 / nrg3742), both of which are incorporated here in their entirety, as a reference. Targeted evolution is an engineering approach to the capsid that emulates natural evolution through iterative cycles of genetic diversification and selection processes, thus allowing the accumulation of beneficial mutations that progressively improve the function of a biomolecule, such as an AAV-based virion. In this approach, wild-type AAV cap genes are diversified to create large genetic libraries that are packaged to generate libraries of viral particles, and selective pressure is applied to isolate original variants with superior phenotypes that can overcome barriers to application of genes.
[008] The AAV variants were revealed, for example, in United States patent numbers 9,193,956, 9,186. 419, 8,632,764, 8,663,624, 8,927,514, 8,628,966, 8,263,396, 8,734,809, 8,889,641, 8,632,764, 8,691,948, 8,299,295, 8,802,440, 8,445,267, 8,906,307, 8,574,583, 8,067,015, 7,588,772, 7,867,484, 8,163,543, 8,283,151, 8,999,678, 7,892,809, 7,906,111, 7,259,151, 7,629,322, 7,220. 577, 8,802,080, 71998951, 8,318,480, 8,962,332, 7,790,449, 7,282,199, 8,906,675, 8,524,446, 7,712,893, 6,491,907, 8,637,255, 7,186,522, 7,105. 345, 6,759,237, 6,984,517, 6,962,815, 7,749,492, 7,259,151, and 6,156,303, United States publication numbers 2013/0295614, 2015/0065562, 2014/0364338, 2013/0323226, 2014 / 0359799, 2013/0059732, 2014/0037585, 2014/0056854, 2013/0296409, 2014/0335054 2013/0195801, 2012/0070899, 2011/0275529, 2011/0171262, 2009/0215879, 2010/0297177, 2010/0297177, 2010/0203083, 2009 / 0317417, 2009/0202490, 2012/0220492, 2006/0292117, and 2004/0002159, European publication numbers 2692731A1, 2383346B1, 2359865B1, 2359866B1, 2359867B1, and 2357010B1, 1791858B1, 1668143B1, 1668143B1, 1660678 341068B1, 2338900B1, 1456419B1, 1310571B1, 1456383B1, 1633772B1, and 1135468B1, international publication numbers (PCT) WO 2014/124282, WO 2013/170078, WO 2014/160092, WO 2014/103957, WO 2014/052789, WO 2013/174760 , WO 2013/123503, WO 2011/038187, WO 2008 / 124015e WO 2003/054197, however, none of these references reveal the modalities and / or characteristics and / or composition of the structures of matter of the AAV variants described by the present invention.
[009] All documents and references cited in the present invention and in the referenced patent documents, are hereby incorporated by way of reference. SUMMARY OF THE INVENTION
[0010] The variant AAV adeno-associated virus capsid protein is provided by the present invention having one or more modifications in the amino acid sequence in relation to a parental AAV capsid protein, when present in an AAV virion, confers increased infectivity. of one or more types of retinal cells compared to the infectivity of retinal cells by an AAV virus comprising the unmodified parental AAV capsid protein. Recombinant AAV virions and pharmaceutical compositions thereof are also provided comprising a variant AAV capsid protein as described by the present invention, methods of making proteins and rAAV variant capsid virions and methods for using these proteins and rAAV capsid virions. in research and clinical practice, for example in the application of nucleic acid sequences to one or more cells of the retina for the treatment of disorders and diseases of the retina.
[0011] In some aspects of the disclosure, variant adeno-associated virus (AAV) capsid proteins are provided, these variant AAV capsid proteins having one or more modifications in the amino acid sequence relative to a parental AAV capsid that, when present in an AAV virion, it imparts increased infectivity to one or more types of retinal cells (eg, a photoreceptor cell) (eg, rods; cones), a retinal ganglional cell (RGC), a glial cell (for example, a Müller glial cell, a microglial cell), a bipolar cell, an amacrine cell, a horizontal cell and / or a retinal pigment epithelium cell (RPE)) compared to the infectivity of the retinal cells by one AAV virus comprising a parental AAV capsid protein that does not comprise modification of the amino acid sequence.
[0012] In some aspects of the disclosure, recombinant AAV virions (rAAV are provided, these rAAV virions comprising a variant capsid protein as described by the present invention, wherein the rAAV virions exhibit an increased infectivity of one or more types of retinal cells eg a photoreceptor cell) (eg rods; cones), a retinal ganglion cell (RGC), a glial cell (eg a Müller glial cell, a microglial cell), a cell bipolar, an amacrine cell, a horizontal cell and / or a retinal pigment epithelium cell (RPE) with respect to the infectivity of the retinal cell by an AAV virion comprising a corresponding unmodified parental AAV capsid protein. In some embodiments, the rAAV virus exhibits increased infectivity of all cells in the retina relative to the AAV virus comprising the parental AAV capsid protein. In other embodiments, the rAAV virus exhibits an increased infectivity of certain types of retinal cells, but not others in relation to the AAV virus comprising the parental AAV capsid protein. Put another way, the rAAV virus exhibits an increased infectivity that is preferred for certain types of retinal cells, but not for others, for example, rAAV demonstrates a preferentially increased infectivity of one or more types of cells selected from photoreceptor cells , retinal ganglion cells, glial cells, bipolar cells, horizontal cell of amacrine cells and / or retinal pigment epithelium cells (RPE), but do not demonstrate an increased infectivity of all cell types.
[0013] In some embodiments, the rAAV virion comprises a heterologous nucleic acid. In some of these embodiments, the heterologous nucleic acid encodes an RNA that encodes a polypeptide. In other such embodiments, the heterologous nucleic acid sequence encodes an RNA that does not encode a polypeptide, for example, the heterologous nucleic acid sequence, an RNA interference agent, a guide RNA for a nuclease, etc.
[0014] Pharmaceutical compositions comprising the infectious rAAV virions in question and a pharmaceutically acceptable carrier are also provided herein.
[0015] Also provided is the use of a rAAV virion comprising a variant capsid protein as disclosed by the present invention in a method of delivering a heterologous nucleic acid to a target cell (such as a retinal cell) by contact of the target cell with the rAAV virus. In some embodiments, the target cell in vivo, such as in the eye of an individual in need of treatment for an eye disease. In other embodiments, the target cell is in vitro.
Also provided herein are methods of treating an eye disease by administering to an individual in need of such treatment an effective amount of rAAV virus comprising a variant capsid protein as described by the present invention or a pharmaceutical composition comprising an amount effectiveness of rAAV virions.
Also provided herein are an isolated nucleic acid comprising a sequence encoding a variant AAV capsid protein as disclosed by the present invention and a host cell comprising the isolated nucleic acid. In yet other embodiments, the isolated nucleic acid and / or the isolated host cell comprises rAAV.
[0018] In some respects, the variant AAV capsid protein comprises an insertion of about 5 amino acids to about 20 amino acids (a "heterologous peptide" or "peptide insert") in the GH loop of the capsid protein, relative to to a corresponding parental AAV capsid protein, where the variant capsid protein, when present in an AAV virion, confers an increased infectivity of a retinal cell compared to the infectivity of a retinal cell by a virus of AAV comprising the corresponding parental AAV capsid protein. In some embodiments, the peptide comprises the sequence selected from the group consisting of QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), ASDSTKA (SEQ ID NO: 15), NQDYTKT (SEQ ID NO: 16 ), HDITKNI (SEQ ID NO: 17), HPDTTKN (SEQ ID NO: 18), HQDTTKN (SEQ ID NO: 19), NKTTNKD (SEQ ID NO: 20), ISNENEH (SEQ ID NO: 21), QANANEN (SEQ ID NO: 22), GKSKVID (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), PNSTHGS (SEQ ID NO: 25), KDRAPST (SEQ ID NO: 26), LAQADTTKNA (SEQ ID NO: 27) , LAISDQTKHA (SEQ ID NO: 28), LGISDQTKHA (SEQ ID NO: 29), LAASDSTKAA (SEQ ID NO: 30), LANQDYTKTA (SEQ ID NO: 31), LAHDITKNIA (SEQ ID NO: 32), LAHPDTTKNA (SEQ ID NO: 32) NO: 33), LAHQDTTKNA (SEQ ID NO: 34), LANKTTNKDA (SEQ ID NO: 35), LPISNENEHA (SEQ ID NO: 36), LPQANANENA (SEQ ID NO: 37), LAGKSKVIDA (SEQ ID NO: 38), LATNRTSPDA (SEQ ID NO: 39), LAPNSTHGSA (SEQ ID NO: 40) and LAKDRAPSTA (SEQ ID NO: 41). In some embodiments, the peptide essentially consists of the sequence selected from the group consisting of QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), ASDSTKA (SEQ ID NO: 15), NQDYTKT (SEQ ID NO: 16), HDITKNI (SEQ ID NO: 17), HPDTTKN (SEQ ID NO: 18), HQDTTKN (SEQ ID NO: 19), NKTTNKD (SEQ ID NO: 20), ISNENEH (SEQ ID NO: 21), QANANEN ( SEQ ID NO: 22), GKSKVID (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), PNSTHGS (SEQ ID NO: 25), KDRAPST (SEQ ID NO: 26), LAQADTTKNA (SEQ ID NO: 27) ), LAISDQTKHA (SEQ ID NO: 28), LGISDQTKHA (SEQ ID NO: 29), LAASDSTKAA (SEQ ID NO: 30), LANQDYTKTA (SEQ ID NO: 31), LAHDITKNIA (SEQ ID NO: 32), LAHPDTTKNA (SEQ ID NO: 33), LAHQDTTKNA (SEQ ID NO: 34), LANKTTNKDA (SEQ ID NO: 35), LPISNENEHA (SEQ ID NO: 36), LPQANANENA (SEQ ID NO: 37), LAGKSKVIDA (SEQ ID NO: 38) , LATNRTSPDA (SEQ ID NO: 39), LAPNSTHGSA (SEQ ID NO: 40) and LAKDRAPSTA (SEQ ID NO: 41). In some respects, the variant AAV capsid protein comprises one or more amino acid substitutions in relation to a corresponding parental AAV capsid protein, where the variant capsid protein, when present in an AAV virion, confers increased infectivity. of a retinal cell compared to the infectivity of a retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
[0019] In related aspects, the variant AAV capsid protein comprises a peptide insertion and or more amino acid substitutions in relation to a corresponding parental AAV capsid protein, wherein the variant capsid protein, when present in a AAV virion, confers increased infectivity of a retinal cell compared to the infectivity of a retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
[0020] Also presented by the present invention is a variant AAV capsid protein comprising the heterologous peptide LAISDQTKHA (SEQ ID NO: 28) and a P34A substitution for AAV2.
[0021] Also presented by the present invention is a variant AAV capsid protein comprising the heterologous peptide LAISDQTKHA (SEQ ID NO: 28) and amino acid substitutions N312K, N449D, N551S, I698V and L735Q in relation to AAV2.
[0022] Methods of making and / or releasing an rAAV comprising a variant AAV capsid as disclosed by the present invention are also presented by the present invention. In addition, kits comprising a rAAV comprising a variant AAV capsid as disclosed by the present invention and for use in the methods disclosed by the present invention are provided by the present invention.
[0023] In other embodiments, the AAV virion comprising the variant capsid protein in the preceding paragraphs may incorporate any of the previous or subsequently disclosed modalities. Indeed, it is appreciated that certain features of the invention, which are, for clarity, described in the context of separate modalities, can also be provided in combination in a single modality. On the other hand, several features of the invention, which are briefly described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination. All combinations of the modalities relating to the invention are specifically covered by the invention and are disclosed by the present invention as if each of the combinations were disclosed individually and explicitly. In addition, all subcombination of the various embodiments and elements thereof are also specifically covered by the invention and are disclosed by the present invention only as if each and all of such subcombination were individually and explicitly disclosed herein by the present invention.
[0024] The Summary of the Invention is not intended to define the Claims nor is it intended to limit the scope of the invention in any way.
[0025] Other features and advantages of the invention disclosed herein will be evident from the following figures, detailed description and Claims. BRIEF DESCRIPTION OF THE FIGURES
[0026] The invention is better understood from the following detailed description when read in conjunction with the accompanying drawings. The patent file or patent application contains at least one drawing executed in color. Copies of this patent publication or patent application with colored design (s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various characteristics of the drawings are not to scale. On the other hand, the dimensions of the various characteristics are arbitrarily expanded or reduced for clarity. The figures are included in the following drawings.
[0027] FIG. 1 represents modalities of a directed evolution methodology. Step (a) represents the generation of a viral capsid library comprising combinations of DNA mutation techniques and cap genes. Step (b) represents the packaging of the viruses in such a way that each viral particle is composed of a mutant capsid involving the cap gene that encodes and purifies that capsid. The capsid library is then placed under selective pressure in vitro or in vivo. In this aspect of targeted evolution technology, tissues or cellular material of interest are harvested to isolate AAV variants that have successfully infected that target and successful viruses are recovered. Step (c) represents the enrichment of stage 1 of successful clones through repeated selection. Step (d) represents the enrichment of stage 2 of selected caps genes that are subjected to further diversification and additional selection steps to iteratively increase viral fitness. Step (e) represents the variants, identified as occurrences during Vector Selection Stages 1 and 2, which will be manufactured as recombinant AAV vectors and characterized as to the level of transduction of various types of cells and tissue targets. By the nature of the directed evolution process of AAV, variants that are revealed by the present invention have already demonstrated the ability to transduce retinal cells and release a genome (the genome encoding the cap variant gene) during the selection process.
[0028] FIG. 2 provides a flat retinal assembly scheme showing where samples from which viral genomes are amplified are collected across a wide area of the retina.
[0029] FIG. 3 shows a PCR amplification of viral genomes of the ganglion cell layer (GCL), inner nuclear layer (INL), photoreceptor / outer nuclear layer (ONL) and retinal tissue of the retinal pigment epithelium layer of a representative selection cycle . Both the right eye (upper image) and the left eye (lower image) were injected with a library. The internal (in) retina, the middle (average) retina and the external / peripheral (external) retina were sampled. Bands inside red boxes represent successful amplification of viral genomes.
[0030] FIGS. 4A-4D represents the frequency of motifs within the sequencing analysis. Figure 4A provides the analysis of
[0031] FIG. 5 provides a representative three-dimensional model of AAV2 containing a random heptamer following amino acid 587.
[0032] FIG. 6 provides an alignment of SEQ ID NOS: 1-11 of wild-type AAV that shows amino acid sites between and across the wild-type (naturally occurring) AAV1, AAV2, AAV3A, AAV3B and AAV4-10 serotypes.
[0033] FIG. 7 provides fundus fluorescence images taken with Heidelberg Spectralis TM of the retina of an African Green monkey after intravitreal administration of 2x1011 vector genomes (vg) of AAV2 applying a GFP transgene under the control of the CMV promoter (AAV2.CMV.GFP ). The images were taken at baseline (A) and at 14 days (B), 28 days (C) and 42 days (D) after injection.
[0034] FIG. 8 provides fundus fluorescence images taken with a Heidelberg Spectralis TM from the retina of an African Green monkey after intravitreal administration of 2x1011 vector genomes (vg) of the new AAV variant LAISDQTKHA + P34A applying a GFP transgene under the control of the promoter of CMV (LAISDQTKHA + P34A.CMV.GFP). The images were taken at baseline (A) and at 14 days (B), 28 days (C) and 42 days (D) after injection.
[0035] FIG. 9 provides fundus fluorescence images taken with a Heidelberg Spectralis TM from the retina of Cinomolgos monkeys after intravitreal administration of the new AAV variant LAISDQTKHA + P34A applying a GFP transgene under the control of the CAG promoter (LAISDQTKHA + P34A.CAG.EGFP) . (A) A monkey's retina injected intravitreally with 2x1011 vg of vector, captured 14 days (A1), 21 days (A2) and 28 days (A3) after injection. (A) The retina of a monkey injected intravitreally with 1x1012 vg of vector, captured 14 days (A1) and 21 days (A2) after the injection.
[0036] FIGS. 10A-10E provide the results of immunohistochemical analysis of the retina of a monkey axially injected with 1x1012 vg of the new variant of AAV LAISDQTKHA + P34A applying a GFP transgene under the control of the CAG promoter, analyzed three weeks after injection. All immunohistochemistry is provided along with the fluorescence image of the fundus, with a red box to indicate approximately where the retinal analysis was performed. Figure 10A: The robust retinal pigment epithelium (RPE) and photoreceptor transduction were observed with the use of a specific antibody for GFP (red). The immunostaining of cone photoreceptors using an M / L opsin antibody is shown in white. Figures 10B and 10C: The robust stem and cone photoreceptor (Figure 10B) and RPE transduction (Figure 10C) were observed by direct EGFP fluorescence (green) and by immunohistochemistry using a specific antibody for GFP ( red). Melanosomes in the RPE appear black in the image. Figure 10D: The transduction of cone photoreceptors (identified by opsin M / L, white) and ganglion cells of the retina (RGC) and around the fovea was observed by direct fluorescence of EGFP (green) and by immunohistochemistry using of a specific antibody to GFP (red). The images in the middle panels are a larger (63X) magnification of the area denoted by a white box on the left panel. Figure 10E: The retinal ganglion cell (RGC) transduction and the retinal ganglion cell layer was observed by direct EGFP fluorescence (right panels, green; lower right panel is a 63X magnification of the upper right panel); the upper left panel shows the region under bright field lighting.
[0037] FIGS. 11A-11F provides data on the in vitro transduction of retinal pigment epithelial cells (RPE) by recombinant AAV virus comprising the new AIS capsid LAISDQTKHA + P34A variant and a GFP transgene under the control of the CAG promoter. Cells that have been differentiated into RPE cells from a human embryonic stem cell line (Figures 11A and 11C) or from human pluripotent stem cells (FB-iPSC) (Figures 11B and 11D) have been infected with the new variant of LAISDQTKHA + P34A.CAG.GFP or AAV2.CAG.GFP wild type control. Figures 11A and 11B: Immunofluorescence imaging of cell cultures 7 days after infection in an MOI of 500 demonstrates that the new AAV variant capsid (left panels) transduces RPE cells better than the wild-type AAV2 capsid (right panels). Figures 11C and 11D: Quantification of the percentage of GFP positive RPE cells in each culture by flow cytometry reveals that the new AAV capsid variant provides a significant, dose-dependent improvement in the number of cells transduced in relation to the capsid of wild-type AAV2, regardless of the cell source. Figures 11E and 11F: Quantification of the amount of GFP in each culture using Western blot reveals that the new variant of AAV provides a significant improvement in the expression of the transgene in relation to the wild-type capsid, regardless of the cell source. DETAILED DESCRIPTION
[0038] Before the present methods and compositions are described, it should be understood that this invention is not limited to a particular method or composition described and how it may vary. It should also be understood that the terminology used by the present invention is for the purpose of describing only particular embodiments, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended Claims.
[0039] The invention disclosed by the present invention is illustrated in the figures and description. However, although the particular modalities are illustrated in the figures, there is no intention to limit the invention to the specific modality or modalities illustrated and / or disclosed. On the contrary, the disclosed invention is intended to cover all modifications, alternative and equivalent constructions covered by the spirit and scope of the invention. Thus, the figures are intended to be illustrative and not restrictive.
[0040] When a range of values is provided, it is understood that each intervening value, for the tenth of the unit of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of that range is also specifically revealed. Each minor range between any declared value or intervening value in an established range and any other declared or intervening value in that established range is covered by the scope of the invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the range, and each range in which either or both of the limits are included in the lower ranges is also covered by the invention, subject to any limit specifically excluded in the established range . When the established range includes one or both of the limits, the bands excluding one or both of the included limits are also included in the invention.
[0041] Unless otherwise defined, all technical and scientific terms used by the present invention have the same meaning as that normally understood by one skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned by the present invention are incorporated herein by reference to disclose and describe the methods and / or materials in relation to which the publications are cited. It should be understood that the present disclosure replaces any disclosure of an incorporated publication to the extent that there is a contradiction.
[0042] As will be apparent to those skilled in the art when reading this disclosure, each of the individual modalities described and illustrated by the present invention has discrete components and characteristics that can be readily separated from or combined with the characteristics of any of the other various modalities without departing from the scope or spirit of the present invention. Any recited method can be performed in the order of the recited events or in any other order that is logically possible.
[0043] Note that as used by the present invention and in the appended Claims, the singular forms "one", "one" and "o" include plural referents, unless the context clearly indicates otherwise. Thus, for example, the reference to "a recombinant AAV virus" includes a plurality of such virions and the reference to "photoreceptor cell" includes reference to one or more photoreceptor cells and equivalents thereof known to those skilled in the art, and so on. against. Note also that the Claims can be made to exclude any optional elements. Accordingly, this statement is intended to serve as an antecedent basis for the use of such exclusive terminology as "only", "only" and the like in connection with the mention of Claim elements, or use of a "negative" limitation.
[0044] The publications discussed by the present invention are provided only for their dissemination before the date of filing the present application. Nothing herein should be construed as an admission that the present invention has no right to predate such publication by virtue of the previous invention. In addition, the publication dates provided may differ from the actual publication dates which may require independent confirmation. DEFINITIONS
[0045] Except where otherwise stated, all scientific and technical terms used by the present invention have the same meaning as is commonly understood by the person skilled in the art to which this technology belongs.
[0046] Adeno-associated virus is a non-pathogenic parvovirus composed of a 4.7 kb single-stranded DNA genome within an icosahedral non-enveloped capsid. The genome contains three open reading frames (ORF) flanked by inverted terminal repetitions (ITR) that function as the viral origin and packaging signal. The rep ORF encodes four non-structural proteins that perform functions in viral replication, transcriptional regulation, site-specific integration and assembly of the virion. The cap ORF encodes three structural proteins (VP 1-3) that come together to form a 60-mer viral capsid. Finally, an ORF present as an alternative reading frame within the cap gene produces the assembly activation protein (AAP), a viral protein that locates the AAV capsid proteins to the nucleolus and functions in the capsid assembly process.
[0047] There are several naturally occurring serotypes ("wild type") and more than 100 known AAV variants, each of which differs in the sequence of amino acids, particularly within the hypervariable regions of capsid proteins and thus in their properties of releasing genes. No AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.
[0048] For the purposes of this disclosure, the terminology "AAV" is an abbreviation for adeno-associated virus, including, but not limited to, the virus itself and its derivatives. Unless otherwise indicated, the terminology refers to all subtypes or serotypes and to the recombinant and competent forms for replication. The term “AAV” includes, but is not limited to, AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3A (AAV-3A or AAV3A), AAV type 3B ( AAV-3B or AAV3B), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7) , AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), AAV type 10 (AAV-10 or AAV10 or AAVrh10), Avian AAV, bovine AAV, canine AAV, goat AAV, equine AAV , AAV primate, AAV not primate and AAV sheep. “Primate AAV” refers to AAV that infects primates, “Non-primate AAV” refers to AAV that infects non-primate mammals, “Bovine AAV” refers to AAV that infects bovine mammals, etc.
[0049] The genomic sequences of various AAV serotypes, as well as the sequences of the native terminal repeats (TRs), Rep proteins and capsid subunits are known in the art. These strings can be found in the literature or in public databases, such as GenBank. See, for example, GenBank Access Numbers NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2 (AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3A ), NC_001729.1 (AAV3A), AF028705.1 (AAV3B), NC_001829.1 (AAV4), U89790.1 (AAV4), NC_006152.1 (AA5), AF085716.1 (AAV-5), AF028704.1 ( AAV6), NC_006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8) NC_006261.1 (AAV-8), AY530579.1 (AAV9), AAT46337 (AAV10) and AAO88208 (AAVrh10); the disclosures thereof being incorporated herein as a reference for the teaching of AAV nucleic acid and amino acid sequences. See also, for example, Srivistava et al. (1983) J. Virology 45: 555; Chiorini et al. (1998) J. Virology 71: 6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73: 939; Xiao et al. (1999) J. Virology 73: 3994; Muramatsu et al. (1996) Virology 221: 208; Shade et. al. (1986) J. Virol. 58: 921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33: 375-383; international patent publications WO00 / 28061, WO99 / 61601, WO98 / 11244; and U.S. Patent No. 6,156,303.
The existing protein sequences of cap (capsid) naturally associated with AAV serotypes are known in the art and include those described by the present invention as AAV1 (SEQ ID NO: 1), AAV2 (SEQ ID NO: 2), AAV3A (SEQ ID NO: 3), AAV3B (SEQ ID NO: 4), AAV4 (SEQ ID NO: 5), AAV5 (SEQ ID NO: 6), AAV6 (SEQ ID NO: 7), AAV7 (SEQ ID NO: 8), AAV8 (SEQ ID NO: 9), AAV9 (SEQ ID NO: 10), AAV10 (SEQ ID NO: 11) and AAVrh10 (SEQ ID NO: 12). The terms "variant AAV capsid protein" or "variant AAV" refer to an AAV capsid protein comprising an amino acid sequence that includes at least one modification or substitution (including deletion, insertion, point mutation, etc.) with respect to a natural existence or sequences of wild-type AAV capsid proteins, for example, as set out in SEQ ID NO: 12, of the present invention. A variant AAV capsid protein can have about 80% identity or more than the amino acid sequence of a wild type capsid protein, for example, 85% identity or more, 90% identity or more or 95% identity or more of the amino acid sequence of the wild type capsid protein, for example, 98% or 99% identity with the wild type capsid protein. A variant AAV capsid protein may not be a wild type capsid protein.
[0051] For the purposes of this disclosure, "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one, AAV capsid protein and an encapsulated AAV polynucleotide.
[0052] For the purposes of this disclosure, the terminology “rAAV” is an abbreviation that refers to the recombinant adeno-associated virus. “Recombinant”, when applied to a polynucleotide, means that the polynucleotide is the product of several stages of cloning, restriction or ligation combinations, and other procedures that result in a construction that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms include replicas of the original polynucleotide construct and descendants of the original virus construct, respectively.
[0053] The term "rAAV vector" encompasses rAAV virions (i.e., viral rAAV particles) (for example, an infectious rAAV virus), which by definition include an rAAV polynucleotide; and also encompasses polynucleotides that encode rAAV (e.g., a single-stranded polynucleotide that encodes rAAV (ss-rAAV), a double-stranded polynucleotide that encodes rAAV (ds-rAAV), e.g., plasmids that encode rAAV and the like).
[0054] If an AAV virus comprises a heterologous polynucleotide (i.e., a polynucleotide that is not a wild-type AAV genome, for example, a transgene to be delivered to a target cell, an RNAi agent or CRISPR agent for be delivered to a target cell, etc.), is typically referred to as a "recombinant AAV virus (rAAV)" or a "rAAV viral particle". In general, the heterologous polynucleotide is flanked by at least one, and in general, by two repeated inverted AAV terminal sequences (ITRs).
[0055] The term "packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle. The AAV “rep” and “cap” genes refer to polynucleotide sequences that encode adeno-associated virus replication and encapsidation proteins. AAV rep and cap are called AAV “packaging genes”.
[0056] The term "helper virus" for AAV refers to a virus that allows AAV (for example, wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such AAV helper viruses are known in the art, including adenoviruses, herpesviruses and poxviruses, such as vaccinia. Adenoviruses cover several different subgroups, although subgroup C type 5 Adenovirus is most commonly used. Numerous human, non-human adenoviruses in mammals and birds are known and are available from depositories such as ATCC. Viruses in the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudo-rabies virus (PRV); which are also available from depositaries like the ATCC.
[0057] The terminology “helper virus function (s)” refers to the function (s) encoded in a helper virus genome that allows replication and packaging of the AAV (in conjunction with other requirements for replication and packaging described by the present invention). As described by the present invention, the "helper virus function" can be provided in several ways, including providing helper virus or providing, for example, polynucleotide sequences that encode the necessary function (s) to a cell producer in trans. For example, a plasmid or other expression vector comprising nucleotide sequences that encode one or more adenoviral proteins is transfected into a producer cell together with an rAAV vector.
[0058] The term “infectious” virus or viral particle is one that comprises a competently assembled viral capsid and is capable of applying a polynucleotide component to a cell for which the viral species is tropic. The term does not necessarily imply any ability to replicate the virus. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods for determining the ratio of infectious viral particles to total viral particles are known in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11: S337 (describing an infectious TCID50 titration assay); and Zolotukhin et al. (1999) Gene Ther. 6: 973. See also the examples.
[0059] The term "tropism", as used by the present invention, refers to the preferential targeting by a virus (for example, an AAV) of cells of a particular host species or of particular cell types within a host species. For example, a virus that can infect heart, lung, liver and muscle cells has a broader (that is, increased) tropism compared to a virus that can infect only lung and muscle cells. Tropism can also include dependence on a virus on particular types of host cell surface molecules. For example, some viruses can infect only cells with surface glycosaminoglycans, while other viruses can infect only cells with sialic acid (such dependencies can be tested using several cell lines deficient in certain classes of molecules as potential host cells for viral infection). In some cases, the tropism of a virus describes the relative preferences of the virus. For example, a first virus can infect all types of cells, but it is much more successful in infecting these cells with superficial glycosaminoglycans. A second virus can be considered to have a similar (or identical) tropism to the first virus if the second virus also prefers the same characteristics (for example, the second virus is also more successful in infecting these cells with superficial glycosaminoglycans), even if absolute transduction efficiencies are not similar. For example, the second virus may be more efficient than the first virus in infecting each type of cell tested, but if the relative preferences are similar (or identical), the second virus can still be considered to have a similar (or identical) tropism . as the first virus. In some embodiments, the tropism of a virion comprising a variant AAV capsid protein in question is not altered in relation to a naturally occurring virus. In some embodiments, the tropism of a virion comprising a variant AAV capsid protein in question is expanded (that is, broader) relative to a naturally occurring virus. In some embodiments, the tropism of a virion comprising a variant AAV capsid protein in question reduced from a naturally occurring virion.
[0060] The term “replication-competent” virus (for example, a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and is also capable of replicating in an infected cell (that is, in the presence auxiliary virus or auxiliary virus working). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors, as described by the present invention, are incompetent for replication in mammalian cells (especially in human cells) due to the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any sequences of AAV packaging genes in order to minimize the possibility that competent AAVs are generated for replication by recombination between AAV packaging genes and an input rAAV vector. In many embodiments, the rAAV vector preparations as described by the present invention are those that contain few or no replication-competent AAVs (rcAAV, also called RCA) (for example, less than about 1 rcAAV per 102 rAAV particles, less than about 1 rcAAV per 104 rAAV particles, less than about 1 rcAAV per 10 rAAV particles, less than about 1 rcAAV per 1012 rAAV particles, or without rcAAV).
[0061] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or the like. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and can be disrupted by non-nucleotide components. If present, changes in the nucleotide structure can be seen before or after the polymer assembly. The term polynucleotide, as used by the present invention, refers interchangeably to double-stranded and single-stranded molecules. Except where otherwise specified or required, any embodiment comprising a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or envisaged to form the double-stranded form.
[0062] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids is the same when comparing the two sequences. The similarity between sequences can be determined in several different ways. To determine the identity of the sequence, the sequences can be aligned using computer methods and programs, including BLAST, available on the Internet at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow for gaps in the sequence. Smith-Waterman is a type of algorithm that allows gaps in the alignments of the sequences. See Meth. Mol. Biol. 70: 173-187 (1997). In addition, the GAP program using the Needleman and Wunsch alignment method can be used to align strings. See J. Mol. Biol. 48: 443-453 (1970).
[0063] The term "gene" refers to a polynucleotide that performs a function of some kind in the cell. For example, a gene can contain an open reading frame that is capable of encoding a gene product. An example of a gene product is a protein, which is transcribed and translated from the gene. Another example of a gene product is an RNA, for example, a functional RNA product, for example, an aptamer, an interfering RNA, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a non-coding RNA ( ncRNA), a guide RNA for nucleases, etc., which is transcribed, but not translated.
[0064] The terminology "gene expression product" or "gene product" is a molecule resulting from the expression of a particular gene, as defined above. Genetic expression products include, for example, a polypeptide, an aptamer, an interfering RNA, a messenger RNA (mRNA), an rRNA, a tRNA, a non-coding RNA (ncRNA) and the like.
[0065] The term "siRNA agent" ("small interference" or "short interference RNA" (or siRNA)) is a nucleotide RNA duplex that is targeted for genetic interest (a "target gene"). A "RNA duplex" refers to the structure formed by the complementary pairing between two regions of an RNA molecule, forming a region of double stranded RNA (dsRNA). SiRNA is "targeted" to a gene in which the nucleotide sequence of the duplex part of siRNA is complementary to a nucleotide sequence of the target gene. In some embodiments, the length of the siRNAs duplex is less than 30 nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length . In some embodiments, the length of the duplex is 19-25 nucleotides in length. In some embodiments, siRNA-mediated gene targeting is accomplished through the use of DNA-directed RNA interference (ddRNAi) which is a gene silencing technique that uses DNA constructs to activate endogenous RNA (RNAi) interference pathways of an animal cell. Such DNA constructs are designed to express self-complementing double-stranded RNANs, typically short-clip RNAs (shRNAs), which once processed cause a target gene or genes to be silenced. Any RNA, including endogenous viral mRNAs or RNAs, can be silenced by designing constructs to express double-stranded RNA to the desired mRNA target. In this way, the RNA duplex portion of a siRNA agent can be part of a short clamp-like structure referred to as shRNA. In addition to the duplex part, the clamp-like structure may contain a loop part placed between the two sequences that form the duplex. The handle can vary in length. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 11 or 13 nucleotides in length. The clamp-like structure may also contain 3 'or 5' protruding parts. In some embodiments, the overhang is a 3 'or a 5' overhang 0, 1, 2, 3, 4 or 5 nucleotides in length. In general, the level of the expression product (for example, mRNA, polypeptide, etc.) of a target gene is reduced by a siRNA agent (for example, a siRNA, a shRNA, etc.) that contains specific nucleotide sequences double-stranded cells that are complementary to at least a 19-25 nucleotide length segment (for example, a 20-21 nucleotide sequence) of the target gene transcript, including the 5 'untranslated (UT) region, ORF or the 3 'UT region. In some embodiments, short interference RNAs are about 19-25 nm in length. See, for example, PCT applications WOO / 44895, W099 / 32619, WO01 / 75164, WO01 / 92513, WO01 / 29058, WO01 / 89304, WO02 / 16620, and WO02 / 29858; and U.S. Patent Publications No. 20040023390 for descriptions of siRNA technology. The siRNA and / or shRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a minimal synthetic polyadenylation signal.
[0066] The terminology "antisense RNA" encompasses RNA that is complementary to a product of genetic expression. For example, an antisense RNA targeting a specific mRNA is an RNA-based agent (or it may be a modified RNA) that is complementary to the mRNA, where the hybridization of the antisense RNA to the mRNA alters mRNA expression (for example, by changing the stability of the RNA, changing the translation of the RNA, etc.). Also included in "antisense RNA" are the nucleic acids that encode an antisense RNA.
[0067] With respect to “CRISPR agents / Cas9”, the term “CRISPR” encompasses systems of short and regularly interspersed palindromic repetitions / associated CRISPR- (Cas) that evolved to provide bacteria and archaea with adaptive immunity against viruses and plasmids using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. The Cas9 protein (or functional equivalent and / or variant thereof, that is, a protein similar to Cas9) naturally contains DNA endonuclease activity that depends on the association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA ( also called guide RNAs). In some cases, the two molecules are covalently linked to form a single molecule (also called a single guide RNA (“sgRNA”)). Thus, the Cas9 or Cas9-like protein associates with a DNA targeting RNA (term that encompasses the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 protein or similar to Cas9 and orients the protein to a target nucleic acid sequence.
[0068] If the Cas9 or Cas9-like protein maintains its natural enzymatic function, it will cleave the target DNA to create a double strand break, which can lead to genome alteration (ie editing: deletion, insertion (when a polynucleotide donor is present), substitution, etc.), thus altering gene expression. Some variants of Cas9 (variants that fall under the term similar to Cas9) have been altered in such a way that they have decreased DNA cleavage activity (in some cases, they cleave a single strand instead of both strands of the target DNA, while in other cases, they have been severely reduced to no DNA cleavage activity). Cas9-like proteins with decreased DNA cleavage activity (even without DNA cleavage activity) can still be targeted to target DNA to block RNA polymerase activity. Alternatively, the Cas9 or Cas9-like protein can be modified by fusing a VP64 transcription activation domain to the Cas9 protein and encoding the fusion protein with a fusion protein with an MS2-P65-HSF1 helper protein and a single guide RNA comprising aptamers of MS2 RNA in the tetra-loop and cell loop to form a Synergistic Activation Mediator complex (Cas9-SAM) in the cell that activates transcription. Thus, enzymatically inactive Cas9-like proteins can be directed to a specific location in a target DNA by a DNA targeting RNA, in order to block or activate the transcription of the target DNA. The term "CRISPR / Cas9 agents", as used by the present invention, encompasses all forms of CRISPR / Cas9, as described above or as is known in the art.
[0069] Detailed information on CRISPR agents can be found, for example, in (a) Jinek et. al., Science. 2012 Aug 17; 337 (6096): 816-21: “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”; (b) Qi et al., Cell. 2013 Feb 28; 152 (5): 1173-83: “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression”, and (c) US patent application number 13 / 842,859 and PCT application number PCT / US13 / 32589 ; all of which are incorporated herein by reference in their entirety. Thus, the term "CRISPR agent" as used by the present invention encompasses any agent (or nucleic acid encoding that agent), comprising naturally occurring and / or synthetic sequences, which can be used in the Cas9-based system (for example, example, Cas9 or similar to Cas9 and a protein, any component of a DNA targeting RNA, for example, a crRNA-like RNA, tracrRNA-like RNA, a single guide RNA, etc., a donor polynucleotide and the like) .
[0070] By "zinc finger nucleases" (ZFNs) we mean artificial DNA endonucleases generated by fusion of a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be designed to target the desired DNA sequences and this allows the zinc finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit the target DNA in the cell (for example, the cell's genome) inducing double strand breaks. For more information on the use of ZFNs, see, for example: Asuri et al., Mol Ther. 2012 Feb; 20 (2): 329-38; Bibikova et al. Science. 2003 May 2; 300 (5620): 764; Wood et al. Science. 2011 Jul 15; 333 (6040): 307; Ochiai et al. Genes Cells. 2010 Aug; 15 (8): 875-85; Takasu et. al., Insect Biochem Mol Biol. 2010 Oct; 40 (10): 759-65; Ekker et al., Zebrafish 2008 Summer; 5 (2): 121-3; Young et al., Proc Natl Acad Sci U S. A. 2011 Apr 26; 108 (17): 7052-7; Goldberg et al., Cell. 2010 Mar 5; 140 (5): 678-91; Geurts et al., Science. 2009 Jul 24; 325 (5939): 433; Flisikowska et al., PLoS One. 2011; 6 (6): e21045. doi: 10.1371 / journal.pone.0021045. Epub 2011 Jun 13; Hauschild et al., Proc Natl Acad Sci U S. A. 2011 Jul 19; 108 (29): 12013-7; and Yu et al., Cell Res. 2011 Nov; 21 (11): 1638-40; all of which are incorporated herein by reference for their teachings related to ZFNs. The term "ZFN agent" encompasses a zinc finger nuclease and / or a polynucleotide comprising a nucleotide sequence that encodes a zinc finger nuclease.
[0071] The terminology "effector nuclease similar to transcription activator" or "TALEN" agents refers to effector nucleases similar to transcription activators (TALENs) are artificial DNA endonucleases generated by the fusion of a TAL DNA binding effector domain (similar to transcription activator) to a DNA cleavage domain. TALENS can be manipulated quickly to link almost any desired DNA sequence and, when introduced into a cell, TALENs can be used to edit the target DNA in the cell (for example, the cell's genome) inducing double strand breaks. For more information on the use of TALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul 7; 29 (8): 731-4; Wood et al. Science. 2011 Jul 15; 333 (6040): 307; Tesson et al. Nat Biotechnol. 2011 Aug 5; 29 (8): 695-6; and Huang et. al., Nat Biotechnol. 2011 Aug 5; 29 (8): 699-700; all of which are incorporated herein by reference for their teaching related to TALENs. The term "TALEN agent" encompasses a TALEN and / or a polynucleotide comprising a nucleotide sequence that encodes a TALEN.
[0072] The terminology "control element" or "control sequence" refers to a sequence of nucleotides involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, processing, translation or polynucleotide degradation. The regulation can affect the frequency, speed or specificity of the process and can be of an intensive or inhibitory nature. Control elements known in the art include, for example, regulatory transcription sequences, such as promoters and enhancers. A promoter is a region of DNA capable of, under certain conditions, binding to the RNA polymerase and initiating transcription of a coding region normally located downstream (in the 3 'direction) of the promoter. Promoters may be acting ubiquitously, that is, active in many types of cells, for example, CAG or CMV promoters; or specific tissue or cell, for example, the rho promoter, which is active in stems, or the opsin promoter, which is active in cones.
[0073] The terminology "operationally linked" or "operationally linked" refers to a juxtaposition of genetic elements, in which the elements are in a relationship that allows them to operate as expected. For example, a promoter is operationally linked to a coding region if the promoter helps to initiate transcription of the coding sequence. There may be intervening residues between the promoter and the coding region, as long as this functional relationship is maintained.
[0074] The term "expression vector" encompasses a vector comprising a polynucleotide region that encodes a polypeptide of interest and is used to effect expression of the protein in a desired target cell. An expression vector can also comprise control elements operatively linked to the coding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operationally linked for expression is sometimes referred to as an "expression cassette", a large number of which are known and available in the art or can be readily constructed from components that are available in the art.
[0075] The term "heterologous" means derived from an entity genotypically distinct from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence to which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid sequence that encodes a heterologous gene product is an rAAV that includes a polynucleotide not normally included in a naturally occurring wild-type AAV and the encoded heterologous gene product is a gene product not normally encoded by a naturally occurring wild-type AAV.
[0076] The terminology "genetic alteration" and "genetic modification" (and the grammatical variants thereof) are used interchangeably here to refer to a process in which a genetic element (for example, a polynucleotide) is introduced into a cell other than by mitosis or meiosis. The element can be heterologous to the cell, or it can be an additional copy or improved version of an element already present in the cell. Genetic alteration can be carried out, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, precipitation with calcium phosphate or contact with a polynucleotide-liposome complex. Genetic alteration can also be carried out, for example, by transduction or infection with a DNA or RNA virus or viral vector. Usually, the genetic element is introduced into a chromosome or minicromosome in the cell; but any changes that alter the phenotype and / or genotype of the cell and its progeny are included in this term.
[0077] With regard to cell modification, the terminology "genetically modified" or "transformed" or "transfected" or "transduced" by exogenous DNA (for example, through a recombinant virus) refers to when such DNA was introduced into the cell. The presence of exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the cell's genome. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.
[0078] As used by the present invention, a cell is referred to as "stably" altered, transduced, genetically modified or transformed with a genetic sequence if the sequence is available to perform its function during prolonged cell culture in vitro and / or for an extended period of time in vivo. In general, such a cell is “inherited” altered (genetically modified) insofar as a genetic change is introduced that is also inherited by the progeny of the altered cell.
[0079] The terms "polypeptide", "peptide" and "protein" are used interchangeably here to refer to polymers of amino acids of any length. The terms also cover a polymer of amino acids that has been modified; for example, formation of disulfide bonds, glycosylation, lipidation, phosphorylation or conjugation with a label component. Polypeptides such as antiangiogenic polypeptides, neuroprotective polypeptides and the like, when discussed in the context of applying a gene product to an individual mammal, and compositions for it, refer to the respective intact polypeptide, or to any genetically modified fragment or derivative thereof. , which retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding antiangiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in applying a gene product to an individual mammal (which may be referred to as "transgenes" to be applied to a recipient cell), include polynucleotides that encode the intact polypeptide or any genetically modified fragment or derivative that has the desired biochemical function.
[0080] As used by the present invention, a plasmid, nucleic acid, vector, virus, virion, host cell, protein or other "isolated" substance refers to a preparation of the substance devoid of at least some of the other components that may also be present. present where the substance or similar substance occurs naturally or is initially prepared from. Thus, for example, an isolated substance can be prepared using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured against a second potentially interfering substance present in the source mixture. Increasing enrichment of the modalities of this disclosure are increasingly isolated. A plasmid, nucleic acid, vector, virus, host cell or other isolated substance is, in some embodiments, purified, for example, from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.
[0081] As used by the present invention, the terms "treatment", "treat" and the like, refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or it can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment", as used by the present invention, encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease (and / or symptoms caused by the disease) from occurring in an individual who may be predisposed to the disease or at risk of acquiring the disease, but have not yet been diagnosed as having the disease; (b) inhibiting the disease (and / or symptoms caused by the disease), that is, preventing its development; and (c) alleviating the disease (and / or symptoms caused by the disease), that is, causing the disease to regress (and / or symptoms caused by the disease), that is, improving the disease and / or one or more symptoms of the disease . For example, the compositions and methods in question can be directed towards the treatment of retinal disease. Non-limiting methods for assessing retinal diseases and their treatment include measuring retinal function and its changes, for example, changes in visual acuity (for example, corrected visual acuity, ambulation, navigation, object detection and discrimination), changes in the visual field (for example, static perimetry and kinetic of the visual field), clinical examination (slit lamp examination of the anterior and posterior segments of the eye), electrophysiological responsiveness to all light and dark wavelengths (for example, all forms of electroretinography (ERG) and standard], all forms of visual evoked potential (VEP), electro-oculography (EOG), color vision, dark adaptation and / or contrast sensitivity, measuring changes in anatomy or health with use of anatomical and / or photographic measurements, for example, Optical Conference Tomography), fundus photography, adaptive optics, laser scanning ophthalmoscopy, fluorescence and / or autoflu orescence, eye motility measurement and eye movements (for example, nys tagmus, fixation preference and stability), measuring reported results (changes reported by the patient in visual and non-visually guided activities and behaviors, results reported by the patient [PRO], assessments based on quality of life questionnaires, daily activities and measures of neurological function (eg functional magnetic resonance imaging (MRI)).
[0082] The terms "individual", "host", "subject" and "patient" are used interchangeably here, and refer to a mammal, including, but without limitation, humans; non-human primates, including apes; sport animals of mammals (eg, horses); mammal farm animals (for example, sheep, goats, etc.); pets (dogs, cats, etc.); and rodents (for example, mice, rats, etc.).
[0083] In some embodiments, the individual is a human who has previously been naturally exposed to AAV and, as a result, houses anti-AAV antibodies (ie, neutralizing AAV antibodies). In some embodiments, the individual is a human who was previously administered an AAV vector (and as a result may harbor anti-AAV antibodies) and needs vector readministration to treat a different condition or to further treat the same condition. Based on positive results in clinical trials involving the application of the AAV gene to, for example, liver, muscle and retina - all tissues affected by neutralizing antibodies against this vehicle - there are many such therapeutic applications / targets for disease.
[0084] The term "effective amount" as used by the present invention is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For the purposes of this disclosure, an effective amount of a compound (for example, an infectious rAAV virion) is an amount that is sufficient to palliate, improve, stabilize, reverse, prevent, delay, or delay progression (and / or symptoms associated with ) a particular disease state (for example, a retinal disease). Accordingly, an effective amount of an infectious rAAV virus is an amount of the infectious rAAV virus that is capable of effectively delivering a heterologous nucleic acid to a target cell (or target cells) of the individual. Effective amounts can be determined preclinically, for example, by detecting in the cell or tissue the gene product (RNA, protein) that is encoded by the heterologous nucleic acid sequence using techniques that are well known in the art, for example, RT-PCR , western blot, ELISA, fluorescence or other reporter readings and the like. Effective amounts can be determined clinically, for example, by detecting a change in disease onset or progression using methods known in the art, for example, fundus autofluorescence, fluorescein angiography, OCT, microperimetry, adaptive optics, etc. as known in the art.
[0085] The terminology “retinal cell” refers to any of the types of cells that make up the retina, such as, but not limited to, retinal ganglion cells (RG), amacrine cells, horizontal cells, bipolar cells, photoreceptors, Müller glial cells, microglial cells and retinal pigment epithelium (RPE). The term "photoreceptor cells" refers in the present invention to, but is not limited to, stem cells or "stems" and conical cells or "cones". The term “Müller cells” or “Müller glia” refers to the glial cells that support neurons in the vertebrate retina.
[0086] The term “directed evolution” refers to a capsid engineering methodology, in vitro and / or in vivo, that emulates natural evolution through iterative cycles of genetic diversification and selection processes, accumulating beneficial mutations that improve progressively the function of a biomolecule. Targeted evolution often involves an in vivo method referred to as "biopanning" for the selection of AAV variants from a library, which have a more efficient level of infectivity for a type of cell or tissue of interest. DETAILED DESCRIPTION
[0087] Adeno-associated viruses (AAVs) are a parvovirus family with a 4.7 kb single-stranded DNA genome contained within an non-enveloped capsid. The viral genome of a naturally occurring AAV has 2 inverted terminal repeats (ITR) - which function as the origin of viral replication and packaging signal - flanking 2 primary open reading frames (ORF): rep (which encodes proteins that work in the viral replication, transcriptional regulation, specific site integration and assembly of the virion) and cap. The cap ORF encodes 3 structural proteins that come together to form a 60 mer viral capsid. Many naturally occurring AAV variants and serotypes have been isolated and none have been associated with human disease.
[0088] Recombinant versions of AAV can be used as gene application vectors, where a marker or therapeutic gene of interest is inserted between the ITRs in place of rep and cap. These vectors have shown that they transduce both cells into divided and undivided cells in vitro and in vivo and can result in stable transgenic expression for years in post-mitotic tissue. See, for example, Knipe DM, Howley PM. Fields ’Virology. Lippincott Williams & Wilkins, Philadelphia, PA, USA, 2007; Gao G-P, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A 2002; 99: 11854-9; Atchison RW, Casto BC, Hammon WM. Adenovirus-Associated Defective Virus Particles. Science 1965; 149: 754-6; Hoggan MD, Blacklow NR, Rowe WP. Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics. Proc Natl Acad Sci U S A 1966; 55: 1467-74; Blacklow NR, Hoggan MD, Rowe WP. Isolation of adenovirus-associated viruses from man. Proc Natl Acad Sci U S A 1967; 58: 1410-5; Bantel-Schaal U, zur Hausen H. Characterization of the DNA of a defective human parvovirus isolated from a genital site. Virology 1984; 134: 52-63; Mayor HD, Melnick JL. Small deoxyribonucleic acid-containing viruses (picodnavirus group). Nature 1966; 210: 331-2; Mori S, Wang L, Takeuchi T, Kanda T. Two novel adeno-associated viruses from Cinomolgos monkey: pseudotyping characterization of capsid protein. Virology 2004; 330: 375-83; Flotte TR. Gene therapy progress and prospects: recombinant adeno-associated virus (rAAV) vectors. Gene Ther 2004; 11: 805-10.
[0089] Recombinant AAV (referred to here simply as "AAV") has provided promising results in an increasing number of clinical trials. However, there are impediments to the application of genes that may limit the usefulness of AAV, such as anti-capsid immune responses, low transduction of certain tissues, an inability to apply to specific types of cells and a relatively low load capacity. In many situations, there are insufficient knowledge of mechanisms to effectively enable rational design with the ability to improve AAV. Alternatively, targeted evolution emerged as a strategy to create new variants of AAV that meet specific biomedical needs. Targeted evolution strategies take advantage of the processes of diversification and genetic selection to allow the accumulation of beneficial mutations that progressively improve the function of a biomolecule. In this process, wild-type AAV cap genes are diversified to create large genetic libraries that are packaged to generate libraries of viral particles, and selective pressure is applied to isolate new variants that can overcome barriers to gene application. It is important to note that the mechanistic basis underlying a gene application problem need not be known for the directed evolution of function, which can, thus, accelerate the development of improved vectors.
[0090] Typically, the variants disclosed by the present invention were generated through the use of an AAV library and / or libraries. Such an AAV library or libraries is / are generated (s) by mutating the cap gene, the gene that encodes the structural proteins of the AAV capsid, through a range of known directed evolution techniques available to the person skilled in the art. in the field of viral genome engineering. See, for example, Bartel et al. Am. Soc. Gene Cell Ther. 15th Annu. Meet. 20, S140 (2012); Bowles, D. et al. J. Virol. 77, 423-432 (2003); Gray et al. Mol. Ther. 18, 570-578 (2010); Grimm, D. et al. J. Virol. 82, 5887-5911; Koerber, J. T. et al. Mol. Ther. 16, 1703-1709 (2008); Li W. et al. Mol. Ther. 16, 1252-1260 (2008); Koerber, J. T. et al. Methods Mol. Biol. 434, 161-170 (2008); Koerber, J. T. et al. Hum. Gene Ther. 18, 367-378 (2007); and Koerber, J. T. et al. Mol. Ther. 17, 2088-2095 (2009). Such techniques, without limitation, are as follows: i) prone to PCR errors to introduce random point mutations into the AAV cap open reading frame (ORF) at a predetermined, modifiable rate; ii) Viral recombination in vitro or in vivo or “DNA mix” to generate random chimeras of AAV cap genes to produce a gene library with multiple AAV serotypes; iii) insertions of random peptides in defined sites of the capsid by ligation of degenerated oligonucleotides in the cap ORF; iv) defined insertions of sequences encoding peptides at random sites of the AAV cap ORF using transposon mutagenesis; v) replacement of AAV capsid surface loops by libraries of bioinformatically designed peptide sequences based on the conservation level of each amino acid position among the AAV serotypes and natural variants to generate "loop-exchange" libraries; vi) random substitution of amino acids in degeneration positions among AAV serotypes to generate libraries of ancestral variants (Santiago-Ortiz et al., 2015); and a combination of such techniques.
[0091] The shuffling of DNA generates chimeras that combine their parenting properties in unique and often beneficial ways; however, some may be unable to package which, in effect, reduces library diversity. The concentration of library diversity is achieved through peptide insertion techniques such as, but not limited to, iii-iv) above. The library's diversity also focuses on techniques such as v) above, and this concentration is directed to multiple hypervariable regions, which are found in exposed handles on the surface, of the AAV capsid. Although many of the techniques generate capsids from variants with only a small area of the mutated capsid, these techniques can be combined with additional mutagenesis strategies to modify the entire capsid.
[0092] Once the AAV library or libraries is / are generated, the viruses are then packaged in such a way that each AAV particle is comprised of a mutant capsid involving a cap gene encoding that capsid and purified. The library variants are then subjected to known and readily available in vitro and / or in vivo selective pressure techniques for those skilled in the art in the field of AAV. See, for example, N. et al. Nature Biotech. 24, 198-204 (2006); Dalkara, D. et al. Sci. Transl. Med. 5, 189ra76 (2013); Lisowski, L. et al. Nature. 506, 382-286 (2013); Yang, L. et al. PNAS. 106, 3946-3951 (2009); Gao, G. et al. Mol. Ther. 13, 77-87 (2006); and Bell, P. et al. Hum. Gene. The R. 22, 985-997 (2011). For example, but not limited to, AAV variants can be selected using i) affinity columns in which the elution of different fractions produces variants with altered binding properties; ii) primary cells - isolated from tissue samples or immortal cell lines that mimic the behavior of cells in the human body - that produce AAV variants with increased efficacy and / or tissue specificity; iii) animal models - that mimic a clinical gene therapy environment - that produce AAV variants that have successfully infected the target tissue; iv) human xenograft models that produce AAV variants that have infected grafted human cells; and / or a combination of techniques for selecting them.
[0093] Once the viruses are selected, they can be recovered by known techniques, such as, but not limited to, adenovirus-mediated replication, PCR amplification, new generation sequencing and cloning and the like. The virus clones are then enriched through repeated cycles of the selection techniques and the AAV DNA is isolated to recover the selected variant cap genes of interest. Such selected variants can be subjected to further modification or mutation and, as such, serve as a new starting point for additional selection steps to iteratively increase AAV viral fitness. However, in certain cases, successful capsids have been generated without further mutation.
[0094] The AAV variants revealed by the present invention were generated at least in part through the use of in vivo directed evolution methodology, such as the techniques described above, involving the use of primate retinal screens after intravitreal administration. Accordingly, the AAV variant capsids disclosed by the present invention comprise one or more modifications in the amino acid sequence that confer more efficient transduction of primate retinal cells than a corresponding parental AAV capsid protein. As used by the present invention, a "corresponding parent AAV capsid protein" refers to an AAV capsid protein of the same wild type serotype or AAV variant as the variant AAV capsid protein in question, but which does not comprise the one or more amino acid sequence modifications of the variant AAV capsid protein in question.
[0095] In some embodiments, the variant AAV capsid protein in question comprises a heterologous peptide of about 5 amino acids to about 20 amino acids inserted by covalent bond into a GH loop of the AAV capsid protein or loop IV, in relative to a corresponding parental AAV capsid protein. By "GH loop", or loop IV, of the AAV capsid protein is meant the solvent accessible part called in the art as the GH loop, or loop IV, of the AAV capsid protein. For the AH capsid GH loop / IV loop, see, for example, van Vliet et al. (2006) Mol. Ther. 14: 809; Padron et al. (2005) J. Virol. 79: 5047; and Shen et al. (2007) Mol. Ther. 15: 1955. Thus, for example, the insertion site may be within amino acids 411-650 of an AAV VP1 capsid protein. For example, the insertion site can be within amino acids 571-612 of AAV1 of VP1, amino acids 570-611 of VP1 of AAV2, within amino acids 571-612 of VP1 of AAV3A, within amino acids 571- 612 of VP1 of AAV3B , within amino acids 569-610 of VP1 of AAV4, in amino acids 560-601 of VP1 of AAV5, in amino acids 571 to 612 of VP1 of AAV6, in amino acids 572 to 613 of VP1 of AAV7, in amino acids 573 to 614 of VP1 of of AAV8, within amino acids 571 to 612 of VP1 of AAV9, or within amino acids 573 to 614 of VP1 of AAV10, or the corresponding amino acids of any of the variants thereof. Those skilled in the art would know, based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes, in which an insertion site "corresponding to the AAV2 amino acids" would be in a capsid protein of any given AAV serotype. . See also Figure 6 for an alignment of SEQ ID NOS: 1-11 of wild-type AAV that provides amino acid sites between and through the wild-type (naturally occurring) AAV1, AAV2, AAV3A, AAV3B and AAV4-10 serotypes.
[0096] In certain embodiments, the insertion site is a unique insertion site between two adjacent amino acids located between amino acids 570-614 of VP1 of any wild type AAV serotype or AAV variant, for example, the insertion site is between two adjacent amino acids located at amino acids 570-610, amino acids 580-600, amino acids 570-575, amino acids 575-580, amino acids 580-585, amino acids 585-590, amino acids 590-600, or amino acids 600-614 of VP1 of any AAV serotype or variant. For example, the insertion site can be between amino acids 580 and 581, amino acids 581 and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589 , or amino acids 589 and 590. The insertion site can be between amino acids 575 and 576, amino acids 576 and 577, amino acids 577 and 578, amino acids 578 and 579, or amino acids 579 and 580. The insertion site can be between amino acids 590 and 591, amino acids 591 and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids 594 and 595, amino acids 595 and 596, amino acids 596 and 597, amino acids 597 and 598, amino acids 598 and 599, or amino acids 599 and 600. For example, the insertion site can be between AAV2 amino acids 587 and 588, between AAV1 amino acids 590 and 591, between AAV3A amino acids 588 and 589, between AAV3B amino acids 588 and 589, between AAV3B amino acids amino acids 584 and 585 of AAV4, between amino acids 575 and 576 of AAV5, between amino acids acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.
[0097] In some embodiments, a peptide insert disclosed by the present invention has a length of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids or 20 amino acids. In another embodiment, a peptide insert disclosed by the present invention comprises from 1 to 4 spacer amino acids at the amine terminal (N-terminus) and / or at the carboxyl terminus (C-terminus) of any of the peptide inserts disclosed by the present invention. Examples of spacer amino acids include, but are not limited to, leucine (L), alanine (A), glycine (G), serine (S), threonine (T) and proline (P). In certain embodiments, a peptide insert comprises 2 spacer amino acids at the N-terminal and 2 spacer amino acids at the C-terminal. In other embodiments, a peptide insert comprises 2 spacer amino acids at the N-terminal and 1 spacer amino acids at the C-terminal.
[0098] The peptide insertions disclosed by the present invention have not been previously described and / or inserted into an AAV capsid. Without the desire to be bound by theory, the presence of any of the disclosed peptide inserts can act to decrease the affinity of the variant capsid for heparin sulfate, which probably reduces the binding to the extracellular matrix in front of the primate's retina. In addition, the peptide insertion patterns disclosed by the present invention can confer increased transduction of primate retinal cells by adding a cell surface receptor binding domain.
[0099] In some preferred embodiments, the insertion peptide comprises an amino acid sequence of any of the formulas below.
[00100] In some respects, an insertion peptide can be a peptide 7 to 10 amino acids in length, of Formula 1a: Y1Y2X1X2X3X4X5X6X7Y3 where each of Y1-Y3, if present, is selected independently from Ala, Leu, Gly, Ser , Thr, Pro X 1 is selected from Gln, Asn, His, Ile and Ala X 2 is selected from Ala, Gln, Asp, Ser, Lys and Pro X 3 is selected from Asp, Ile, Thr and Asn X4 is selected from Thr, Ser, Tyr, Gln, Glu, Ala and X 5 is selected from Thr, Lys and Asn X6 is selected from Lys, Asn and Glu X7 is selected from Asn, Thr, lie, His, Asp and Ala.
[00101] In certain embodiments, the Formula 1a insertion peptide comprises an amino acid sequence selected from QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), ASDSTKA (SEQ ID NO: 15), NQDYTKT ( SEQ NO: 16), HDITKNI (SEQ ID NO: 17), HPDTTKN (SEQ ID NO: 18), HQDTTKN (SEQ ID NO: 19), NKTTNKD (SEQ ID NO: 20), ISNENEH (SEQ ID NO: 21) and QANANEN (SEQ ID NO: 22).
[00102] In other respects, an insertion peptide can be a peptide of 7 to 10 amino acids in length, of Formula 1b: Y1Y2X1X2X3X4X5X6X7Y3 where each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser , Thr, Pro X1 is selected from Gln, Asn, His and Ile X2 is selected from Ala, Gln, Asp and Ser X3 is selected from Asp and Ile X4 is selected from Thr, Tyr and Gln X5 is selected from Thr and Lys X6 is selected from Lys and Asn X7 is selected from Asn, Thr, Ile and His
[00103] In certain embodiments, the Formula 1b insertion peptide comprises an amino acid sequence selected from QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), NQDYTKT (SEQ NO: 16), HDITKNI (SEQ ID NO: 17) and HQDTTKN (SEQ ID NO: 19).
[00104] In other respects, an insertion peptide can be a peptide of 7 to 10 amino acids in length, of Formula 1c. Y1Y2X1X2AspX3ThrLysX4Y3 where each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro X1 is selected from Gln, Asn, His and Ile X2 is selected from Ala, Gln and Ser X3 is selected among Thr, Tyr and Gln X4 is selected from Asn, Thr and His
[00105] In certain embodiments, the Formula 1c insertion peptide comprises an amino acid sequence selected from QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), NQDYTKT (SEQ NO: 16) and HQDTTKN (SEQ ID NO: 19).
[00106] In other respects, an insertion peptide can be a peptide 7 to 10 amino acids in length, of Formula 1d: Y1Y2X1X2AspX3ThrThrX4Y3 where each of Y1-Y3, if present, is selected independently from Ala, Leu, Gly, Ser , Thr, Pro X1 is selected from Gln and Ile X2 is selected from Ala and Ser X3 is selected from Thr and Gln X4 is selected from Asn and His
[00107] In certain embodiments, the Formula 1d insertion peptide comprises an amino acid sequence selected from QADTTKN (SEQ ID NO: 13) and ISDQTKH (SEQ ID NO: 14).
[00108] In other respects, an insertion peptide can be a peptide of 7 to 11 amino acids in length, of Formula Ie. Y1Y2X1X2AsnX3AsnGluX4Y3 Where each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro X1 is selected from Gln and Ile X2 is selected from Ala and Ser X3 is selected from Glu and Ala X4 is selected from Asn and His
[00109] In other embodiments, a Formula 1e insertion peptide comprises an amino acid sequence selected from ISNENEH (SEQ ID NO: 21), and QANANEN (SEQ ID NO: 22).
[00110] In yet another embodiment, an insertion peptide can be a peptide of 7 to 11 amino acids in length, of Formula IIa: Y1Y2X1X2DX3TKX4Y3. wherein each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro X1 is selected from Q, N, A, H, and I; X2 is selected from Q, A, P, and S; X3 is selected from T, Y, S, and Q; and X4 is selected from T, N, A and H.
[00111] In another embodiment of a peptide insertion of an amino acid sequence of the formula X1X2DX3TKX4, the peptide insertion is selected from the group consisting of QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), ASDSTKA, NQDYTKT (SEQ NO: 16), HQDTTKN (SEQ ID NO: 19) and HPDTTKN (SEQ ID NO: 18).
[00112] In some of these modalities, an insertion peptide can be a peptide of 7 to 11 amino acids in length, of Formula IIb: Y1Y2X1X2DX3TKX4Y3
[00113] Where each of Y1-Y3, if present, is selected independently from Ala, Leu, Gly, Ser, Thr, Pro X1 is selected from N, A and H; X2 is selected from Q, P, and S; X3 is selected from T, Y, and S; and X4 is selected from T, N and A.
[00114] In another embodiment of a peptide insertion of an amino acid sequence of the formula X1X2DX3TKX4, the peptide insertion is selected from the group consisting of ASDSTKA, NQDYTKT (SEQ NO: 16), HQDTTKN (SEQ ID NO: 19) and HPDTTKN (SEQ ID NO: 18).
[00115] In other embodiments, the insertion peptide comprises an amino acid sequence selected from KDRAPST (SEQ ID NO: 26), TNRTSPD (SEQ ID NO: 24), PNSTHGS (SEQ ID NO: 25) and GKSKVID (SEQ ID NO : 23).
[00116] In some embodiments, the insertion peptide comprises an amino acid sequence selected from ASDSTKA (SEQ ID NO: 15), QANANEN (SEQ ID NO: 22), QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: : 14), NQDYTKT (SEQ ID NO: 16), HDITKNI (SEQ ID NO: 17), HPDTTKN (SEQ ID NO: 18), HQDTTKN (SEQ ID NO: 19), NKTTNKD (SEQ ID NO: 20), ISNENEH (SEQ ID NO: 21), GKSKVID (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), PNSTHGS (SEQ ID NO: 25) and KDRAPST (SEQ ID NO: 26)
[00117] In other preferred embodiments, the insertion peptide has 1 to 3 spacer amino acids (Y1-Y3) at the amino and / or carboxyl terminus of an amino acid sequence selected from QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), ASDSTKA (SEQ ID NO: 15), NQDYTKT (SEQ ID NO: 16), HDITKNI (SEQ ID NO: 17), HPDTTKN (SEQ ID NO: 18), HQDTTKN (SEQ ID NO: 19) , NKTTNKD (SEQ ID NO: 20), ISNENEH (SEQ ID NO: 21), QANANEN (SEQ ID NO: 22), GKSKVID (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), PNSTHGS (SEQ ID NO: 24) NO: 25) and KDRAPST (SEQ ID NO: 26). In certain such modalities, the insertion peptide is selected from the group consisting of: LAQADTTKNA (SEQ ID NO: 27), LAISDQTKHA (SEQ ID NO: 28), LGISDQTKHA (SEQ ID NO: 29), LAASDSTKAA (SEQ ID NO: 29) : 30), LANQDYTKTA (SEQ ID NO: 31), LAHDITKNIA (SEQ ID NO: 32), LAHPDTTKNA (SEQ ID NO: 33), LAHQDTTKNA (SEQ ID NO: 34), LANKTTNKDA (SEQ ID NO: 35), LPISNENEHA (SEQ ID NO: 36), LPQANANENA (SEQ ID NO: 37), LAGKSKVIDA (SEQ ID NO: 38), LATNRTSPDA (SEQ ID NO: 39), LAPNSTHGSA (SEQ ID NO: 40) and LAKDRAPSTA (SEQ ID NO: 41).
[00118] In some embodiments, the AAV variant capsid protein in question does not include any other amino acid sequence modifications other than a peptide insert of about 5 amino acids to about 20 amino acids in the GH loop, or in the loop of IV. For example, in some embodiments, the AAV variant capsid protein comprises a peptide insert comprising an amino acid sequence selected from the group consisting of QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), ASDSTKA (SEQ ID NO: 15), NQDYTKT (SEQ ID NO: 16), HDITKNI (SEQ ID NO: 17), HPDTTKN (SEQ ID NO: 18), HQDTTKN (SEQ ID NO: 19), NKTTNKD (SEQ ID NO: 20), ISNENEH (SEQ ID NO: 21), QANANEN (SEQ ID NO: 22), GKSKVID (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), PNSTHGS (SEQ ID NO: 25), KDRAPST ( SEQ ID NO: 26), LAQADTTKNA (SEQ ID NO: 27), LAISDQTKHA (SEQ ID NO: 28), LGISDQTKHA (SEQ ID NO: 29), LAASDSTKAA (SEQ ID NO: 30), LANQDYTKTA (SEQ ID NO: 31) ), LAHDITKNIA (SEQ ID NO: 32), LAHPDTTKNA (SEQ ID NO: 33), LAHQDTTKNA (SEQ ID NO: 34), LANKTTNKDA (SEQ ID NO: 35), LPISNENEHA (SEQ ID NO: 36), LPQANANENA (SEQ ID NO: 37), LAGKSKVIDA (SEQ ID NO: 38), LATNRTSPDA (SEQ ID NO: 39), LAPNSTHGSA (SEQ ID NO: 40) and LAKDRAPSTA (SEQ ID NO: 41), and the AAV variant capsid does not includes any other sub amino acid substitutions, insertions or deletions (that is, the AAV variant capsid protein comprises said insertion and, on the other hand, is identical to the corresponding AAV capsid protein). In other words, the AAV variant capsid protein comprising said insertion is otherwise identical to the parental AAV capsid protein in which the peptide has been inserted. As another example, the AAV variant capsid protein in question comprises a peptide insert having an amino acid sequence selected from QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), ASDSTKA (SEQ ID NO: 15), NQDYTKT (SEQ ID NO: 16), HDITKNI (SEQ ID NO: 17), HPDTTKN (SEQ ID NO: 18), HQDTTKN (SEQ ID NO: 19), NKTTNKD (SEQ ID NO: 20), ISNENEH ( SEQ ID NO: 21), QANANEN (SEQ ID NO: 22), GKSKVID (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), PNSTHGS (SEQ ID NO: 25), KDRAPST (SEQ ID NO: 26) ), LAQADTTKNA (SEQ ID NO: 27), LAISDQTKHA (SEQ ID NO: 28), LGISDQTKHA (SEQ ID NO: 29), LAASDSTKAA (SEQ ID NO: 30), LANQDYTKTA (SEQ ID NO: 31), LAHDITKNIA (SEQ ID NO: 32), LAHPDTTKNA (SEQ ID NO: 33), LAHQDTTKNA (SEQ ID NO: 34), LANKTTNKDA (SEQ ID NO: 35), LPISNENEHA (SEQ ID NO: 36), LPQANANENA (SEQ ID NO: 37) , LAGKSKVIDA (SEQ ID NO: 38), LATNRTSPDA (SEQ ID NO: 39), LAPNSTHGSA (SEQ ID NO: 40) and LAKDRAPSTA (SEQ ID NO: 41), where the peptide insert is located between amino acids 587 and 588 of VP1 of the capsid of AAV2 or the corresponding amino acids of a VP1 of another parental AAV, for example, between amino acids 588 and 589 of VAV of AAV1, AAV3A, AAV3B, AAV6 or AAV9, between amino acids 586 and 587 of VP1 of AAV4, between amino acids 577 and 578 of VAV of AAV5, between amino acids 589 and 590 of VP1 of AAV7, between amino acids 590 to 591 of VP1 of AAV8 or AAV10, etc., where the variant AAV capsid protein sequence is otherwise , identical to the corresponding parental AAV capsid protein sequence, for example, SEQ ID NOs: 1-12.
[00119] In other embodiments, the variant AAV capsid protein in question, in addition to comprising a peptide insert, for example, as disclosed herein or as known in the art, comprises about 1 to about 100 amino acid substitutions or deletions , for example, 1 to about 5, about 2 to about 4, about 2 to about 5, about 5 to about 10, about 10 to about 15, about 15 at about 20, about 20 to about 25, about 25-50, about 50-100 amino acid substitutions or deletions compared to the parental AAV capsid protein. Thus, in some embodiments, a variant capsid protein in question comprises an amino acid sequence with a sequence identity of 85% or more, 90% or more, 95% or more, or 98% or more, for example, or 99% identity to the corresponding parent AAV capsid, for example, a wild type capsid protein, as set out in SEQ ID NOs: 1-12.
[00120] In another embodiment, the one or more amino acid substitutions are in amino acid residue (s) 1, 15, 34, 57, 66, 81, 101, 109, 144, 164, 176 188, 196, 226, 236, 240, 250, 312, 363, 368, 449, 456, 463, 472, 484, 524, 535, 551, 593, 698, 708, 719, 721, and / or 735 da AAV2 VP1 capsid protein as numbered before insertion of the peptide, or the corresponding amino acid residue (s) of another AAV capsid protein. In some of these modalities, one or more amino acid substitutions are selected from the group consisting of M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, and L735Q of peptide, or the corresponding amino acid residue (s) of another AAV capsid protein.
[00121] In a preferred embodiment, a variant AAV capsid protein comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from ISDQTKH (SEQ ID NO: 14), LGISDQTKHA (SEQ ID NO: 29) and LAISDQTKHA (SEQ ID NO: 28), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the substitution corresponding in another parental serotype of AAV (that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R , S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, I5S, V5, , V719M, S721L, L735Q and a combination thereof. In some embodiments, one or more amino acid substitutions are selected from the group consisting of: M1L + L15P + P535S, P34A, P34A + S721L, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, Q164K + V708I, T176P, L188I, S196Y, G226E, G236V, I240T, N312K, N312K + N449D + D472N + N551S + I698V + L735Q, P363L, R484C + V708I, T456K and V708I. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype.
[00122] In a particularly preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LAISDQTKHA (SEQ ID NO : 28) or LGISDQTKHA (SEQ ID NO: 29) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid, and further comprises a substitution of amino acid P34A at residue 34 with respect to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding residue of another AAV capsid. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more, sequence identity of amino acids over the entire length of the amino acid sequence shown in SEQ ID NO: 2 or the corresponding parental AAV capsid. In a particularly preferred embodiment, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least 98% sequence identity or is 100 % identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKAAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAISDQTKHARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENS KR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 42)
[00123] In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the LAISDQTKHA amino acid sequence (SEQ ID NO : 28) or LGISDQTKHA (SEQ ID NO: 29) between amino acids 587 and 588 of the AAV2 capsid protein or at the corresponding position in the capsid protein of another AAV serotype and comprises an N312K amino acid substitution compared to the sequence of AAV2 capsid amino acids (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype and optionally further comprises amino acid substitutions N449D, D472N, N551S, I698V and / or L735Q compared to the amino acid sequence of capsids of AAV2 or the corresponding substitutions in another parental AAV serotype. In a particularly preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LAISDQTKHA (SEQ ID NO: 28) or LGISDQTKHA (SEQ ID NO: 29) between amino acids 587 and 588 of AAV2 of AAV2 capsid of AAV2 or the corresponding position in the capsid protein of another serotype and comprises amino acid substitutions N312K, N449D, D472N, N551S, I698V and L735Q compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or substitutions in the corresponding residues in another parental AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In a particularly preferred embodiment, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least 98% identity or sequence is 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLKFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSE YQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYF PSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTD TPSGTTTQSRLQFSQAGASDIRNQSRNWLPGPCYRQQRVSKTSADNNNSE YSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEK TSVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAISDQTKHARQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGL KHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVE IEWELQKENSK RWNPEVQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNQ (SEQ ID NO: 43)
[00124] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578. from AAV5, amino acids 589 and 590 from AAV7, or amino acids 590 to 591 from AAV8 or AAV10, the peptide insert comprising a selected amino acid sequence from ISDQTKH (SEQ ID NO: 14), LGISDQTKHA (SEQ ID NO : 29) and LAISDQTKHA (SEQ ID NO: 28), and b) a replacement of valine by isoleucine at amino acid 709 of AAV3A or AAV3B, a replacement of alanine by isoleucine at position 709 of AAV1 or AAV6, a replacement of asparagine by isoleucine at amino acid 707 of AAV4 or amino acid 709 of AAV9 or a substitution of threonine for isoleucine in amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10 or a substitution of glutamine for isoleucine in amino acid 697 of AAV5 and optionally, on the other hand, identical to any r one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LAISDQTKHA (SEQ ID NO: 28 ) or LGISDQTKHA (SEQ ID NO: 29) between amino acids 587 and 588 of AAV2 capsid and b) a substitution of amino acid valine for isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, where the variant capsid protein it comprises from 2 to 5 from 5 to 10, or from 10 to 15 amino acid substitutions.
[00125] In yet another embodiment, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LAISDQTKHA (SEQ ID NO: 28) or LGISDQTKHA (SEQ ID NO: 29) between amino acids 587 and 588 of AAV2 capsid and b) a substitution of amino acid valine for isoleucine in amino acid 708 compared to the amino acid sequence of AAV2 and is, on the other hand similarly to the amino acid sequence of SEQ ID NO: 2.
[00126] In yet another embodiment, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LAISDQTKHA (SEQ ID NO: 28) or LGISDQTKHA (SEQ ID NO: 29) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the capsid from Variant AAV has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYS TPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLGISDQTKHARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 44)
[00127] In a preferred embodiment, a variant AAV capsid protein comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from QADTTKN (SEQ ID NO: 13) and LAQADTTKNA (SEQ ID NO: 27), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype (or that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I59, E19 preferably selected from S109T, P250S, A524T, A593E, I698V, V708I, and / or V719M. The peptide insertion site is preferably located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. In a particularly preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO: 27) between AAV2 capsid amino acids 587 and 588 or the corresponding position in the capsid protein of another AAV serotype and comprises an I698V amino acid substitution compared to the AAV2 amino acid sequence or the corresponding substitution in another AAV parental serotype, wherein the substituted amino acid (s) does not occur naturally in the corresponding position. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In some embodiments, the corresponding amino acid substitution is an I699V amino acid substitution compared to the AAV3A, AAV3B or AAV9 capsid amino acid sequence, an I687V substitution compared to the amino acid sequence of AAV5, an I700V substitution compared to the AAV7 amino acid sequence, an I701V substitution compared to the AAV8 or AAV10 amino acid sequence. In a particularly preferred embodiment, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least 98% sequence identity or is 100 % identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAQADTTKNARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENS KR WNPEVQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 45)
[00128] In other particularly preferred embodiments, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO : 27) between amino acids 587 and 588 of AAV2 capsid or the corresponding position in the capsid protein of another AAV serotype and comprises an amino acid substitution V719M and, optionally, an amino acid substitution V708I compared to the amino acid sequence of AAV2 or the corresponding substitution in another parental serotype of AAV, in which the substituted amino acid (s) do not naturally occur in the corresponding position.
[00129] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578. from AAV5, amino acids 589 and 590 from AAV7, or amino acids 590 to 591 from AAV8 or AAV10, the peptide insert comprising an amino acid sequence selected from QADTTKN (SEQ ID NO: 13) and LAQADTTKNA (SEQ ID NO : 27), and b) a substitution of valine for isoleucine at amino acid 709 of AAV3A or AAV3B, a substitution of alanine for isoleucine at position 709 of AAV1 or AAV6, a substitution of asparagine for isoleucine at amino acid 707 of AAV4 or amino acid 709 of AAV9 or a substitution of threonine for isoleucine at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10 or a substitution of glutamine for isoleucine at amino acid 697 of AAV5. In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578 from AAV5, amino acids 589 and 590 from AAV7 or amino acids 590 to 591 from AAV8 or AAV10, the peptide insert comprising an amino acid sequence selected from QADTTKN (SEQ ID NO: 13) and LAQADTTKNA (SEQ ID NO: 27), and eb ) a substitution of serine amino acids for threonine at position 109 compared to the amino acid sequence of AAV1, AAV3A, AAV3B, AAV4, AAV7, AAV8, AAV9 or AAV10 or at position 108 compared to the amino acid sequence of AAV5 or AAV6. In preferred embodiments, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO: 27) between the amino acids 587 and 588 of the AAV2 capsid and comprises a substitution of serine for threonine at amino acid 109 (S109T) or a substitution of amino acid valine for isoleucine at amino acid 708 (V708I) in comparison with the amino acid sequence of AAV2, where the protein of variant capsid comprises from 1 to 5, from 5 to 10, or from 10 to 15 amino acid substitutions and, preferably, at least about 85%, at least about 90%, at least about 95%, at least at least about 98% or greater amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO: 2. In other preferred embodiments, the variant AAV capsid comprises a peptide insert comprising comprising the amino acid sequence QADTTKN (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO: 27) between amino acids 587 and 588 of the AAV2 capsid or the corresponding position in the protein of capsid from another AAV serotype and comprises a substitution of serine for threonine at amino acid 109 and a substitution of amino acid valine for isoleucine at amino acid 708 compared to the amino acid sequence of AAV2.
[00130] In yet another embodiment, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO: 27) between amino acids 587 and 588 of AAV2 capsid and b) at least one amino acid substitution, wherein the variant capsid amino acid sequence does not comprise a substitution of isineucine for valine amino acid at amino acid 708 compared to amino acid sequence of AAV2. and does not comprise a substitution of serine for threonine at amino acid 109 as compared to the amino acid sequence of AAV2.
[00131] In yet another embodiment, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO: 27) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence of SEQ ID NO: 2.
[00132] In another preferred embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence selected from HDITKNI (SEQ ID NO: 17), IAHDITKNIA (SEQ ID NO: 60) and LAHDITKNIA (SEQ ID NO: 32), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental serotype of AAV (that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q , Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, R389S, N449D, T456K, A463, , I698V, V708I, V719M, S721L, L735Q and a combination thereof. In some embodiments, the AAV capsid protein comprises one or more amino acid substitutions selected from S109T, R389S, A593E and / or V708I. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. In a preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence HDITKNI (SEQ ID NO: 17) or comprising, consisting essentially of, or consisting of the amino acid sequence IAHDITKNIA (SEQ ID NO: 60) or LAHDITKNIA (SEQ ID NO: 32) between amino acids 587 and 588 of AAV2 capsid and comprises an amino acid substitution S109T compared to the amino acid sequence of AAV2 capsid or the corresponding substitution in another parental AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2.
[00133] In yet another embodiment, the variant capsid comprises a) a peptide insert comprising the amino acid sequence HDITKNI (SEQ ID NO: 17) or comprising, consisting essentially of, or consisting of the amino acid sequence IAHDITKNIA (SEQ ID NO : 60) or LAHDITKNIA (SEQ ID NO: 32) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the AAV capsid variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYS TPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAHDITKNIARQAAT ADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKH PPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRW NPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 46)
[00134] In other embodiments, the variant capsid comprises a) a peptide insert comprising, consisting essentially of, or consisting of the LAHDITKNIA amino acid sequence between amino acids 587 and 588 of AAV2 capsid and b) at least one amino acid substitution, wherein the variant capsid amino acid sequence does not comprise a substitution of the amino acid valine for isoleucine at amino acid 708 compared to the amino acid sequence of AAV2. In still other embodiments, the variant capsid comprises a) a peptide insert comprising the amino acid sequence DITKNIA (SEQ ID NO: 61) or comprising, consisting essentially of, or consisting of the sequence of amino acid sequences IAHDITKNIA (SEQ ID NO: 60) or LAHDITKNIA (SEQ ID NO: 32) between amino acids 587 and 588 of AAV2 capsid and b) a substitution of V708I compared to the amino acid sequence of AAV2. In other embodiments, the variant capsid comprises a) a peptide insert comprising, consisting essentially of, or consisting of the LAHDITKNIA amino acid sequence (SEQ ID NO: 32) between amino acids 587 and 588 of AAV2 capsid and b) two or more amino acid substitutions, wherein the variant capsid amino acid sequence comprises a substitution of the amino acid valine for isoleucine at amino acid 708 compared to the amino acid sequence of AAV2.
[00135] In another preferred embodiment, a variant AAV capsid protein comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from NQDYTKT (SEQ ID NO : 16) and LANQDYTKTA (SEQ ID NO: 31), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype ( that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K , T176P, L188I, S196Y, G226E, G236V, I240T, P250S, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698, themselves. In some embodiments, the AAV capsid protein comprises one or more amino acid substitutions selected from S109T, S109T + S463Y, D368H and V708I. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. In a preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence NQDYTKT (SEQ ID NO: 16) or comprising, consisting essentially of, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO: 31) between the AAV2 capsid amino acids 587 and 588 and comprise a V708I amino acid substitution compared to the AAV2 capsid amino acid sequence or the corresponding substitution in another AAV parental serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In yet another embodiment, the variant capsid comprises a) a peptide insert comprising the amino acid sequence NQDYTKT (SEQ ID NO: 16) or comprising, consisting essentially of, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO: 31) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNN WGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLANQDYTKTARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 47)
[00136] In other embodiments, the variant capsid comprises a) a peptide insert comprising the amino acid sequence NQDYTKT (SEQ ID NO: 16) or comprising, consisting essentially of, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO: 31) between amino acids 587 and 588 of AAV2 capsid and b) an amino acid substitution S109T compared to the sequence of SEQ ID NO: 2 and, optionally, an amino acid substitution S463Y, where the variant capsid is at least about 85%, at least about 90%, at least about 95%, at least about 98% identical to the entire length of the amino acid sequence shown in SEQ ID NO: 2. In related embodiments, the variant capsid comprises a) a peptide insert comprising the amino acid sequence NQDYTKT (SEQ ID NO: 16) or comprising, consisting essentially of, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO: 31) between amino acids 587 and 588 of c apsid of AAV2 and b) an amino acid substitution S109T compared to the amino acid sequence of SEQ ID NO: 2 and is otherwise identical to the amino acid sequence of SEQ ID NO: 2.
[00137] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578 from AAV5, amino acids 589 and 590 from AAV7 or amino acids 590 to 591 from AAV8 or AAV10, the peptide insert comprising a selected amino acid sequence from NQDYTKT (SEQ ID NO: 16) and LANQDYTKTA (SEQ ID NO: 31 ), and b) a substitution of the amino acid asparagine for lysine at position 313 compared to the amino acid sequence of AAV1 or AAV6, or at position 314 compared to the amino acid sequence AAV9, or a substitution of serine for lysine at position 312 of AAV3A or AAV3B or at position 315 of AAV8 or AAV10, or a substitution of arginine for lysine at position 303 of AAV4 or AAV5, or at position 314 of AAV7. In another embodiment, the variant capsid comprises a) a peptide insert comprising the amino acid sequence NQDYTKT (SEQ ID NO: 16) or comprising, consisting essentially of, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO: 31) between AAV2 capsid amino acids 587 and 588 and b) an N312K amino acid substitution, wherein the variant capsid protein comprises from 1 to 5, from 5 to 10, or from 10 to 15 amino acid substitutions.
[00138] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from PNSTHGS (SEQ ID NO : 25) and LAPNSTHGSA (SEQ ID NO: 40), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype ( that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K , T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, S7, E7, E7, E7, E7 combination thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. In a preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence PNSTHGS (SEQ ID NO: 25) or comprising, consisting essentially of, or consisting of the amino acid sequence LAPNSTHGSA (SEQ ID NO: 40) between amino acids 587 and 588 of AAV2 capsid and comprises an amino acid substitution V708I compared to the AAV2 capsid amino acid sequence or the corresponding substitution in another parental AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In yet another embodiment, the variant capsid comprises a) a peptide insert comprising the amino acid sequence PNSTHGS (SEQ ID NO: 25) or comprising, consisting essentially of, or consisting of the amino acid sequence LAPNSTHGSA (SEQ ID NO: 40) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNN WGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAPNSTHGSARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 48)
[00139] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from NKTTNKDA (SEQ ID NO : 62) and LANKTTNKDA (SEQ ID NO: 35), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype ( that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K , T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, S7, E7, E7, E7, E7 combination thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. In a preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence NKTTNKDA (SEQ ID NO: 62) or comprising, consisting essentially of, or consisting of the amino acid sequence LANKTTNKDA (SEQ ID NO: 35) between amino acids 587 and 588 of AAV2 capsid and comprises an amino acid substitution N449D compared to the AAV2 capsid amino acid sequence or the corresponding substitution in another parental AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In yet another embodiment, the variant capsid comprises a) a peptide insert comprising the amino acid sequence NKTTNKDA (SEQ ID NO: 62) or comprising, consisting essentially of, or consisting of the amino acid sequence LANKTTNKDA (SEQ ID NO: 35) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRP KRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLANKTTNKDARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 49)
[00140] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from TNRTSPD (SEQ ID NO : 24) and LATNRTSPDA (SEQ ID NO: 39), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype ( that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K , T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, LM7, E9 themselves. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. In a related embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert located between amino acids 588 and 589 of AAV1 VP1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578. from AAV5, amino acids 589 and 590 from AAV7, or amino acids 590 to 591 from AAV8 or AAV10, the peptide insert comprising an amino acid sequence selected from TNRTSPD (SEQ ID NO: 24) and LATNRTSPDA (SEQ ID NO: 39 ), and b) a substitution of valine for isoleucine at amino acid 709 of AAV3A or AAV3B, a substitution of alanine for isoleucine at position 709 of AAV1 or AAV6, a substitution of asparagine for isoleucine at amino acid 707 of AAV4 or amino acid 709 of AAV9 or one replacement of threonine with isoleucine at amino acid 710 of AAV7 or amino acid 711 from AAV8 or AAV10 or a replacement of glutamine with isoleucine at amino acid 697 of AAV5. In other embodiments, the variant capsid protein comprises a) a peptide insert comprising, consisting essentially of, or consisting of the amino acid sequence LATNRTSPDA (SEQ ID NO: 39) between amino acids 587 and 588 of AAV2 capsid and b) a substitution of amino acid valine for isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises from 1 to 5, from 5 to 10, or from 10 to 15 amino acid substitutions. In yet another embodiment, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence TNRTSPD (SEQ ID NO: 24) between amino acids 587 and 588 of AAV2 capsid and b) a substitution of valine amino acid for isoleucine at amino acid 708 compared to the AAV2 amino acid sequence. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2.
[00141] In yet another embodiment, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence TNRTSPD (SEQ ID NO: 24) or comprising, consisting essentially of, or consisting of the amino acid sequence LATNRTSPDA (SEQ ID NO: 39) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWG FRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLATNRTSPDARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 50)
[00142] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from GKSKVID (SEQ ID NO : 23) and LAGKSKVIDA (SEQ ID NO: 38), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype ( that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K , T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, S7, E7, E7, E7, E7 combination thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In some embodiments, the variant AAV capsid comprises a peptide insert located between amino acids 587 and 588 of AAV2 capsid comprising the amino acid sequence GKSKVID (SEQ ID NO: 23) or comprising, consisting essentially of, or consisting of the LAGKSKVIDA amino acid sequence. (SEQ ID NO: 38) and is otherwise identical to the amino acid sequence of SEQ ID NO: 2. In other embodiments, the variant AAV capsid comprises a) a peptide insert comprising, consisting essentially of, or consisting of in the LAGKSKVIDA amino acid sequence (SEQ ID NO: 38) between amino acids 587 and 588 of AAV2 capsid and comprises at least one amino acid substitution.
[00143] In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAGKSKVIDARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNP EIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 51)
[00144] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence selected from ASDSTKA (SEQ ID NO : 15) and LAASDSTKAA (SEQ ID NO: 30), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype ( that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K , T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, S7, E7, E7, E7, E7 combination thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In yet another embodiment, the variant capsid comprises a peptide insert comprising the amino acid sequence ASDSTKA (SEQ ID NO: 15) or comprising, consisting essentially of, or consisting of the amino acid sequence LAASDSTKAA (SEQ ID NO : 30) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGF RPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAASDSTKAARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 52)
[00145] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from KDRAPST (SEQ ID NO : 26) and LAKDRAPTSA (SEQ ID NO: 41), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype ( that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K , T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, S7, E7, E7, E7, E7 combination thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In yet another embodiment, the variant capsid comprises a peptide insert comprising the amino acid sequence KDRAPST (SEQ ID NO: 26) or comprising, consisting essentially of, or consisting of the amino acid sequence LAKDRAPTSA (SEQ ID NO : 41) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGF RPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAKDRAPSTARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 53)
[00146] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence selected from HQDTTKN (SEQ ID NO : 19) and LAHQDTTKNA (SEQ ID NO: 34), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype ( that is, different from AAV2), in which the substituted amino acids do not occur naturally in one or more of the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V7 Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In yet another embodiment, the variant capsid comprises a peptide insert comprising the amino acid sequence HQDTTKN (SEQ ID NO: 19) or comprising, consisting essentially of, or consisting of the amino acid sequence LAHQDTTKNA (SEQ ID NO : 34) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGF RPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAHQDTTKNARQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGL KHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 54)
[00147] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from ISNENEH (SEQ ID NO : 21) and LPISNENEHA (SEQ ID NO: 36), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype ( that is, different from AAV2), where the substituted amino acids do not occur naturally in one or more of the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V7 Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In yet another embodiment, the variant capsid comprises a peptide insert comprising the amino acid sequence ISNENEH (SEQ ID NO: 21) or comprising, consisting essentially of, or consisting of the amino acid sequence LPISNENEHA (SEQ ID NO : 36) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGF RPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLPISNENEHARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 55)
[00148] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from QANANEN (SEQ ID NO : 22) and LPQANANENA (SEQ ID NO: 37), and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype ( that is, different from AAV2), in which the substituted amino acid (s) do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K , T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, S7, E7, E7, E7, E7 combination thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In yet another embodiment, the variant capsid comprises a peptide insert comprising the amino acid sequence QANANEN (SEQ ID NO: 22) or comprising, consisting essentially of, or consisting of the amino acid sequence LPQANANENA (SEQ ID NO : 37) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGF RPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLPQANANENARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 56)
[00149] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from HPDTTKN (SEQ ID NO : 18) and LAHPDTTKNA (SEQ ID NO: 33) and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype (or different from AAV2), in which the substituted amino acids do not occur naturally in the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236 , I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, a combination of the same L735Q. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position on the capsid protein of another AAV serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more, amino acid sequence identity over the entire length of the sequence amino acid shown in SEQ. ID NO: 2. In yet another embodiment, the variant capsid comprises a peptide insert comprising the amino acid sequence HPDTTKN (SEQ ID NO: 18) or comprising, consisting essentially of, or consisting of the amino acid sequence LAHPDTTKNA (SEQ ID NO : 33) between amino acids 587 and 588 of AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity or 100% identical to the following amino acid sequence:
[00150] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHK DDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDN PYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKT APGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPL GQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWM GDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNR FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLT STVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAV GRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLI DQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRV SKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQS GVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNL AHPDTTKNARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGH FHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSV EIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYL trnL (SEQ ID NO: 57).
[00151] In several respects, a variant AAV capsid protein is provided comprising one or more amino acid substitutions with respect to a corresponding parental AAV capsid protein, wherein the variant capsid protein, when present in a virion of AAV, confers an increased infectivity of a retinal cell compared to the infectivity of a retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
[00152] In some embodiments, a variant AAV capsid protein comprises a P34A amino acid substitution compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or a P33A amino acid substitution compared to the amino acid sequence of AAV5 capsid (SEQ ID NO: 6). In some preferred embodiments, the variant capsid protein comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more, amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6 and comprises an amino acid substitution P34A or P33A compared to the amino acid sequence of AAV2 or AAV5 capsid, respectively. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence comprising an amino acid substitution P34A compared to the amino acid sequence shown in SEQ ID NO: 2 and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In related embodiments, the variant capsid protein comprises an amino acid substitution P34A compared to the amino acid sequence of SEQ ID NO: 2, wherein the variant capsid protein comprises from 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of an AAV2 capsid protein shown in SEQ ID NO: 2.
[00153] In other embodiments, a variant AAV capsid protein comprises an amino acid substitution at amino acid 164 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (that is, different from AAV2), where the substituted amino acid does not occur naturally in the corresponding position. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more of amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO 2 and comprises an amino acid substitution at amino acid 164 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2). In some embodiments, the rAAV virion comprises a substitution of the amino acid glutamine for lysine at amino acid 164 as compared to the amino acid sequence of AAV1, AAV2 or AAV6 or at amino acid 165 as compared to the amino acid sequence of AAV7, AAV8 or AAV10; or comprises a substitution of serine for lysine at amino acid 160 of AAV5 or comprises a substitution of alanine for lysine at amino acid 164 of AAV9. In related embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 164 (for example, Q164K) compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2), wherein the capsid protein of variant comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of an AAV2 capsid protein shown in SEQ ID NO: 2. In some preferred embodiments, the capsid protein variant comprises an amino acid sequence comprising a Q164K amino acid substitution compared to the amino acid sequence shown in SEQ ID NO: 2 and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In other embodiments , the variant capsid protein comprises the amino acid substitutions Q164K and V708I compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding substitutions in another parental serotype of AAV (that is, different from AAV2) and is at least 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more, amino acid sequence identity for the entire length of the amino acid sequence shown in SEQ ID NO: 2
[00154] In other embodiments, a variant AAV capsid protein comprises an amino acid substitution at amino acid 698 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (that is, different from AAV2), where the substituted amino acid does not occur naturally in the corresponding position. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more of amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO 2 and comprises an amino acid substitution at amino acid 698 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2). In some embodiments, the rAAV virion comprises an amino acid substitution isoleucine for valine at amino acid 698 compared to the amino acid sequence of AAV2 or at amino acid 699 compared to the amino acid sequence of AAV3A, AAV3B or AAV9, or at amino acid 687 of AAV5, or at amino acid 700 of AAV7, or at amino acid 701 of AAV8 or AAV10. In related embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 699 (for example, I698V) compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2), wherein the capsid protein of variant comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of an AAV2 capsid protein shown in SEQ ID NO: 2. In some preferred embodiments, the capsid protein variant comprises an amino acid sequence comprising an I698V amino acid substitution compared to the amino acid sequence shown in SEQ ID NO: 2 and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2.
[00155] In other embodiments, a variant AAV capsid protein comprises an amino acid substitution at amino acid 109 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (that is, different from AAV2). In some preferred embodiments, the variant capsid protein comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more of amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO 2 and comprises an amino acid substitution at amino acid 109 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2). In some embodiments, the variant capsid protein comprises a substitution of amino acids from serine to threonine at position 109 compared to the amino acid sequence of AAV1, AAV3A, AAV3B, AAV4, AAV7, AAV8, AAV9 or AAV10 or at position 108 in compared to the amino acid sequence of AAV5 or AAV6. In related embodiments, the variant capsid protein comprises an S109T amino acid substitution compared to the AAV2 amino acid sequence, wherein the variant capsid protein comprises from 1 to 5, from 5 to 10, or from 10 to 15 amino acid substitutions. In other related embodiments, the variant capsid protein comprises an amino acid substitution S109T and an amino acid substitution A593E compared to the amino acid sequence of AAV2. In some embodiments, the variant capsid protein comprises amino acid substitutions S109T and A493V and, optionally, A593E and / or V708I compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding substitutions in another parental serotype of AAV (ie different from AAV2) and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more, of amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO 2. In other embodiments, the variant capsid protein comprises amino acid substitutions S109T, A493V, A593E and V708I compared to AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding substitutions in another parental AAV serotype (i.e., different from AAV2) and is at least 85%, at least about 90%, at least about 95%, at least about 98%, or by at least about 99% or more of amino acid sequence identity for the entire length of the amino acid sequence shown in SEQ ID NO: 2 In other preferred embodiments, the variant capsid protein comprises amino acid substitutions S109T and V708I in compared to the AAV2 capsid amino acid sequence and is at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99%, or more, of amino acid sequence identity in relation to the total length of the amino acid sequence shown in SEQ ID NO 2 or is otherwise identical to the amino acid sequence of SEQ ID NO: 2.
[00156] In other embodiments, a variant AAV capsid protein comprises an amino acid substitution at amino acid 593 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (that is, different from AAV2). In some preferred embodiments, the variant capsid protein comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more of amino acid sequence identity across the length of the amino acid sequence shown in SEQ ID NO 2 and comprises an amino acid substitution at amino acid 593 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2). In some embodiments, the variant capsid protein comprises a substitution of amino acid glycine for glutamate at amino acid 594 compared to the amino acid sequence of AAV1, AAV3A, AAV6 or AAV9, or at amino acid 583 of AAV5, or at amino acid 596 of AAV8 or AAV10, or a substitution of amino acid arginine for glutamate at amino acid 594 of AAV3B, or a substitution of amino acid aspartate for glutamate at amino acid 592 of AAV4 or a substitution of amino acid glutamine for glutamate at position 595 of AAV7. In other embodiments, the variant capsid protein comprises an A593E amino acid substitution compared to the AAV2 amino acid sequence and does not comprise one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence: I19V, V369A, K26R, N215D, G355S, V46A and S196P. In related embodiments, the variant capsid protein comprises amino acid substitutions A593E and N596D compared to the amino acid sequence of AAV2 and is at least about 85%, at least about 90%, at least about 95%, at least about 98% or less, about 99% identity with the total length of the amino acid sequence shown in SEQ ID NO 2. In other embodiments, the variant capsid comprises amino acid substitutions A593E and N596D compared to amino acid sequence of AAV2 and is otherwise identical to the amino acid sequence of AAV2. In other embodiments, the variant capsid comprises the amino acid substitutions A593E and V708I compared to the amino acid sequence of AAV2 and is at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% identity to the total length of the amino acid sequence shown in SEQ ID NO 2. In other embodiments, the variant capsid comprises amino acid substitutions A593E and V708I compared to the sequence of AAV2 amino acids and is otherwise identical to the AAV2 amino acid sequence.
[00157] In other embodiments, a variant AAV capsid protein comprises an amino acid substitution at amino acid 708 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (that is, different from AAV2), where the substituted amino acid does not occur naturally in the corresponding position. Preferably, the rAAV virus does not comprise a substitution of proline for serine at amino acid 250 as compared to AAV2 or a corresponding amino acid in another parent AAV serotype. In some embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% or more of amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO: 2 and comprises an amino acid substitution at amino acid 708 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) . In preferred embodiments, the variant capsid protein comprises a substitution of valine for isoleucine (V708I) at amino acid 708 compared to the AAV2 capsid amino acid sequence and is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more, of amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO: 2 or otherwise, identical to the amino acid sequence of SEQ ID NO: 2, wherein the variant capsid protein does not comprise a P250S amino acid substitution. In some embodiments, the variant capsid protein comprises a substitution of valine for isoleucine at amino acid 709 of AAV3A or AAV3B, a substitution of alanine for isoleucine at position 709 of AAV1 or AAV6, a substitution of asparagine for isoleucine at amino acid 707 of AAV4 or amino acid 709 of AAV9 or a substitution of threonine for isoleucine at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10 or a substitution of glutamine for isoleucine at amino acid 697 of AAV5. In related embodiments, the variant capsid protein comprises an amino acid substitution V708I compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10 or 10 to 15 substitutions amino acid and where the variant capsid protein does not comprise a P250S amino acid substitution. In other embodiments, the variant capsid protein comprises an amino acid substitution V708I and also comprises an amino acid substitution A593E and / or S109T compared to the amino acid sequence of AAV2. In other related embodiments, the variant capsid comprises amino acid substitutions of V708I and A593E compared to the AAV2 amino acid sequence, wherein the variant capsid protein is otherwise identical to the AAV2 amino acid sequence. In other related embodiments, the variant capsid comprises amino acid substitutions of V708I and S109T compared to the AAV2 amino acid sequence, wherein the variant capsid protein is otherwise identical to the AAV2 amino acid sequence. In other embodiments, the variant capsid protein comprises amino acid substitutions V708I and V719M compared to the amino acid sequence of AAV2 and is at least about 85%, at least about 90%, at least about 95%, at least at least about 98% or at least about 99% or more of the amino acid sequence identity to the total length of the amino acid sequence shown in SEQ ID NO 2 or is otherwise identical to the amino acid sequence of SEQ ID NO: 2. In other embodiments, the variant capsid protein comprises amino acid substitutions V708I and R733C compared to the amino acid sequence of AAV2 and is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more, identity of the amino acid sequence in relation to the total length of the amino acid sequence shown in SEQ ID NO 2, or is otherwise identical to the amino acid sequence those of SEQ ID NO: 2. In other embodiments, the variant capsid protein comprises amino acid substitutions V708I and G727D compared to the amino acid sequence of AAV2 and is at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99%, or more, of the identity of the amino acid sequence in relation to the total length of the amino acid sequence shown in SEQ ID NO 2, or otherwise similarly to the amino acid sequence of SEQ ID NO: 2.
[00158] In other embodiments, a variant AAV capsid protein comprises an amino acid substitution at amino acid 196 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (that is, different from AAV2), where the substituted amino acid does not occur naturally at the corresponding position and is optionally different from proline. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99 % or more of amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO: 2 and comprises an amino acid substitution at amino acid 196 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2 ) and is optionally different from an S196P replacement. In preferred embodiments, the variant capsid protein comprises a substitution of the amino acid serine for tyrosine at amino acid 196 of AAV2 or AAV9 or at amino acid 197 of AAV7, AAV8 or AAV10 or at amino acid 186 of AAV5; or a substitution of alanine for tyrosine at amino acid 196 of AAV1 or AAV6; or a substitution of methionine for tyrosine at amino acid 191 of AAV4; or a substitution of threonine for tyrosine at amino acid 196 of AAV3A or AAV3B. In a related embodiment, the variant capsid protein comprises an amino acid sequence comprising an S196Y amino acid substitution compared to the amino acid sequence shown in SEQ ID NO: 2 and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2 In related embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 196 other than an S196P substitution (for example, it comprises an S196Y substitution) compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2), where the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of an AAV2 capsid protein shown in SEQ ID NO: 2.
[00159] In other embodiments, a variant AAV capsid protein comprises an amino acid substitution at amino acid 175 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (that is, different from AAV2), where the substituted amino acid does not occur naturally in the corresponding position. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more of amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO 2 and comprises an amino acid substitution at amino acid 175 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2). In some embodiments, the variant capsid comprises an amino acid substitution Q175H compared to the amino acid sequence of AAV2 as shown in SEQ ID NO: 2 or a replacement of glutamine with histidine in the corresponding position in another parental serotype of AAV. In related embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 175 (for example, Q175H) compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2), wherein the capsid protein of variant comprises 1 to 5, 5 to 10 or 10 to 15 amino acid substitutions compared to the amino acid sequence of an AAV2 capsid protein shown in SEQ ID NO: 2.
[00160] In other embodiments, a variant AAV capsid protein comprises an amino acid substitution at amino acid 64 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (that is, different from AAV2), where the substituted amino acid does not occur naturally in the corresponding position. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more of amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO 2 and comprises an amino acid substitution at amino acid 64 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2). In some embodiments, the rAAV virion comprises a P64S amino acid substitution compared to the AAV2 amino acid sequence as shown in SEQ ID NO: 2 or a proline substitution for serine in the corresponding position in another AAV parental serotype. In related embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 64 (for example, P64S) compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2), wherein the capsid protein of variant comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of an AAV2 capsid protein shown in SEQ ID NO: 2.
[00161] In other embodiments, a variant AAV capsid protein comprises an amino acid sequence of at least 85%, at least 90%, at least 95% or at least 98% identity with a wild-type AAV capsid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12 and also comprises i) one or more amino acid substitutions selected from the group consisting of P34A, S109T + V708I, A593E + N596D, V708I + V719M, V708I + G727D, S109T + A493V + + A593E + V708I, V708I + R733C, Q164K, and I698V and / or (ii) a selected peptide insert from the group that consists of QT (QTT) ID NO: 13), ISDQTKH (SEQ ID NO: 14), ASDSTKA (SEQ ID NO: 15), NQDYTKT (SEQ ID NO: 16), HDITKNI (SEQ ID NO: 17), PQANANEN (SEQ ID NO: 63) , TNRTSPD (SEQ ID NO: 24), PNSTHGS (SEQ ID NO: 25), KDRAPST (SEQ ID NO: 26), HQDTTKN (SEQ ID NO: 19), HPDTTKN (SEQ ID NO: 18), NKTTNKD (SEQ ID NO: 18) NO: 20), GKSKVID (SEQ ID NO: 23), PISNENEH (SEQ ID NO: 64), LAQADTTKNA (SEQ ID NO: 27), LAISDQTKHA (SEQ ID NO: 28), LGISDQTKHA (SEQ ID NO: 29), LAASDSTKAA (SEQ ID NO: 30), LAHDITKNIA (SEQ ID NO: 32), LPQANANENA (SEQ ID NO: 37), LANQDYTKTA (SEQ ID NO: 31), LATNRTSPDA (SEQ ID NO: 39), LAPNSTHGSA (SEQ ID NO: 40), LAKDRAPSTA (SEQ ID NO: 41), LAHQDTTKNA (SEQ ID NO: 34), LAHPDTTKNA (SEQ ID NO: 33), LANKTTNKDA (SEQ ID NO: SEQ ID NO: 33) : 35), LAGKSKVIDA (SEQ ID NO: 38), and LPISNENEHA (SEQ ID NO: 36). In some embodiments, the variant AAV capsid comprises one or more specified amino acid substitutions and / or peptide inserts and is otherwise identical to a sequence selected from the group consisting of SEQ ID NOS: 1-12.
[00162] In some embodiments, a variant AAV capsid protein is an ancestral capsid protein. An ancestral capsid protein means an evolutionary ancestor of a capsid protein that is found in nature today, for example: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11, AAV12 , AAV13, which is generated in silico by random substitution of amino acids in positions of degeneration among the AAV capsid proteins that are found in nature today. A non-limiting example of an ancestral capsid is provided below, where the degeneration positions (residues 264, 266, 268, 448, 459, 460, 467, 470, 471, 474, 495, 516, 533, 547, 551, 555, 557, 561, 563, 577, 583, 593, 596, 661, 662, 664, 665, 710, 717, 718, 719, 723) are marked as an “X”:
[00163] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQD DGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNP YLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTA PGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPL GEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWL GDRVITTSTRTWALPTYNNHLYKQISSXSXGXTNDNHYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNL TSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAV GRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLI DQYLYYLXRTQSTGGTAGXXELLFSQXGPXXMSXQAKNWLPGPCYRQQR VSKTLXQNNNSNFAWTGATKYHLNGRXSLVNPGVAMATHKDDEXRFFPSS GVLIFGKXGAGXNNTXLXNVMXTXEEEIKTTNPVATEXYGVVAXNLQSSNT APXTGXVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GLKHPPPQILIKNTPVPANPPXXFXXAKFASFITQYSTGQVSVEIEWELQKE NSKRWNPEIQYTSNYAKSXNVDFAVXXXGVYXEPRPIGTRYLTRNL (SEQ ID NO: 58)
[00164] In some embodiments, the ancestral capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% or more, amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the ancestral capsid protein comprises an amino acid sequence having at least about 85%, at least at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more, of amino acid sequence identity over the entire length of the AAV2 amino acid sequence, for example , as presented in SEQ ID NO: 2. In some embodiments, the ancestral capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% or more of id amino acid sequence entity with the total length of the amino acid sequence of the ancestral sequence disclosed in SEQ ID NO: 58 or SEQ ID NO: 2 and comprises one or more amino acid residues selected from the group consisting of: Alanine (A) in 264, Alanine (A) in 266, Serina (S) in 268, Alanine (A) in 448, Threonine (T) in 459, Arginine (R) in 460, Alanine (A) in 467, Serina (S) in 470 , Asparagine (N) in 471, Alanine (A) in 474, Serine (S) in 495, Asparagine (D) in 516, Asparagine (D) in 533, Glutamine (Q) in 547, Alanine (A) in 551, Alanine (A) in 555, Glutamic acid (E) in 557, Methionine (M) in 561, Serine (S) in 563, Glutamine (Q) in 577, Serine (S) in 583, Valine (V) in 593, Threonine (T) in 596, Alanine (A) in 661, Valine (V) in 662, Threonine (T) in 664, Proline (P) in 665, Threonine (T) in 710, Aspartic acid (D) in 717, Asparagine (N) in 718, Glutamic acid (E) in 719, and Serine (S) in 723. In some preferred embodiments, the capsid protein of variant comp breaks down an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, in some cases 100% identity amino acid sequence with the total length of the following amino acid sequence and comprises one or more amino acid residues selected from the group consisting of: Alanine (A) in 264, Alanine (A) in 266, Serine (s) in 268, Alanine ( A) at 448, Threonine (T) at 459, Arginine (R) at 460, Alanine (A) at 467, Serine (s) at 470, Asparagine (N) at 471, Alanine (A) at 474, Serine (s) ) at 495, asparagine (D) at 516, asparagine (D) at 533, glutamine (Q) at 547, alanine (A) at 551, alanine (A) at 555, glutamic acid (E) at 557, methionine (M ) at 561, serine (s) at 563, glutamine (Q) at 577, Serine (s) at 583, Valine (V) at 593, Threonine (T) at 596, Alanine (A) at 661, Valine (V) at 662, Threonine (T) at 664, Proline (P) at 665, Threonine (T) at 710, Aspartic Acid (D) at 717, Asparagine (N) at 718, glutamic acid (E) 719 and Serine (s) 723:
[00165] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQK QDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGD NPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAK TAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQ PLGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTW LGDRVITTSTRTWALPTYNNHLYKQISSASAGSTNDNHYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANN LTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQA VGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPL IDQYLYYLARTQSTGGTAGTRELLFSQAGPSNMSAQAKNWLPGPCYRQQR VSKTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFPS SGVLIFGKQGAGANNTALENVMMTSEEEIKTTNPVATEQYGVVASNLQSSN TAPVTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GLKHPPPQILIKNTPVPANPPAVFTPAKFASFITQYSTGQVSVEIEWELQKEN SKRWNPEIQYTSNYAKSTNVDFAVDNEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 59).
[00166] In other embodiments, a variant AAV capsid protein comprises an amino acid sequence of at least 85%, at least 90%, at least 95% or at least 98% identical to a selected wild-type AAV capsid sequence of the group consisting of the ancestral variant disclosed here as SEQ ID NO: 58, comprises one or more amino acid residues selected from the group consisting of: Alanine (A) in 264, Alanine (A) in 266, Serine (S) in 268, Alanine (A) in 448, Threonine (T) in 459, Arginine (R) in 460, Alanine (A) in 467, Serine (S) in 470, Asparagine (N) in 471, Alanine (A) in 474, Serina (S) in 495, Asparagine (D) in 516, Asparagine (D) in 533, Glutamine (Q) in 547, Alanine (A) in 551, Alanine (A) in 555, Glutamic acid (E) in 557, Methionine (M) in 561, Serine (S) in 563, Glutamine (Q) in 577, Serine (S) in 583, Valine (V) in 593, Threonine (T) in 596, Alanine (A) in 661, Valina ( V) in 662, Threonine (T) in 664, Proline (P) in 665, Threonine (T) in 710, Acid aspartic (D) in 717, Asparagine (N) in 718, Glutamic acid (E) in 719, and Serine (S) in 723; and also comprises i) one or more amino acid substitutions selected from the group consisting of P34A, S109T + V708I, A593E + N596D, V708I + V719M, V708I + G727D, S109T + A493V + A593E + V708I, V708I + R733C, Q164 I698V and / or (ii) a peptide insert selected from the group consisting of QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), ASDSTKA (SEQ ID NO: 15), NQDYTKT (SEQ ID NO: 16), HDITKNI (SEQ ID NO: 17), PQANANEN (SEQ ID NO: 63), TNRTSPD (SEQ ID NO: 24), PNSTHGS (SEQ ID NO: 25), KDRAPST (SEQ ID NO: 26), HQDTTKN ( SEQ ID NO: 19), HPDTTKN (SEQ ID NO: 18), NKTTNKD (SEQ ID NO: 20), GKSKVID (SEQ ID NO: 23), PISNENEH (SEQ ID NO: 64), LAQADTTKNA (SEQ ID NO: 27) ), LAISDQTKHA (SEQ ID NO: 28), LGISDQTKHA (SEQ ID NO: 29), LAASDSTKAA (SEQ ID NO: 30), LAHDITKNIA (SEQ ID NO: 32), LPQANANENA (SEQ ID NO: 37), LANQDYTKTA (SEQ ID NO: 31), LATNRTSPDA (SEQ ID NO: 39), LAPNSTHGSA (SEQ ID NO: 40), LAKDRAPSTA (SEQ ID NO: 41), LAHQDTTKNA (SEQ ID NO: 34), LAHPDTTKNA (SEQ ID NO: 33) , LANKTTNKDA (SEQ ID NO: 35), LAGKSKVIDA (SEQ ID NO: 38), and LPISNENEHA (SEQ ID NO: 36). In some embodiments, the variant AAV capsid comprises one or more specified amino acid substitutions and / or peptide inserts and is otherwise identical to SEQ ID NO: 59.
[00167] The AAV variants disclosed herein were generated through the use of in vivo directed evolution involving the use of primate retinal scans after intravitreal administration. In some embodiments, the variant capsid proteins disclosed by the present invention, when present in an AAV virus, confer increased transduction of a retinal cell as compared to the transduction of the retinal cell by an AAV virus comprising the capsid protein corresponding parental AAV or wild-type AAV. For example, in some embodiments, the variant capsid proteins disclosed by the present invention, when present in an AAV virion, confer more efficient transduction of primate retinal cells than AAV virions comprising the corresponding parental AAV capsid protein. or wild-type AAV capsid protein, for example, cells absorb more AAV virions comprising the variant AAV capsid protein in question than the AAV virions comprising the parent AAV or wild-type AAV capsid protein . In some of such embodiments, the variant AAV virion or variant rAAV exhibits at least 2 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times , or more than 50-fold, increased retinal cell transduction compared to retinal cell transduction by a wild-type AAV rAAV or virion comprising the corresponding parental AAV capsid protein. In certain such embodiments, the variant capsid proteins disclosed by the present invention, when present in an AAV virion, confer broader transduction of primate retinal cells than AAV virions comprising the corresponding parental AAV capsid protein or the wild-type AAV capsid protein. In other words, the variant AAV virion transduces cell types not transduced by virions comprising the corresponding parental AAV capsid protein and, consequently, more cell types in the retina than the corresponding parental AAV virus. In some embodiments, the variant AAV virus preferably transduces a retinal cell, for example, a rAAV virus in question infects a retinal cell 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 50 times, or more than 50 times, than another cell in the retina or a non-retinal cell, for example, a cell outside the eye. In some embodiments, the transduced retinal cell is a photoreceptor cell (for example, rods; cones). In some embodiments, the retinal cell is a retinal ganglion cell (RGC). In some embodiments, the retinal cell is a retinal epithelial cell (RPE cell). In some embodiments, the retinal cell is a Müller glial cell. In some embodiments, the retinal cell is a microglial cell. In some embodiments, the retinal cell is an amacrine cell. In some embodiments, the retinal cell is a bipolar cell. In some embodiments, the retinal cell is a horizontal cell. An increase in retinal cell transduction, for example, increased transduction efficiency, broader transduction, more preferred transduction, etc., can be readily evaluated in vitro or in vivo by any number of methods in the art for measuring expression genic. For example, AAV can be packaged with a genome comprising an expression cassette comprising a reporter gene, for example, a fluorescent protein, under the control of a ubiquitous or tissue-specific promoter, and the extent of transduction assessed by detecting the fluorescent protein , for example, fluorescent microscope. As another example, AAV can be packaged with a genome comprising a bar-encoded nucleic acid sequence, and the extent of transduction assessed by detecting the nucleic acid sequence, for example, by PCR. As another example, AAV can be packaged with a genome comprising an expression cassette comprising a therapeutic gene for the treatment of a retinal disease, and the extent of transduction assessed by detecting the retinal disease treatment in a distressed patient who has been administered to AAV.
[00168] Eye diseases that can be treated with the use of a variant rAAV vector or virion and / or method disclosed herein include, but are not limited to, monogenic diseases, complex genetic diseases, acquired diseases and traumatic injuries. Examples of monogenic diseases include, but are not limited to, Bardet-Biedl syndrome; Batten's disease; Crystalline dystrophy of Bietti; choroidideremia; chorioretinal atrophy; chorioretinal degeneration; dystrophies of cones or rod cones (autosomal dominant, autosomal recessive and X-linked); congenital stationary night blindness (autosomal dominant, autosomal recessive and linked to the X chromosome); color vision disorders, including achromatopsia (including ACHM2, ACHM3, ACHM4 and ACHM5), protanopia, deuteranopia and tritanopia; Friedreich's ataxia; Leber's congenital amaurosis (autosomal dominant and autosomal recessive), including, but not limited to, LCA1, LCA2, LCA3, LCA4, LCA6, LCA7, LCA8, LCA12 and LCA15; Leber's Hereditary Optical Neuropathy; macular dystrophy (autosomal dominant and autosomal recessive), including, but not limited to, acute macular degeneration, better vitelliform macular dystrophy, standard dystrophy, North Carolina macular dystrophy, inherited druses, Sorsby fundus dystrophy, lifted malattia and genetically determined retinopathy prematurity; eye disease of retinal development; ocular albinism; optical atrophies (autosomal dominant, autosomal recessive and X-linked); retinitis pigmentosa (autosomal dominant, autosomal recessive, X-linked and inherited mitochondrial features), examples of which include RP1, RP2, RP3, RP10, RP20, RP38, RP40 and RP43; X-linked retinoschisis; Stargardt's disease; and Usher syndrome, including, but not limited to, USH1B, USH1C, USH1D, USH1F, USH1G, USH2A, USH2C, USH2D and USH3. Examples of complex genetic diseases include, but are not limited to, glaucoma (open angle, angle closure, low tension, normal tension, congenital, neovascular, pigment, pseudoexfoliation); age-related forms and other forms of macular degeneration, both exudative and non-exudative (autosomal dominant and autosomal recessive), such as acute macular degeneration, vitelliform macular degeneration; retinopathy of prematurity; and Vogt Koyanagi-Harada syndrome (VKH). Examples of acquired diseases include, but are not limited to, acute macular neuroretinopathy; anterior ischemic optic neuropathy and posterior ischemic optic neuropathy; Behçet's disease; branch retinal vein occlusion; choroidal neovascularization; diabetic retinopathy, including proliferative diabetic retinopathy and associated complications; diabetic uveitis; edema, such as macular edema, cystoid macular edema and diabetic macular edema; disorders of the epiretinal membrane; macular telangiectasis; multifocal choroiditis; diabetic retinal dysfunction not retinopathy; ocular tumors; optical atrophies; retinal detachment; retinal disorders, such as central retinal vein occlusion, proliferative vitreoretinopathy (PVR), arterial retinal and venous occlusive disease, vascular occlusion, uveitic retinal disease; uveal effusion; infectious and infiltrative disease of the retina; optic nerve diseases, such as acquired optic atrophy. Examples of traumatic injuries include, but are not limited to, histoplasmosis; trauma to the optic nerve; eye trauma that affects a posterior ocular site or location; retinal trauma; viral infection of the eye; viral infection of the optic nerve; a posterior eye condition caused by or influenced by laser eye treatment; posterior eye conditions caused by or influenced by photodynamic therapy; photocoagulation, radiation retinopathy; and sympathetic ophthalmia.
[00169] In another embodiment, a variant capsid disclosed herein comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes a gene product, such as, without limitation, an interfering RNA, a long non-coding RNA, a non-coding RNA short, an antisense RNA, an aptamer, a polypeptide, a secreted antibody, a single chain antibody, a VHH domain, a soluble receptor, an antibody, a knottin, a DARPin, a centurin, a chaperone, a specific site nuclease which provides knock-down specific site of gene function or a modified modified site nuclease which provides specific activation of the transcription gene.
A variant of the rAAV variant disclosed herein comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes a gene product. In some embodiments, the gene product is an interfering RNA. In some embodiments, the gene product is a long non-coding RNA. In some embodiments, the gene product is a short non-coding RNA. In some embodiments, the gene product is an antisense RNA. In some embodiments, the gene product is an aptamer. In some embodiments, the gene product is a polypeptide. In some embodiments, the gene product is a secreted antibody. In some embodiments, the gene product is a single chain antibody. In some embodiments, the gene product is a VHH domain. In some embodiments, the gene product is a soluble receptor. In some embodiments, the gene product is an amphibody. In some embodiments, the gene product is a knottin. In some embodiments, the gene product is a DARPin. In some embodiments, the gene product is a centurin. In some embodiments, the gene product is a chaperone. In some embodiments, the gene product is a specific site nuclease that provides a specific knock-down site of the gene's function.
[00171] The use of the gene product includes, but is not limited to, improving the level of a factor in a cell, improving the level of a factor in a neighboring cell by secreting a factor, decreasing the level of a factor in a cell, or decreasing the level of a factor in a neighboring cell by secreting a factor. The gene product can be designed to supplement the level of a defect in the missing gene product, to decrease the level of a defect in the missing gene product, to introduce a new support gene product, to supplement the level of a gene gene product support, decrease the level of an impaired gene product, or both, decrease the level of an impaired gene product and introduce or supplement the level of a support gene product.
[00172] The gene products provided by the AAV variants in question can be used to alter the level of gene products or gene product activity, directly or indirectly linked to retinal diseases and traumas. Genes whose gene products are directly or indirectly linked to genetic diseases include, for example, ADP type 6 ribosylation factor (ARL6); BBAsoma 1 interaction protein (BBIP1); BBSoma 1 protein (BBS1); BBSoma 2 protein (BBS2); BBSoma 4 protein (BBS4); BBSoma 5 protein (BBS5); BBSoma 7 protein (BBS7); BBSoma 9 protein (BBS9); BBSoma 10 protein (BBS10); BBSoma 12 protein (BBS12); 290 kDa centrosomal protein (CEP290); intraflagellar transport protein 172 (IFT172); intraflagellar transport protein 27 (IFT27); inositol polyphosphate-5-phosphatase E (INPP5E); member 13 of subfamily J of the internal potassium rectification channel (KCNJ13); leucine type 1 transcription factor (LZTFL1); McKusick-Kaufman syndrome protein (MKKS); Meckel syndrome type 1 protein (MKS1); nephronophysis protein 3 (NPHP1); colon cancer antigen serologically defined 8 (SDCCAG8); protein 32 containing tripartite motif (TRIM32); tetratricopeptide repeat domain 8 (TTC8); Batten's disease protein (CLN3); cytochrome P450 4V2 (CYP4V2); Rab 1 escort protein (CHM); Protein 13 containing domain (positive regulator) PR (PRDM13); protein-coupled RPE-retinal receptor (RGR); member 1 of the TEA domain family (TEAD1); aryl hydrocarbon type 1 receptor protein (AIPL1); homeobox transcription factor of the cone-rod type-otx photoreceptor (CRX); guanylate cyclase 1A activating protein (GUCA1A); retinal specific guanylate cyclase (GUCY2D); member 3 of the family associated with the phosphatidylinositol transfer membrane (PITPNM3); prominin 1 (PROM1); peripheral (PRPH); periferin 2 (PRPH2); regulatory membrane synaptic exocytosis protein 1 (RIMS1); semaphorin 4A (SEMA4A); human homologue of the protein C. elegans unc119 (UNC119); ATP binding cassette carrier - retina (ABCA4); ADAM 9 metallopeptidase domain (ADAM9); activation transcription factor 6 (ATF6); chromosome 21 open reading frame 2 (C21orf2); chromosome 8 open reading frame 37 (C8orf37); calcium channel; voltage dependent; alpha 2 / delta 4 subunit (CACNA2D4); member of family 1 related to cadherin (protocaderin 21) (CDHR1); ceramide kinase type protein (CERKL); alpha subunit of the cation channel linked to the photoreceptor cGMP cone (CNGA3); beta 3 subunit of the cation channel linked by cyclic cone nucleotides (CNGB3); cyclin M4 (CNNM4); guanine nucleotide-binding protein (protein G); alpha 2 transduction activity polypeptide (GNAT2); member V of the potassium channel subfamily 2 (KCNV2); phosphodiesterase 6C (PDE6C); phosphodiesterase 6H (PDE6H); centriole proteome centriolar protein B (POC1B); RAB28 member of the RAS oncogene family (RAB28); homeobox transcription factor 2 of the anterior neural fold and retina (RAX2); 11-cis retinol dehydrogenase 5 (RDH5); protein 1 interacting with RP GTPase regulator (RPGRIP1); member 5 of the tubulin tyrosine ligase-like family (TTLL5); L-type voltage-dependent calcium channel alpha-1 subunit (CACNA1F); GTPase regulator of retinitis pigmentosa (RPGR); baston transducin alpha subunit (GNAT1); beta phosphodiesterase subunit of rods cGMP (PDE6B); rhodopsin (RHO); calcium binding protein 4 (CABP4); G 179 protein coupled receptor (GPR179); rhodopsin kinase (GRK1); metabotropic glutamate receptor 6 (GRM6); protein 3 from the leucine-rich repeating immunoglobulin-like transmembrane domains (LRIT3); arrestin (antigen-s) (SAG); 24th family of solute carriers (SLC24A1); transient receptor potential calcium channel, subfamily M, member 1 (TRPM1); nictalopin (NYX); green cone opsine (OPN1LW); red cone opsine (OPN1MW); blue cone opsine (OPN1SW); frataxin (FXN); inosine monophosphate dehydrogenase 1 (IMPDH1); orthodentula homeobox 2 protein (OTX2); crumbs counterpart 1 (CRB1); death domain-containing protein 1 (DTHD1); growth differentiation factor 6 (GDF6); homologous Chlamydomonas transport protein intraflagellar 140 (IFT140); protein B1 containing IQ motif (IQCB1); lebercillin (LCA5); lecithin retinol acyltransferase (LRAT); nicotinamide adenylyltransferase 1 nucleotide (NMNAT1); RD3 protein (RD3); retinol dehydrogenase 12 (RDH12); 65 kD protein specific to the retinal pigment epithelium (RPE65); spermatogenesis-associated protein 7 (SPATA7); tubby type protein (TULP1); mitochondrial genes (KSS, LHON, MT-ATP6, MT-TH, MT-LT1, MT-TP, MT-TS2, mitochondrial encoded NADH [MT-ND] dehydrogenases); bestrofin 1 (BEST1); protein 5 collagen related to tumor necrosis and C1q (C1QTNF5); fibrillin-like extracellular matrix protein 1 containing EGF (EFEMP1); elongation of the protein of very long fatty acids (ELOVL4); retina fascina homologue 2, actin aggregating protein (FSCN2); guanylate cyclase activating protein 1B (GUCA1B); hemicentin 1 (HMCN1); proteoglycan 1 of the interphotoreceptor matrix (IMPG1); protein 1 similar to retinitis pigmentosa 1 (RP1L1); tissue inhibitor of metalloproteinases-3 (TIMP3); complement factor H (CFH); complement factor D (CFD); complement of component 2 (C2); complement of component 3 (C3); complement factor B (CFB); modulator of autophagy regulated by damage to DNA 2 (DRAM2); chondroitin sulfate proteoglycan 2 (VCAN); mitofusin 2 (MFN2); member 1 of group F of the nuclear receptor subfamily (NR2F1); optic atrophy 1 (OPA1); transmembrane protein 126A (TMEM126A); homologue A of internal mitochondrial membrane translocase 8 (TIMM8A); carbonic anhydrase IV (CA4); hexokinase 1 (HK1); kelch-like protein 7 (KLHL7); group E3 of nuclear receptor subfamily 2 (NR2E3); leucine neural retinal zipper (NRL); member 3 of subfamily W of family 2 of olfactory receptors (OR2W3); pre-mRNA processing factor 3 (PRPF3); pre-mRNA processing factor 4 (PRPF4); pre-mRNA processing factor 6 (PRPF6); pre-mRNA processing factor 8 (PRPF8); pre-mRNA processing factor 31 (PRPF31); membrane protein of the outer segment of the retina 1 (ROM1); retinitis pigmentosa protein 1 (RP1); protein 1 associated with PIM1 kinase (RP9); small 200kDa nuclear ribonucleoprotein (SNRNP200); secreted phosphoprotein 2 (SPP2); topoisomerase I arginine / serine-rich protein (TOPORS); ADP- ribosylation factor-like binding protein 2 (ARL2BP); structure 71 for open reading of chromosome 2 (C2orf71); clarin-1 (CLRN1); alpha subunit of the rod-controlled cGMP (CNGA1) channel; beta subunit of the rod-controlled cGMP channel (CNGB1); cytochrome P450 4V2 (CYP4V2); dehydrodolichyl diphosphate synthetase (DHDDS); box DEAH 38 polypeptide (DHX38); subunit 1 of the ER membrane protein complex (EMC1); spacemaker / eyes closed counterpart (EYS); family with sequence similarity 161 of member A (FAM161A); 125 receptor coupled to G protein (GPR125); Heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT); NAD specific (+) isocitrate dehydrogenase 3 beta (IDH3B); proteoglycan 2 of the interphotoreceptor matrix (IMPG2); KIAA1549 protein (KIAA1549); kizuna centrosomal protein (KIZ); male germ cell associated kinase (MAK); tyrosine kinase receptor of the c-mer proto-oncogene (MERTK); mevalonate kinase (MVK); NIMA (never in mitosis of gene A) related to kinase 2 (NEK2); neuronal differentiation protein 1 (NEUROD1); alpha phosphodiesterase subunit cGMP (PDE6A); gamma rods specific to 6G phosphodiesterase cGMP (PDE6G); progressive rod degeneration protein (PRCD); retinol-binding protein 3 (RBP3); retinaldehyde 1 binding protein (RLBP1); member 14 of the family 7 of solute carriers (SLC7A14); usherin (USH2A); zinc finger protein 408 (ZNF408); zinc finger protein 513 (ZNF513); oral-facial-digital syndrome protein (OFD1); retinitis pigmentosa 2 (RP2); retinoschinine (RS1); protein 12 containing the abhydrolase domain (ABHD12); cadherin-like gene 23 (CDH23); 250 kDa centrosomal protein (CEP250); member 2 of the calcium and integrin binding family (CIB2); whirlina (DFNB31); homologue of susceptibility to monogenic audiogenic fainting (GPR98); histidyl-tRNA synthetase (HARS); myosin VIIA (MYO7A); protocaderin 15 (PCDH15); harmonin (USH1C); human mouse support protein homologue containing ankyrin repeats and SAM domain (USH1G); dystrophin (DMD); norrin (NDP); phosphoglycerate kinase (PGK1); calpain 5 (CAPN5); frizzled-4 Wnt receptor homolog (FZD4); integral membrane protein 2B (ITM2B); protein 5 related to the low density lipoprotein receptor (LRP5); micro RNA 204 (MIR204); retinoblastoma protein 1 (RB1); tetrasopin 12 (TSPAN12); chromosome 12 open reading frame 65 (C12orf65); cadherin 3 (CDH3); membrane-related frizzled protein (MFRP); ornithine aminotransferase (OAT); phospholipase A2 group V (PLA2G5); retinol binding protein 4 (RBP4); regulator of G 9 protein signaling (RGS9); regulator of protein G signaling binding protein 9 (RGS9BP); ARMS2; rodent repair deficiency complementation group 6 protein with cross-excision repair complementation (ERCC6); fibulin 5 (FBLN5); HtrA serine peptidase 1 (HTRA1); type 3 toll receiver (TLR3); and type 4 toll receiver (TLR4).
[00173] The genes whose gene products induce or promote apoptosis are here called "pro-apoptotic genes" and the products of those genes (mRNA; protein) are called "pro-apoptotic gene products". Pro-apoptotic targets include, for example, Bax gene products; Bid gene product offer; Bak gene products; Bad gene products; Bcl-2; Bcl-X1. Anti-apoptotic gene products include X-linked apoptosis inhibitor.
[00174] The genes whose gene products induce or promote angiogenesis are here called "pro-angiogenic genes" and the products of those genes (mRNA; protein) are called "pro-angiogenic gene products". Pro-angiogenic targets include, for example, vascular endothelial growth factor (VEGFa, VEGFb, VEGFc, VEGFd); vascular endothelial growth factor 1 receptor (VEGFR1); vascular endothelial growth factor 2 receptor (VEGFR2); Fms-related tyrosine kinase 1 (Fltl); placental growth factor (PGF); Platelet-derived growth factor (PDGF); angiopoietins; sonic hedgehog. The genes whose gene products inhibit angiogenesis are here called "antiangiogenic genes" and the products of those genes (mRNA; protein) are called "antiangiogenic gene products". Antiangiogenic gene products include endostatin; tumstatin; angiostatin; pigment epithelium-derived factor (PEDF) and fusion proteins or antibodies that are specific for target pro-angiogenics and / or their receptors, for example, the anti-VEGF fusion proteins sFLT1 or Eylea, the specific VEGF LucentisTM antibodies and AvastinTM, etc.
[00175] The genes whose gene products function as immunomodulators, for example, complement factors, toll-like receptors, are called "immunomodulatory genes". Exemplary immunomodulatory genes include cytokines, chemokines and the fusion proteins or antibodies that are specific to them and / or their receptors, for example, the anti-IL-6 fusion protein RilonaceptTM, the H Factor specific antibody. Complement lampamizumab, etc. Genes whose gene products function as neuroprotective factors, for example, platelet-derived growth factor receptor (PDGFR); glial-derived neurotrophic factor (GDNF); rod-derived cone viability factor (RdCVF); fibroblast growth factor (FGF); neurturin (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain-derived neurotrophic factor (BDNF); epidermal growth factor. Genes whose gene products function as light-responsive opsins, for example, opsin; rhodopsin; channel rhodopsin; halo rhodopsin.
[00176] In some cases, a gene product of interest is a specific site endonuclease that provides for the specific knock-down site of the gene's function, for example, where the endonuclease knocks out an allele associated with a retinal disease. For example, when a dominant allele encodes a defective copy of a gene that, when wild-type, is a structural protein of the retina and / or provides normal retinal function, a specific site endonuclease can be targeted to the defective allele and knocked out in the defective allele.
[00177] In addition to knocking out a defective allele, a specific site nuclease can also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele. Thus, for example, a rAAV virion in question can be used to deliver either a specific site endonuclease that knocks out a defective allele, or can be used to provide a functional copy of the defective allele, resulting in the repair of the defective allele, thereby providing the production of a functional retinal protein (for example, functional retinoschin, functional RPE65, functional periphery, etc.). See, for example, Li et al. (2011) Nature 475: 217. In some embodiments, an rAAV virion disclosed herein comprises a heterologous nucleotide sequence that encodes a specific site endonuclease; and a heterologous nucleotide sequence that encodes a functional copy of a defective allele, where the functional copy encodes a functional retinal protein. Functional retinal proteins include, for example, retinoschinoin, RPE65, protein 1 interacting with the retinitis pigmentosa GTPase regulator (RGPR), periphery, periphery-2 and the like.
[00178] Site specific endonucleases that are suitable for use include, for example, meganucleases; zinc finger nucleases (ZFNs); transcription activator effector nucleases (TALENs); and short palindromic repetitions interspersed regularly / associated with CRISPR (Cas), where such specific site endonucleases are non-naturally occurring and are modified to target a specific gene. Such site-specific nucleases can be manipulated to target specific sites within a genome, and the non-homologous end junction can then repair the break by inserting or deleting multiple nucleotides. Such site-specific endonucleases (also called "INDELs") then throw the protein out of the structure and effectively knock out the gene. See, for example, U.S. Patent Publication No. 2011/0301073.
[00179] In some embodiments of the variant rAAV vector disclosed here, a nucleotide sequence encoding a gene product of interest is operably linked to a constitutive promoter. Suitable constitutive promoters include, for example, the cytomegalovirus (CMV) promoter (Stinski et al. (1985) Journal of Virology 55 (2): 431-441), the early CMV enhancer / chicken β-actin promoter (CBA) / rabbit β-globin intron (CAG) (Miyazaki et al. (1989) Gene 79 (2): 269-277, CBSB (Jacobson et al. (2006) Molecular Therapy 13 (6): 1074- 1084), human elongation factor 1α (EF1α) promoter (Kim et al. (1990) Gene 91 (2): 217-223), human phosphoglycerate kinase (PGK) promoter (Singer-Sam et al. (1984) Gene 32 (3): 409-417, mitochondrial heavy chain promoter (Loderio et al. (2012) PNAS 109 (17): 6513- 6518), ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101 105) In other embodiments, a nucleotide sequence that encodes a gene product of interest is operationally linked to an inducible promoter In some cases, a nucleotide sequence that encodes a gene product of interest is operationally linked to an element r egulator specific for tissue type or specific for cell type. For example, in some cases, a nucleotide sequence encoding a gene product of interest is operationally linked to a specific photoreceptor regulatory element (for example, a specific photoreceptor promoter), for example, a regulatory element that confers selective expression of the gene operably linked in a photoreceptor cell. Suitable photoreceptor-specific regulatory elements include, for example, a rhodopsin promoter; a rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44: 4076); a promoter of the beta phosphodiesterase gene (Nicoud et al. (2007) J. Gene Med.9: 1015); a promoter of the retinitis pigmentosa gene (Nicoud et al. (2007) supra); a retinoid interphotoreceptor binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); a promoter for the IRBP gene (Yokoyama et al. (1992) Exp. Eye Res. 55: 225), a promoter for the opsin gene (Tucker et al. (1994) PNAS 91: 2611-2615), a promoter for the gene for retinosqusina (Park et al. (2009) Gene Therapy 16 (7): 916-926), a promoter of the CRX homeodomain protein gene (Furukawa et al. (2002) The Journal of Neuroscience 22 (5): 1640- 1647), guanine nucleotide binding protein (GNAT1) alpha transduction activity polypeptide 1 (Lee et al. (2010) Gene Therapy 17: 1390-1399), a retinal-specific leucine zipper protein gene promoter neural (NRL) (Akimoto et al. (2006) PNAS 103 (10): 3890-3895), human cone arrestin promoter (hCAR) (Li et al. (2002) Biochemistry and Molecular Biology 43: 1375-1383) , and the promoters PR2.1, PR1.7, PR1.5 and PR1.1 (Ye et al. (2016) Human Gene Therapy 27 (1): 72-82)). In some cases, a nucleotide sequence that encodes a gene product of interest is operationally linked to a specific regulatory element of the retinal pigment epithelium cell (for example, a specific RPE promoter), for example, a regulatory element that confers selective expression of the operably linked gene in an RPE cell. Suitable RPE specific regulatory elements include, for example, a RPE65 gene promoter (Meur et al. (2007) Gene Therapy 14: 292-303), a cell retinaldehyde-binding protein (CRALBP) gene promoter (Kennedy et al. (1998) Journal of Biological Chemistry 273: 5591-5598), a promoter of the pigment epithelium-derived factor gene (PEDF aka serpin F1) (Kojima et al. (2006) Molecular and Cellular Biochemistry 293 (1- 2): 63-69), and a vitelliform macular dystrophy (VMD2) promoter (Esumi et al. (2004) The Journal of Biological Chemistry 279 (18): 19064-19073). In some cases, a nucleotide sequence that encodes a gene product of interest is operationally linked to a specific regulatory element in Müller's glial cells (for example, a specific glial promoter), for example, a regulatory element that confers expression selection of the gene operably linked in a glial cell of the retina. Suitable glial-specific regulatory elements include, for example, a promoter of glial fibrillary acidic protein (GFAP) (Besnard et al. (1991) Journal of Biological Chemistry 266 (28): 18877-18883). In some cases, a nucleotide sequence that encodes a gene product of interest is operationally linked to a specific regulatory element of bipolar cells (for example, a specific bipolar promoter), for example, a regulatory element that confers selective expression of the gene operationally bound in a bipolar cell. Suitable specific bipolar regulatory elements include, for example, a GRM6 promoter (Cronin et al. (2014) EMBO Molecular Medicine 6 (9): 1175-1190).
[00180] For the purposes of the invention, the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence that encodes a variant AAV capsid protein as described above. An isolated nucleic acid can be an AAV vector, for example, a recombinant AAV vector.
[00181] The present disclosure also provides a method of treating a retinal disease, the method comprising administering to an individual in need of an effective amount of a variant rAAV virion comprising a transgene of interest as described above and disclosed. on here. One skilled in the art would readily be able to determine an effective amount of the rAAV virus in question and that the disease was treated by testing a change in one or more functional or anatomical parameters, for example, visual acuity, visual field, electrophysiological responsiveness to light and darkness, color vision, contrast sensitivity, anatomy, retinal and vasculature health, ocular motility, fixation preference and stability.
[00182] Non-limiting methods for assessing retinal function and its changes include assessing visual acuity (eg, best corrected visual acuity, ambulation, navigation, object detection and discrimination), assessment of the visual field (eg , static and kinetic perimetry of the visual field), conducting a clinical examination (for example, examination of a slit lamp in the anterior and posterior segments of the eye), assessment of electrophysiological responsiveness to all light and dark wavelengths (for example , all forms of electroretinography (ERG) [full field, multifocal, standard], all forms of evoked visual potential (VEP), electro-oculography (EOG), color vision, dark adaptation and / or contrast sensitivity) . Non-limiting methods for assessing the anatomy and health of the retina and its changes include Optical Coherence Tomography (OCT), fundus photography, adaptive optical scanning ophthalmoscopy (AO-SLO), fluorescence and / or autofluorescence; measurement of eye motility and eye movements (eg nystagmus, preferably, fixation and stability), measurement of reported results (changes reported by the patient in visual and non-visually oriented activities and activities, results reported by the patient [PRO], assessments based on a quality of life questionnaire, daily activities and measurements of neurological function (for example, functional magnetic resonance imaging (MRI)).
[00183] In some embodiments, an effective amount of the rAAV virus in question results in a decrease in the rate of loss of retinal function, anatomical integrity, or retinal health, e.g. a decrease of 2 times, 3 times, 4 times or 5 times or more in the rate of loss and, consequently, in the progression of the disease, for example, a decrease of 10 times or more in the rate of loss and, consequently, in the progression of disease. In some embodiments, the effective amount of the rAAV virus in question results in a gain in visual function, retinal function, an improvement in ocular motility and / or an improvement in neurological function, for example, a 2-fold, 3-fold improvement, 4 times or 5 times or more in retinal function, retinal health or anatomy, and / or improvement in eye motility, for example, a 10-fold or more improvement in retinal function, anatomy or health of the retina, and / or improves ocular motility. As will be readily appreciated by the person skilled in the art, the dose required to achieve the desired treatment effect will typically be in the range of 1 x 108 to about 1 x 1015 recombinant virions, typically referred to by those skilled in the art as 1 x 10 8 at about 1 x 1015 “vector genomes”.
[00184] A rAAV virion in question can be administered via intraocular injection, for example, by intravitreal injection, by subretinal injection, by supracoroidal injection, or by any other convenient mode or route of administration that will result in the application of the rAAV virus to the eye. Other convenient manostats or routes of administration include, without limitation, intravenous, intra-arterial, periocular, intracameral, subconjunctival and sub-joint injections and topical and intranasal administration. When administered via intravitreal injection, the rAAV virion in question is able to move through the vitreous and cross the inner limiting membrane (also referred to here as an internal limiting membrane, or “ILM”; a thin, transparent acellular membrane on the surface of the retina that forms the boundary between the retina and the vitreous body, formed by astrocytes and the Müller cell extremity feet), and / or moves through the layers of the retina more efficiently, compared to the capacity of a virion to AAV comprising the corresponding parental AAV capsid protein.
[00185] A variant capsid protein disclosed herein is isolated, for example, purified. In some embodiments, a variant capsid protein disclosed here is included in an AAV vector or in a recombinant AAV virus (rAAV). In other embodiments, such variant AAV vectors and / or variant AAV viruses are used in an in vivo or ex vivo method of treating eye disease in the primate retina.
[00186] The present description further provides host cells such as, without limitation, isolated (genetically modified) host cells comprising a nucleic acid in question. A host cell according to the invention disclosed herein, can be an isolated cell, such as a cell from an in vitro cell culture. Such a host cell is useful for producing a variant of the rAAV variant in question, as described herein. In one embodiment, that host cell is stably and genetically modified with a nucleic acid. In other embodiments, a host cell is transiently and genetically modified with a nucleic acid. Such a nucleic acid is introduced stably or transiently into a host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection and the like. For stable transformation, a nucleic acid will generally further include a selectable marker, for example, any of several well-known selectable markers, such as neomycin resistance and the like. Such a host cell is generated by introducing a nucleic acid into any of a variety of cells, for example, mammalian cells, including, for example, murine cells and primate cells (for example, human cells). Exemplary mammalian cells include, but are not limited to, primary cells and cell lines, where exemplary cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, fibroblasts C3H10T1 / 2, CHO cells and the like. Examples of host cells include, without limitation, HeLa cells (for example, American Type Culture Collection (ATCC) No. CCL-2), CHO cells (for example, ATCC No. CRL9618, CCL61, CRL9096), 293 cells (for example, ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (for example, ATCC No. CRL-1658), Huh-7 cells, BHK cells (for example, ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney cells (HEK) ( ATCC No. CRL1573), HLHepG2 cells and the like. A host cell can also be prepared using a baculovirus to infect insect cells, such as Sf9 cells, that produce AAV (see, for example, US Patent No. 7,271.20; US Patent Application No. 122 / 297,958). In some embodiments, a genetically modified host cell includes, in addition to a nucleic acid comprising a nucleotide sequence that encodes a variant AAV capsid protein, as described above, a nucleic acid that comprises a nucleotide sequence that encodes one or more proteins rep of AAV. In other embodiments, a host cell further comprises a variant rAAV vector. A variant of a variant rAAV can be generated using such host cells. Methods for generating an rAAV virus are described, for example, in U.S. Patent Publication No. 2005/0053922 and U.S. Patent Publication No. 2009/0202490.
[00187] The present disclosure further provides a pharmaceutical composition comprising: a) the variant rAAV virion, as described above and disclosed herein; and b) a pharmaceutically acceptable carrier, diluent, excipient or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient or buffer is suitable for use in a human or non-human patient. Such excipients, vehicles, diluents and buffers include any pharmaceutical agent that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail here. Pharmaceutically acceptable excipients have been widely described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., Eds., 7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. In some aspects of the present invention, the present invention provides a pharmaceutical composition comprising about 1 x 108 to about 1 x 1015 recombinant viruses or 1 x 108 to about 1 x 1015 vector genomes, wherein each said recombinant virus comprises a genome that encodes one or more gene products.
[00188] The following examples are presented to provide the skilled person with a complete description and disclosure for guidance on how to make and use the variant AAV capsids disclosed herein, and are not intended to limit the scope of the invention disclosed herein. In addition, the following examples are not intended to represent that the experiments below are all or the only experiments performed. EXAMPLES
[00189] The following examples are presented in order to provide those skilled in the art with a complete description and disclosure of how to make and use the present invention, and are not intended to limit the scope of what the inventors consider to be their invention, nor do they intend represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy in relation to the numbers used (for example, quantities, temperature, etc.), but some errors and experimental deviations must be accounted for. Unless otherwise stated, the parts are parts by weight, the molecular weight is the weight average molecular weight, the temperature is in degrees centigrade and the pressure is close to atmospheric.
[00190] General methods in molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. Eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. Eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are hereby incorporated by reference. The reagents, cloning vectors and kits for genetic manipulation referred to in this disclosure are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich and ClonTech. Example 1
[00191] Intravitreal Injection and Tissue Collection. A single male Cinomolgos monkey (macaca fascicularis), 4-10 years old and weighing at least 4 kg through intravitreal injection through the sclera (approximately 3 mm behind the limbus, using an appropriate administration procedure and device, was used) for human use). The animal was anesthetized and administered with 100 μL of topical anesthetic from the library in each eye.
[00192] Euthanasia was performed by trained veterinary personnel, with intravenous injection of 100 mg / kg of sodium pentobarbital on day 14 ± 3. The eyes were nucleated and stored at 4 ° C until dissection.
[00193] Tissue dissection. The eyes were cut along the ora serrata with a scalpel, and the anterior segment was removed. Relief cuts were made on the retina around the fovea to allow for flat mounting of the retina, and the vitreous was removed. Six retinal samples from each quadrant (upper, lower, nasal and temporal) were collected, as shown in Figure 2, and cell material corresponding to the RPE cells, photoreceptors, biopolar cells, amacrine cells, horizontal and / or ganglion cells were isolated .
[00194] Directed Evolution. The directed evolution process is shown in Figure 1A-1E. Briefly, a viral capsid library is created comprising more than 20 proprietary combinations of the DNA mutation and cap genes technique (Figure 1A). The viruses are then packaged (Figure 1B) - such that each particle is made up of a mutant capsid around the cap gene encoding that capsid - and purified. The capsid library is placed under selective pressure in vivo. The tissue or cellular material of interest is harvested to isolate AAV variants that have successfully infected that target, and successful viruses are recovered. Successful clones are enriched through repeated selection (Stage I - Figure 1D). The selected cap genes then undergo proprietary rediversification and are enriched through additional selection steps to iteratively increase viral fitness (Stage 2 - Figure 1D). The variants identified during vector selection stages 1 and 2 demonstrate the ability to transduce primate retinal cells (Figure 1E).
[00195] Successful recovery of AAV Capsid Genomes: Cycles 1-6. Capsids recovered from each selection cycle were used to pack the injected library to start the subsequent selection cycle. The recovery of tissue capsid genes represents the successful internalization of library vectors in the tissue of interest. After the 4th cycle, additional library diversification was incorporated before packaging the library and injection for the 5th cycle. The recovery of the viral genomes of RPE, PR, inner nuclear layer (INL) and retinal tissue from the ganglion cell layer (GCL) from a representative selection cycle are shown in Figure 3. Bands inside the boxes represent successful recovery of viral genomes.
[00196] Sequencing Analysis: Cycles 3-6. During cycles 3-6, sequencing was performed on individual clones within the library to determine the frequency of variants within the population. The variants were evaluated for the presence of motifs within the sequencing data. The variants were grouped into motifs based on the presence of a unifying variation (for example, a specific point mutation or specific peptide insertion sequence at a consistent location within a capsid) that occurred in multiple sequences. Reasons representing at least 5% of the population sequenced in two or more selection cycles or at least 10% of the population sequenced in one or more selection cycles are represented in Figure 4A (analysis of sequencing of cycle 3), 4B (analysis of sequencing cycle 4), 4C (cycle 5 sequencing analysis) and 4D (cycle 6 sequencing analysis).
[00197] Several representative clones that have been identified as conferring increased retinal cell infectivity are listed in Table 1 below (each clone contains the identified and identical replacement (s) and / or insertion (s) ( s) to SEQ ID NO: 2; sequences and frequency (in parentheses) are listed for each clone): Table 1. Modifications in the amino acid sequence of the AAV VP1 capsid protein that confer greater infectivity of one or more cells of the retina. The substitutions listed in column 2 are based on the amino acid sequence for wild-type AAV2, i.e., in the absence of inserted peptide.











[00198] Also identified as a capsid with an increased infectivity of one or more retinal cells, it was a clone with the following ancestral VP1 capsid sequence:
[00199] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQD DGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNP YLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTA PGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPL GEPPAGPSGLGSGTMAAGGGAPMDNANEGADGVGNASGNWHCDSTWL GDRVITTSTRTWALPTYNNHLYKQISSASAGSTNDNHYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNL TSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAV GRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLI DQYLYYLARTQSTGGTAGTRELLFSQAGPSNMSAQAKNWLPGPCYRQQRV SKTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFPSS GVLIFGKQGAGANNTALENVMMTSEEEIKTTNPVATEQYGVVASNLQSSNT APVTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GLKHPPPQILIKNTPVPANPPAVFTPAKFASFITQYSTGQVSVEIEWELQKEN SKRWNPEIQYTSNYAKSTNVDFAVDNEGVYSEPRPIGTRYLTRNL. (SEQ ID NO: 59)
[00200] This ancestral capsid variant is evolved from the ancestral capsid of SEQ ID NO: 58, in which the degeneration positions (residues 264, 266, 268, 448, 459, 460, 467, 470, 471, 474, 495, 516, 533, 547, 551, 555, 557, 561, 563, 577, 583, 593, 596, 661, 662, 664, 665, 710, 717, 718, 719, 723) evolved to understand Alanine ( A) in 264, Alanine (A) in 266, Serine (S) in 268, Alanine (A) in 448, Threonine (T) in 459, Arginine (R) in 460, Alanine (A) in 467, Serina (S ) in 470, Asparagine (N) in 471, Alanine (A) in 474, Serine (S) in 495, Asparagine (D) in 516, Asparagine (D) in 533, Glutamine (Q) in 547, Alanine (A) in 551, Alanine (A) in 555, glutamic acid (E) in 557, Methionine (M) in 561, Serine (S) in 563, Glutamine (Q) in 577, Serine (S) in 583, Valine (V) in 593, Threonine (T) in 596, Alanine (A) in 661, Valine (V) in 662, Threonine (T) in 664, Proline (P) in 665, Threonine (T) in 710, Aspartic Acid (D) in 717, Asparagine (N) in 718, Glutamic acid (E) in 719, and S erina (S) in 723.
[00201] The variant AAV virions disclosed herein can incorporate reasonable, rational design parameters, characteristics, modifications, advantages and variations, which are easily evident to those skilled in the art in the field of AAV viral vector manipulation. Example 2
[00202] Targeted evolution was employed to discover new variants of adeno-associated viruses (AAV) with application of genes superior to retinal cells after intravitreal administration (IVT), a route of administration with significant advantages over other methods of applying genes to the human eye (Example 1). Cellular tropism after intravitreal administration of the new variant AAV comprising a substitution of P34A and the peptide LAISDQTKHA (SEQ ID NO: 28) inserted at amino acid 588 (LAISDQTKHA + P34A) was evaluated in vivo in non-human primates (NHP) as a representative example the ability of AAV variants containing ISDQTKH (SEQ ID NO: 14) to transduce retinal cells.
[00203] Recombinant AAV virions comprising an AAV2 capsid or the new LAISDQTKHA + P34A variant capsid and a genome comprising a green fluorescent protein (GFP) transgene operably linked to a CMV promoter (AAV2.CMV.GFP and LAISDQTKHA + P34A.CMV.GFP, respectively) or a CAG promoter (AAV2.CAG.EGFP and LAISDQTKHA + P34A.CAG.EGFP, respectively) were manufactured using standard methods. African Green Monkeys (Figures 7, 8) or Cinomolgos (Figure 9) were injected intravitreally with various vector doses ranging from 4x1010 vg to 1x1012 1e12 vg per eye (see figure captions for details) and retinal cell transduction was assessed in life by background fluorescence imaging with a Heidelberg SpectralisTM.
[00204] Intravitreal application of AAV comprising the new variant LAISDQTKHA + P34A resulted in broader and more robust transgenic expression through the NHP retina than AAV2 (Figures 7-9). The images reveal that the new variant AAV capsid provides robust expression within the center of the fovea (an area rich in cones); in the parafoveal ring (an area rich in retinal ganglion cells) and in the periphery (an area rich in many cell types, including rods, Müller glia, amacrine cells, bipolar cells) as early as 2 weeks after injection. In contrast, and consistent with the results reported by others, wild-type AAV2 provides a weaker expression that is primarily in the parafoveal ring and can only be detected at later times. Immunohistochemical analysis of various regions of the retina performed 3 weeks after injection confirmed that many types of retinal cells, including retinal pigment epithelial cells, rod and cone photoreceptors, and retinal ganglion cells, have been successfully transduced through the retina (Figures 10A-10E).
[00205] This study illustrates the application of superior genes by the variant comprising ISDQTKH following a clinically preferred route of administration compared to clinically relevant AAV2. Similar efficacy is achievable with other variants comprising this peptide insertion motif. Likewise, similar efficacy is achievable with other variants disclosed by the present invention that have been identified using the same directed evolutionary approach. Example 3
[00206] Cell tropism of the new AAV variant LAISDQTKHA + P34A for retinal pigment epithelial cells (RPE) and photoreceptor cells (PR) was evaluated in vitro in RPE cells and PR cells generated from pluripotent stem cells induced by fibroblasts (FB-iPSC) or human embryonic stem cells (ESC).
[00207] AAV virions comprising an AAV2 capsid or the new LAISDQTKHA + P34A variant capsid and a genome comprising a green fluorescent protein (EGFP) transgene operably linked to a CAG promoter (AAV2.CAG.EGFP and LAISDQTKHA + P34A .CAG.EGFP, respectively) were manufactured using standard methods. Human EPR cell cultures were generated from the ESI-017 human embryonic stem cell cell line or pluripotent stem cells induced by human fibroblast derivatives (“FB-iPSC”) using a differentiation protocol 45 days. Maturation in RPE cells was confirmed by detecting the expression of mature EPR markers including RPE65 and BEST1; the synthesis of VEGF and PEDF; and the ability to phagocytize external segments of the rods. PR cultures were generated by a multi-stage eye cup formation paradigm and confirmed to understand PRs by detecting the expression of Recoverina and S Opsina after 179 days in culture.
[00208] Regarding AAV2, LAISDQTKHA + P34A provided significantly greater transgenic transduction and expression efficiency in human EPR cultures seven days after infection, as determined by immunofluorescence (Figures 11A-B), flow cytometry (increase of 2 , 7 times; Figures 11C-D) and Western blot analysis (Figures 11E-F). Robust transduction and expression were also achieved with the use of LAISDQTKHA + P34A.CAG.EGFP in human PR cultures at 32 days after infection. This study illustrates the superior ability of variants comprising ISDQTKH (SEQ ID NO: 14) for applying genes to retinal cells.
[00209] The foregoing illustrates merely the principles of the invention. It will be appreciated that those skilled in the art will be able to conceive of various arrangements which, although not explicitly described or shown here, incorporate the principles of the invention and are included within its spirit and scope. In addition, all examples and conditional language cited here are primarily intended to help the reader understand the principles of the invention and the concepts contributed by the inventors to promote the technique, and should be considered without limitation to such examples and conditions specifically recited.
[00210] In addition, all statements that describe the principles, aspects and modalities of the invention, as well as specific examples thereof, are intended to cover their structural and functional equivalents. Additionally, it is intended that such equivalents include the equivalents currently known and the equivalents developed in the future, that is, any developed elements that perform the same function, regardless of the structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described here. On the contrary, the scope and spirit of the present invention are incorporated by the appended Claims.

权利要求:
Claims (28)
[0001]
1. Variant Adeno-Associated Virus (AAV) Capsid Protein, comprising a peptide insert in the GH loop of the capsid protein relative to a corresponding parental AAV capsid protein, characterized in that the peptide insert comprises the sequence of ISDQTKH amino acids (SEQ ID NO: 14) and where the insertion site is located between the amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO: 2) or in the corresponding position in the capsid protein of another serotype AAV.
[0002]
2. Variant Adeno-Associated Virus (AAV) Capsid Protein according to Claim 1, characterized in that the peptide insert comprises the amino acid sequence Y1Y2ISDQTKHY3, wherein each of Y1-Y3 is independently selected from Ala , Leu, Gly, Ser, Thr and Pro.
[0003]
Variant Adeno-Associated Virus (AAV) Capsid Protein according to Claim 2, characterized in that the peptide insert comprises or consists of the LAISDQTKHA amino acid sequence (SEQ ID NO: 28).
[0004]
4. Variant Adeno-Associated Virus (AAV) Capsid Protein according to any one of Claims 1 to 3, characterized in that the capsid protein comprises one or more amino acid substitutions with respect to AAV2 VP1 (SEQ ID NO: 2) or one or more corresponding substitutions in the capsid protein of another AAV serotype.
[0005]
5. Variant Adeno-Associated Virus (AAV) Capsid Protein according to any one of Claims 1 to 4, characterized in that the capsid protein comprises one or more of the following amino acid substitutions with respect to AAV2 VP1 ( SEQ ID NO: 2) or the corresponding substitution (s) in the capsid protein of another AAV serotype: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K , T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, SQ7, E7, E7, E7, E7
[0006]
6. Variant Adeno-Associated Virus (AAV) Capsid Protein according to Claim 5, characterized in that the capsid protein comprises an amino acid substitution P34A with respect to VP1 of AAV2 (SEQ ID NO: 2) or corresponding substitution in the capsid protein of another AAV serotype.
[0007]
Variant Adeno-Associated Virus (AAV) Capsid Protein according to Claim 6, characterized in that the capsid protein comprises the amino acid sequence shown as SEQ ID NO: 42.
[0008]
Variant Adeno-Associated Virus (AAV) Capsid Protein according to Claim 7, characterized in that the capsid protein consists of the amino acid sequence shown as SEQ ID NO: 42.
[0009]
9. Variant Adeno-Associated Virus (AAV) Capsid Protein according to any one of Claims 1 to 8, characterized in that the capsid protein gives an infectious recombinant AAV virus (rAAV) an increased infectivity, of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
[0010]
10. Isolated Nucleic Acid, characterized in that it comprises a nucleotide sequence that encodes a variant AAV capsid protein as defined in any one of Claims 1 to 9.
[0011]
11. Infectious rAAV virion, characterized in that it comprises a variant AAV capsid protein as defined in any one of Claims 1 to 9.
[0012]
Infectious rAAV virion according to Claim 11, characterized in that it further comprises a heterologous nucleic acid that encodes a gene product.
[0013]
13. Infectious rAAV virion according to Claim 12, characterized in that the gene product is a short interfering protein or RNA.
[0014]
Infectious rAAV virion according to Claim 13, characterized in that the variant AAV capsid protein comprises the amino acid sequence shown in SEQ ID NO: 42 and wherein the heterologous nucleic acid comprises a nucleotide sequence that encodes a protein escort protein-1 Rab.
[0015]
Infectious rAAV virion according to Claim 14, characterized in that the variant AAV capsid consists of the amino acid sequence shown as SEQ ID NO: 42 and in which the nucleotide sequence encoding the protein-1 protein of Rab escort is operationally linked to a CAG promoter.
[0016]
16. Infectious rAAV virion according to Claim 13, characterized in that the variant AAV capsid protein comprises the amino acid sequence shown in SEQ ID NO: 42 and wherein the heterologous nucleic acid comprises a nucleotide sequence that encodes a GTPase regulatory protein for retinitis pigmentosa.
[0017]
17. Infectious rAAV virion according to Claim 16, characterized in that the variant AAV capsid protein consists of the amino acid sequence shown as SEQ ID NO: 42.
[0018]
18. Infectious rAAV virion according to Claim 17, characterized in that the nucleotide sequence encoding the retinitis pigmentosa GTPase regulatory protein is operably linked to a rhodopsin kinase promoter.
[0019]
19. Infectious rAAV virion according to Claim 13, characterized in that the variant AAV capsid protein comprises the amino acid sequence shown in SEQ ID NO: 42 and wherein the heterologous nucleic acid comprises a nucleotide sequence that encodes a polypeptide that inhibits the activity of vascular endothelial growth factor (VEGF).
[0020]
20. Infectious rAAV virion according to Claim 19, characterized in that the variant AAV capsid protein consists of the amino acid sequence shown as SEQ ID NO: 42.
[0021]
21. Infectious rAAV virion according to Claim 20, characterized in that the polypeptide is a fusion protein or antibody.
[0022]
22. Pharmaceutical composition, characterized in that it comprises an rAAV virion as defined in any one of Claims 13 to 21 and a pharmaceutically acceptable excipient.
[0023]
23. Use of rAAV Virion, as defined in any one of Claims 13 to 21, and / or Pharmaceutical Composition, as defined in Claim 22, characterized in that it is for the manufacture of a medicament.
[0024]
24. Use of rAAV Virion, as defined in Claim 14 or 15, and / or Pharmaceutical Composition, which comprises an rAAV Viron, as defined in Claim 14 or 15, characterized in that it is for the manufacture of a medicament for the choroidideremia treatment.
[0025]
25. Use of rAAV Virion, as defined in any of Claims 16 to 18, and / or Pharmaceutical Composition, which comprises an rAAV virion, as defined in any of Claims 16 to 18, characterized in that it is for the manufacture of a medicine for the treatment of X-linked retinitis pigmentosa.
[0026]
26. Use of rAAV Virion, as defined in any of Claims 19 to 21, and / or Pharmaceutical Composition, which comprises an rAAV virion, as defined in any of Claims 19 to 21, characterized in that it is for the manufacture of a medicine for the treatment of wet age-related macular degeneration, diabetic retinopathy, diabetic macular edema or choroidal neovascularization.
[0027]
27. Use of rAAV Virion and / or Pharmaceutical Composition according to any one of Claims 23, 24, 25 and 26, characterized in that the rAAV virus or pharmaceutical composition is suitable for administration by injection intravitreal.
[0028]
28. Use of rAAV Virion, and / or Pharmaceutical Composition, according to any one of Claims 23, 24, 25 and 26, characterized in that the drug is for treating a human individual.

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法律状态:
2020-02-11| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2020-05-05| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-07-07| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-10-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-22| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 12/05/2017, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201662336441P| true| 2016-05-13|2016-05-13|

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US62/336,441|2016-05-13|

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US201662378106P| true| 2016-08-22|2016-08-22|

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US201662384590P| true| 2016-09-07|2016-09-07|

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PCT/US2017/032542|WO2017197355A2|2016-05-13|2017-05-12|Adeno-associated virus variant capsids and methods of use thereof|

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