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    • 专利摘要:
    In vitro method to recover the expression of the F5 gene encoding coagulation factor V. The present invention is related to the deficiency of factor V of coagulation and to gene editing (CRISPR), for the in vitro correction of mutations in the F5 gene and the generation of tools with which to cure the disease that today does not have treatment. The invention includes an in vitro method to recover the expression of the F5 gene encoding coagulation factor V, by using the CRISPR/Cas9 methodology that corrects the new pathological mutation described. The invention also includes in vitro cell cultures in which the new mutation has been corrected, as well as kits that include the pairs of guides used in the CRISPR/Cas9 method to recover the expression of the F5 gene with that mutation. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2785323A1
    申请号:ES202030442
    申请日:2020-05-14
    公开日:2020-10-06
    发明作者:Martin Antonio Liras；Ramos Luis Javier Serrano；Noguera Sara Bernal
    申请人:Universidad Complutense de Madrid；Fundacio Institut de Recerca de lHospital de La Santa Creu i Sant Pau；
    IPC主号:
    专利说明:

    [0004] TECHNICAL SECTOR
    [0006] The present invention falls within the field of diseases related to blood coagulation and, more specifically, to the deficiency of factor V, also called parahemophilia or Owren's disease. Likewise, it is related to the application of gene editing for the correction of mutations in the gene that codes for factor V coagulation.
    [0008] BACKGROUND OF THE INVENTION
    [0010] Factor V is an essential protein that participates in coagulation and plays a key role in the so-called blood coagulation cascade due to its procoagulant and anticoagulant activity. 80% of circulating factor V is produced in the liver and the remaining 20% in the alpha granules of platelets. In humans, the gene that expresses this protein ( F5) is approximately 80kb in size and is located on chromosome 1q24.2. The coding sequence of the F5 gene is divided into 25 exons and 24 introns and its cDNA is 6914bp in size. So far, approximately 150 point mutations and small insertions and deletions in the gene have been described. Factor V deficiency, an ultra-rare disease occurring in only 1 to 9 per million, is an autosomal recessive bleeding disorder associated with mutations in the F5 gene . This inherited bleeding disorder is clinically characterized by a heterogeneous spectrum of hemorrhagic manifestations ranging from mucosal or soft tissue bleeding to life-threatening bleeding. Currently, there is no palliative or curative treatment for this disease, only the administration of fresh frozen plasma. This alternative is very short-lived (4 hours) and the dosage is very difficult to adjust to the characteristics of the patient since the concentration of factor V in plasma pools is highly variable depending on the donor (Tabibian S, et al. A Comprehensive Overview of Coagulation Factor V and Congenital Factor V Deficiency. Semin Thrnmb Hemost, 2019; 45: 523-43).
    [0012] In the patent application US2012270708A1 on the treatment of coagulopathies with hyperfibrinolysis, the use of thrombomodulin analogues (new modifications of the protein) is described for the preparation of a medicine with which to treat coagulopathies that present with hyperfibrinolysis, such as related disorders with hemophilia including parahemophilia.
    [0014] WO2010069946A1 describes a method to purify factor V from biological fluids, with the interest of using it in cases of deficiency in this coagulation factor.
    [0016] Patent application WO2008059009A2 refers to a method to prevent and treat bleeding in joints, muscles and soft tissues in hemophiliac patients, by administering factor V resistant to APC (activated protein C), which has the ability to inactivate both factor V as coagulation factor VIII, as a hemostatic.
    [0018] WO2010060035A1 describes therapeutic strategies that use variants derived from factor V present in snake venom, and its derivatives, to modulate the coagulation cascade in patients who require it, since factor V from the venom of some snakes presents around a 44% sequence homology with that of mammalian factor V.
    [0020] Compositions and methods have also been described to inhibit the expression of certain mutant genes of factor V, as in patent application US2011130443A1 in which a double-stranded RNA is described to inhibit the expression of the mutant gene that causes a substitution of G by A at nucleotide 1691 in the factor V gene, which causes an Arg506Gln change.
    [0022] Despite the contributions cited, there is still no treatment for this disease, to a great extent, because some of the solutions described are not economically profitable as they are an ultra-rare disease. In any case, they would be palliative and low-efficacy treatments in severe deficiencies of factor V.
    [0024] EXPLANATION OF THE INVENTION
    [0026] In vitro method to recover the expression of the F5 gene encoding coagulation factor V.
    [0028] Due to the fact that currently in the pharmacological market there is no specific curative or palliative treatment of this pathology, with the exception of generic treatment for various coagulopathies such as fresh frozen plasma, in this invention a gene methodology is used, based on gene editing by means of the CRISPR / Cas9 tool, to provide tools to cure (not palliate) this pathology, acting specifically on the mutation that produces this factor V deficiency in coagulation. It is an in vitro method that is not a method of treating the human or animal body through surgery or therapies performed in the body and it is not intended to modify the genetic identity of the human germ line. The objective is to provide the necessary tools that can later be used to establish a long-term or permanent and individualized curative treatment for each patient.
    [0030] One aspect of the present invention relates to an in vitro method for recovering the expression of the F5 gene encoding coagulation factor V using the CRISPR / Cas9 methodology. This method includes the use of a pair of guides in which each of the guides is between 18 and 22 nucleotides in length and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to the position of the localized mutation. at nucleotide 3279 of the F5 gene . Preferably, a pair of guides characterized by SEQ ID NO: 68-69 or a pair of guides characterized by SEQ ID NO: 70-71 is used. By using the CRISPR / Cas9 methodology with any of these pairs of guides, a mutation in nucleotide 3279 of the F5 gene is corrected that generates a nonsense mutation in the amino acid sequence of factor V that gives rise to a stop codon (from stop) (p.Trp1093 *). This mutation has been identified by sequencing the F5 gene from a patient with severe factor V deficiency; the sequence obtained is characterized by SEQ ID NO: 65.
    [0031] Another aspect of the present invention relates to an in vitro cell culture in which the expression of the F5 gene encoding coagulation factor V has been recovered. In these cells, the mutation of the F5 gene identified at nucleotide 3279 that generated a nonsense mutation in the amino acid sequence of factor V that gives rise to a stop codon (p.Trp1093 *) has been corrected by using the CRISPR / Cas9 methodology including the use of a pair of guides, in which each of the guides has a length of between 18 and 22 nucleotides and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to the position of the mutation located at nucleotide 3279 of the F5 gene . Preferably, the pair of guides characterized by SEQ ID NO: 68-69 or the pair of guides characterized by SEQ ID NO: 70-71 is used. The complete sequence of the mutated F5 gene can be that characterized by SEQ ID NO: 65.
    [0033] Another aspect of the invention refers to a kit comprising a pair of guides, in which each of the guides has a length of between 18 and 22 nucleotides and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to to the position of the mutation located at nucleotide 3279 of the F5 gene . Preferably, it comprises the pair of guides characterized by SEQ ID NO: 68-69 and / or the pair of guides characterized by SEQ ID NO: 70-71. Furthermore, this kit can comprise a Cas9 protein or a polynucleotide encoding the Cas9 protein.
    [0035] BRIEF DESCRIPTION OF THE FIGURES
    [0037] To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is attached as an integral part of said description in which, with an illustrative and non-limiting nature, it has been represented the next:
    [0039] Figure 1. Pathological genetic variants (mutations) found in the patient's F5 gene.
    [0041] Figure 2. Gene editing for correction of mutations using CRISPR / Cas9, according to the state of the art.
    [0042] FIGURE 3. Design of gene editing guides using CRISPR / Cas9, to obtain the mutated cell model and to correct the mutation:
    [0043] A) Obtaining the mutated cell model.
    [0044] B) Reversal and correction of the mutation.
    [0046] PREFERRED EMBODIMENT OF THE INVENTION
    [0048] The present invention is further illustrated by the following examples, which are not intended to be limiting of its scope.
    [0050] Example 1. Hematological and coagulation analysis.
    [0052] Blood samples were taken from a 10-year-old patient and her parents. The girl, from southern Spain, presents a severe deficiency of factor V (FV: C <1%). The other clotting factors have normal levels. When analyzing both the father and the mother of the girl, it turned out that they both have deficiencies in factor V coagulation.
    [0054] For sampling, the approval of the Ethics Committee of the Hospital 12 de Octubre in Madrid was previously obtained and the procedure was followed in accordance with the Declaration of Helsinki.
    [0056] The blood samples were distributed in tubes with 0.105M sodium citrate (1:10). Plasma was obtained by centrifugation at 2000xg for 20 minutes and, subsequently, it was stored in aliquots at -80 ° C until use.
    [0058] The following hematological parameters were analyzed: hemoglobin concentration (Hb), mean corpuscular hemoglobin (HCM), mean corpuscular hemoglobin concentration (MCHC), erythrocyte count, hematocrit, mean corpuscular volume (MCV), reticulocyte count, white blood cell count , platelet count, mean platelet volume (MPV), and platelet count. All these parameters were obtained in an ADVIA®120 Hematology System automatic cell counter; Siemens Healthcare GmbH, Zurich, Switzerland).
    [0060] Blood samples for analysis of platelet function and time of coagulation tubes were taken in vacuum blood collection tubes (Vacutainer, Becton Dickinson) with 3.8% sodium citrate and processed on a Platelet Function Analyzer-100 (PFA-100®; Siemens Healthcare Diagnostics AG, Zurich, Switzerland). Collagen / adenosine-5-diphosphate (ADP) cartridges (Dade® PFA, Siemens Healthcare Diagnostics, AG, Zurich, Switzerland) were used to determine the bleeding time and platelet aggregation. The cartridges were used at room temperature by adding 1 mL of citrated blood to them. The samples were aspirated under constant vacuum through a capillary and a microscopic opening made in a membrane coated with collagen and ADP.
    [0062] All parameters related to hemostasis, such as prothrombin time (PT), prothrombin activity (AP), activated partial thromboplastin time (aTPP) (cephalin time), fibrinogen (F), and the international normalized index (INI), were determined in accordance with the standards defined by the Department of Hematology of the Hospital Universitario de Jaén, Spain. HemosIL® kits (Instrumentation Laboratory; Bedford, Massachusetts, USA) were used according to the manufacturer's instructions.
    [0064] HemosIL® RecombiPlasTin 2G was used to measure prothrombin time (PT) and fibrinogen levels and a highly sensitive thromboplastin reagent, based on recombinant human tissue factor (rhFT), was used. After reconstituting the RecombiPlasTin 2G diluents, the PT reagent included in the RecombiPlasTin 2G kit is converted into a liposomal preparation containing rhFT in a mixture of synthetic phospholipids and combined with calcium chloride, buffer and a preservative. As rhFT does not contain any contaminating clotting factors, RecombiPlasTin 2G is highly sensitive to extrinsic pathway clotting factors, making it particularly suitable for assays with these factors. On the other hand, RecombiPlasTin 2G's formulation makes it insensitive to therapeutic heparin levels. In the PT test, the addition of the reagent to the patient's plasma in the presence of calcium ions activates the extrinsic coagulation pathway. This eventually results in the conversion of fibrinogen to fibrin, with the formation of a solid gel. Fibrinogen was quantified by relating the absorbance, or scattering of light, during clot formation, with a calibrator. TP results are reported in seconds, percent activity, or INI; fibrinogen levels, in g / L.
    [0065] HemosIL® APTT-SP (liquid) was used to determine the aTPP value. The aTPP test uses a contact activator that stimulates factor XIIa production. The activator is a dispersion of colloidal silica with synthetic phospholipids, a buffer and a preservative and provides a contact surface for the interaction of high molecular weight kininogen, kallikrein and factor XIIa. Contact activation occurs at 37 ° C for a specified period of time. The addition of 0.025 M calcium chloride with a preservative triggers a series of reactions that will lead to the formation of clots. Phospholipids are also required to generate the compounds that will act on factor X and prothrombin. Results are expressed in seconds.
    [0067] HemosIL® FV deficient plasma was used for the quantitative determination of factor V activity. Factor V activity in plasma was determined using the modified prothrombin time test performed on the patient's citrate-diluted plasma in the presence of plasma. human immunodepleted with factor V. The correction for prolonged clotting time for deficient plasma was proportional to the concentration (percent activity) of the specific factor (factor V) in the patient's plasma, which can be obtained by drawing a calibration curve. The reference levels of factor V ranged between 60-130% (0.60-1.30 IU).
    [0069] In both the girl and her parents, all hematological parameters, including hemoglobin concentration (Hb), mean corpuscular hemoglobin (HCM), mean corpuscular hemoglobin concentration (MCHC), total erythrocyte count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), reticulocytes (RETIC), leukocyte count (GB), differential GB count, platelet count (PLT), mean platelet volume (VMP) , and platelet (PCT), were within the reference ranges.
    [0071] In the coagulation analysis, the girl showed altered values for both extrinsic and intrinsic coagulation pathways (prothrombin time (PT), prothrombin activity (AP), activated partial thromboplastin time (aTPP) (cephalin time) ), fibrinogen (F), international normalized index (INI), and factor V activity. The patient has a severe factor V deficiency with a factor V activity less than 1% of the reference value. In her parents, alterations in coagulation parameters and factor V activity were in line with the level of involvement of the allele of the F5 gene and the heterozygous state they present.
    [0073] Example 2. DNA extraction.
    [0075] Following informed consent, EDTA anticoagulated blood samples were collected. Genomic DNA was automatically extracted from previously isolated peripheral leukocytes by saline precipitation procedures using Autopure instrumentation (Qiagen). Using this kit, ready-to-use genomic DNA is obtained simply and quickly from 200pL whole blood samples. It is based on a vacuum centrifugation and the previous separation of leukocytes is not necessary. Neither phenol / chloroform extraction nor alcohol precipitation is required. DNA purified for use in PCR is stored between -25 ° C and -15 ° C.
    [0077] Example 3. Analysis of mutations.
    [0079] Primers corresponding to the complete sequence encoding the F5 gene and the adjacent intronic regions were designed, according to entry NC_000001.11 (169511951 169586630, complementary) of the NCBI database, to obtain PCR products (chain reaction of the polymerase) with a size of between 300 and 600 bp, approximately (Table 1).
    [0081] Table 1. Reference of the primers and size of the fragments obtained for the complete sequencing of the F5 gene of the patient under study.
    [0083]
    [0084]
    [0085]
    [0086]
    [0087]
    [0090] PCR amplification was performed in a final volume of 25pL containing 1.5mM-2.5mM MgCl 2 (depending on the fragment to be amplified), 200pM of each dNTP, 0.2pM of each primer, 0.5 units of Taq DNA polymerase (Ecogen) in manufacturer's recommended buffer and 150-200ng of genomic DNA. Thermal cycler conditions were as follows: 94 ° C for 5 minutes, followed by 30 cycles at 94 ° C for 30 seconds (denaturation step); 58 ° C-60 ° C (depending on the fragment to be amplified) for 30 seconds and 72 ° C for 2 minutes (elongation step), followed by a final extension step of 20 minutes at 72 ° C. When the PCR amplification was completed, the remaining dNTPs and primers remaining in the PCR product mix were removed by using the digestion enzymes ExoSAP (exonuclease I and alkaline phosphatase) (Sigma-Aldrich). Once the purification procedure was completed, the bidirectional sequencing reaction of the entire sequence was performed by the Sanger method using the BigDye ™ Terminator v1.1 Cycle Sequencing kit. The sequencing reactions of the amplified fragments were purified using the SEQ96 assembly kit on a vacuum manifold, to remove unincorporated labeled terminators and salts before subjecting the sequencing products to capillary electrophoresis on an ABI Prism 3500 Genetic Analyzer. (Applied Biosystems). The chromatograms of the sequences were analyzed using SeqScape v.2.1.1.
    [0092] To describe mutations and genetic variants, the Human Genome Variation Society (HGVS) nomenclature (den Dunnen JT, et al. HGVS recommendations for the description of sequence variants. 2016 update. Hum Mutation, 2016; 37: 564-) was used. 9). All nucleotide changes identified in the patient with factor V deficiency were analyzed using the Alamut visual computer program v.2.6. software, which integrates genetic and genomic information from different sources in a coherent and convenient environment to describe variants using the HGVS nomenclature and help interpret their pathogenic status. Furthermore, all the changes detected in the girl were studied in the mother and father to determine family segregation.
    [0094] All deleterious mutations and genetic variants were assigned a nucleotide number from the first translated base of the F5 gene according to the reference sequence NM_000130.4 from the NCBI database.
    [0096] In the analysis of the F5 gene mutations of the patient, a total of 24 different sequence variants were identified, including 2 nonsense mutations that give rise to a stop codon, 1 reading frame shift mutation, 6 mutations that give rise to amino acid change, 9 synonymous variations (no translation change) and 7 intron changes (Table 2 and Figure 1).
    [0098] In Figure 1, the two pathogenic variants (mutations) found in the F5 gene of the patient with severe factor V deficiency are shown. They are two nonsense mutations that give rise to two stop codons (CS). One of them, NM_000130.4: c. 3279G> A, p.Trp1093 *, which implies a change from a guanine to an adenine (G> A), and the other, the NM_000130.4: c. 2218C> T, p.Arg740 *, already described previously, which involves a change from a cytosine to a thymine (C> T). The following characteristics are indicated: Msn: Nonsense mutation; (D) domains (A1, A2, B, A3, C1 and C2); exons (E), heavy chain (Hc), light chain (Lc), and post-translational cleavage region (ps); activated protein C inactivation site (APCC); thrombin activation site (TC).
    [0100] Table 2. Genetic variants identified in the patient's F5 gene.
    [0104] *Not applicable.
    [0105] ** Data not available.
    [0107] Of the 24 changes, 17 have a small allelic frequency (FAM)> 5% to that described in the database of 1000 genomes (5 nonsense, 7 synonyms and 5 intronic variants) (Table 2). The remaining seven changes have no FAM value because they are rare sequence variants that are not previously described.
    [0109] In addition, we also detected a correct family segregation of the changes identified in the patient with her parents. In the girl, the two nonsense mutations (p.Arg740 * and p.Trp1093 *) are both in the heterozygous state, one mutation is inherited from her mother (NM_000130.4: c. 2218C> T, p.Arg740 *) and the other is inherited from his father (NM_000130.4: c. 3279G> A, p.Trp1093 *).
    [0111] The type of mutation was correlated with the levels of factor V activity in the patient and in her parents. The c.3279G> A mutation in the father is associated with an activity of 21% and the c.2218C> T mutation in the mother, with a factor V activity of 62.9%.
    [0113] Of all the variants (mutations) found in the F5 gene of the patient with severe factor V deficiency, two are considered pathogenic missense mutations because they meet the criteria for pathogenicity (the very pathogenicity of a stop codon). Of them, NM_000130.4: c. 3279G> A, p.Trp1093 * is new since it is not described so far, while the other one, NM_000130.4: c. 2218C> T, p.Arg740 *, has already been described previously (Lunghi B, et al. A Novel Factor V Nuil Mutation Detected in a Thrombophilic Patient With Pseudo-Homozygous APC Resistance and in an Asymptomatic Unrelated Subject. Blood, 1998; 92: 1463-5 ).
    [0115] Example 4. Gene editing.
    [0117] Once the specific mutation of the patient under study was known, gene editing (correction of the mutation) was applied using the CRISPR / Cas9 technique (Figure 2) in a cell model designed in the laboratory that carried the same mutation of the patient. CRISPR / Cas9 technology (Regularly Clustered and Interleaved Short Palindromic Repeats) represents a significant improvement over other next-generation gene editing tools. The CRISPR / Cas9 system enables site-specific genomic targeting in the genome in virtually any organism.
    [0119] Figure 2 shows the state of the art: the target for CRISPR / cas9 are small DNA sequences, located around the mutation (MUT X -red-), which expresses a non-functional protein (PPT X -red-). It is a gene therapy strategy since it is necessary to introduce the complementary guides (in yellow), which confine the mutation and, by means of a small deletion in the genomic DNA inside the nucleus of the cell, correct the reading frame. The functional protein is indicated as PPT V -green-.
    [0121] The CRISPR / Cas type II system is a prokaryotic adaptive immune response system that uses antisense RNA to guide the nucleation of Cas9 to induce DNA cleavage at a specific site. This DNA damage is repaired by cellular DNA repair mechanisms, either through the homologous or non-homologous DNA repair pathway.
    [0123] The CRISPR / Cas9 system has been leveraged to create a simple, programmable RNA method to mediate genome editing in mammalian cells, and can be used to generate knockout (KO) (insertion / deletion) models. To create genetic alterations, a single guide RNA (sgRNA) is generated to direct the Cas9 nuclease to a specific genomic location. Double chain breaks induced by Cas9 are repaired via the DNA repair pathway.
    [0125] Example 5. Criteria for the design of specific guidelines for CRISPR / Cas9.
    [0127] The specific guidelines necessary for obtaining the mutated cell model and for correcting the mutation (Figure 3) were designed based on the genetic maps of human factor V, prepared thanks to computer programs that show us relevant information about the gene, as well as the complete genetic sequence in the form of cDNA (coding without introns). To design the gene editing guides as accurately as possible, the web application http://crispor.tefor.net/ was used. The selection criteria were based on the length of the sequence to be removed, the "score", the specificity and the possible "off-target". Both the designed guides and the Cas 9 nuclease were purchased from IDT (USA).
    [0129] Example 6. Obtaining the cellular model and correction of the mutation.
    [0131] Both to obtain the cell model and to eliminate the mutation (Figure 3), the HepG2 cell line was used, provided by Dr. José Carlos Segovia from the Center for Energy, Environmental and Technological Research. HepG2 cells belong to an immortal cell line derived from well-differentiated hepatocellular carcinoma. These cells are epithelial in morphology, have a modal chromosome number of 55, and are not tumorigenic in mice. They secrete factor V in addition to a wide variety of important proteins, eg, albumin, and the acute phase proteins fibrinogen, alpha 2-macroglobulin, alpha 1-antitrypsin, transferrin, and plasminogen.
    [0133] For the development of the KO cell model for factor V, the following editing guides were used:
    [0135] 5'-CAATCAGACATTGCCCTCTA-3 '(G1) (SEQ ID NO: 66)
    [0136] 5'-GAGGAATTCTGATTATGGTC-3 '(G2) (SEQ ID NO: 67)
    [0138] In the case of the guides used for the correction of the mutation in the KO model, 2 pairs of guides were used to compare the editing effectiveness of each pair of guides, these being:
    [0139] Couple 1
    [0140] 5'-AAGAAGACTTAAGCATTCGT-3 '(G3) (SEQ ID NO: 68)
    [0141] 5'-TGCCTGACCAGTGTCATTTG-3 '(G4) (SEQ ID NO: 69)
    [0142] Couple 2
    [0143] 5'-TTCCTCAAATGACACTGGTC-3 '(G5) (SEQ ID NO: 70)
    [0144] 5'-CAATGTCTGATTGAGGTCTG-3 '(G6) (SEQ ID NO: 71)
    [0146] The strategy to follow is similar to that of creating the KO model, but in this case restoring the correct reading frame of the gene, and as a consequence, the production of functional factor V.
    [0148] Figure 3 shows:
    [0149] • BD: Domain B. E13: Exon 13. ps: Post-translational division region.
    [0150] • PAM: Protospacer adjacent motif ( protospaceradjacentmotif). It is a 3 base pair DNA sequence immediately after the DNA sequence recognized by the Cas9 nuclease.
    [0151] • Del 35 bp: Deletion (elimination) of a sequence of 35 base pairs, a region that corresponds to the mutated area in the patient with a stop codon (CS, G> A, change from a guanine to an adenine). The alteration of this area by means of the G1 and G2 guides complementary to the mutation, reproduce the mutation in the model.
    [0152] • Del 82 bp: Deletion (deletion) of an 82 base pair sequence, which is complemented by the G3 and G4 guidelines to give a non-pathological sequence of the gene.
    [0153] • Del 49 bp: Deletion (deletion) of a sequence of 49 base pairs, which is complemented by the G5 and G6 guides to give a non-pathological sequence of the gene.
    [0155] The methodology was based on introducing the guides and the Cas9 nuclease inside the cell so that they later reach the nucleus where they are internalized. For this, the method of production of a ribonucleoprotein (RNP), formed from the guides and the Cas9 nuclease, was used. Obtaining this complex was carried out using a thermocycler protocol based on incubating the 2 elements of the guide (crRNA and tracrRNA) at 95 ° C, 5 min and subsequent addition of Cas 9, incubating for 2 minutes at room temperature. Finally, this RNP was introduced through a 4D Nucleofector ™ System (Lonza, Switzerland) and the nucleofection kit "SF line cells" (Lonza, Switzerland) following the protocol of the commercial house to carry out the nucleofection in said cell line. This procedure has less cellular toxicity, better internalization in kernel, higher editing efficiency, less production time and it is cheaper than others.
    [0157] Example 7. Confirmation of the pattern and correction of the mutation.
    [0159] To confirm the cell model mutated after CRISPR / Cas9, the proliferation, morphology and karyotype of HepG2 cells were studied. Flow cytometry was then carried out with HepG2 cell membrane specific markers. And finally, the cDNA for factor V was sequenced in HepG2 cells.
    [0161] In this case, no significant differences were observed between the native HepG2 and KO lines, produced by gene editing, with respect to proliferation data, morphology and expression of membrane markers, verified by flow immunocytometry, which indicates that the guidelines designed and Editing does not affect, at least, genes related to the cell cycle and / or proliferation, cell structure, or membrane proteins, indicating that gene editing may be an alternative due to its few non-specific effects on other genes, in in vitro studies . Regarding DNA sequencing, the efficiency of editing to produce the KO model, with the guidelines and protocols for gene editing used, was around 60% accurate editing, giving rise to a cell culture in which 60% of the The cells were KO for the factor V gene. Subsequently, and to obtain a pure cell line, a growth in colonies was performed to achieve a cell culture where 100% of the cells had the mutation derived from the editing and therefore were KO for the factor V gene.
    [0163] To confirm the correction of the mutation after CRISPR / Cas9, flow cytometry was again carried out and the factor V cDNA of the cell lines was sequenced to check the efficacy of editing and correction (treatment) of the mutation.
    [0165] In this case, a different editing precision was obtained between the different pairs of gene editing guides, namely, with pair 1 the editing efficiency was approximately 40%, while with pair 2, it was 30%. Still being Lower efficiencies than in the previous case of the KO model, a cell-based treatment with a correction efficiency of 30-40% implies the transition from a severe disease state or phenotype to a mild or even asymptomatic state. The decrease in the efficiency in the correction of the KO cell line may also be due to the fact that that area of the DNA has previously undergone another gene editing (to develop the KO model), which can make the CRISPR system lose efficacy, when editing on a previously edited zone.
    权利要求:
    Claims (10)
    [1]
    1. In vitro method to recover the expression of the F5 gene that encodes coagulation factor V by using the CRISPR / Cas9 methodology that includes the use of a pair of guides in which each of the guides has a length of between 18 and 22 nucleotides and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to the position of a mutation located at nucleotide 3279 of the F5 gene .
    [2]
    2. In vitro method according to claim 1, wherein the mutated F5 gene is characterized by SEQ ID NO: 65.
    [3]
    In vitro method according to any of the preceding claims, in which the pair of guides used is the pair of guides characterized by SEQ ID NO: 68-69 or the pair of guides characterized by SEQ ID NO: 70-71.
    [4]
    4. In vitro cell culture in which the expression of the F5 gene that encodes coagulation factor V has been recovered by using the CRISPR / Cas9 methodology, including the use of a pair of guides in which each of the guides it is between 18 and 22 nucleotides in length and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to the position of a mutation located at nucleotide 3279 of the F5 gene .
    [5]
    5. In vitro cell culture according to claim 4, wherein the F5 gene with a mutation at nucleotide 3279 is characterized by SEQ ID NO: 65.
    [6]
    In vitro cell culture according to any of claims 4-5 in which the pair of guides used is the pair of guides characterized by SEQ ID NO: 68-69 or the pair of guides characterized by SEQ ID NO: 70-71 .
    [7]
    7. Kit comprising a pair of guides in which each of the guides is between 18 and 22 nucleotides in length and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to the position of a mutation located in nucleotide 3279 of the F5 gene .
    [8]
    Kit according to claim 7 in which the F5 gene with a mutation at nucleotide 3279 is characterized by SEQ ID NO: 65.
    [9]
    9. Kit according to any of claims 7-8, comprising the pair of guides characterized by SEQ ID NO: 68-69 and / or the pair of guides characterized by SEQ ID NO: 70-71.
    [10]
    10. Kit according to any one of Claims 7-9, which further comprises a Cas9 protein.
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    同族专利:
    公开号 | 公开日
    WO2021229131A1|2021-11-18|
    ES2785323B2|2020-12-18|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP1958648A1|2005-10-28|2008-08-20|Dnavec Corporation|Therapeutic method for blood coagulation disorder|
    US20110130443A1|2005-11-09|2011-06-02|Hans-Peter Vornlocher|Compositions And Methods For Inhibiting Expression Of Factor V Leiden Mutant Gene|
    WO2020022803A1|2018-07-26|2020-01-30|주식회사 툴젠|Gene editing of anticoagulants|
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    PCT/ES2021/070341| WO2021229131A1|2020-05-14|2021-05-13|In vitro method for recovering the expression of the f5 gene encoding coagulation factor v|
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