Gene editing therapy for pyruvate kinase deficiency (PKD)

Abstract

The present invention relates to the treatment of pyruvate kinase deficiency (PKD) using the clustered regularly interspaced short palindromic repeats (CRISPR) system. This technology offers the possibility of designing improved single guide RNAs (sgRNAs), particularly improved crRNAs, that can be associated with tracrRNAs and then incorporated into CRISPR-associated proteins (Cas9) to recognize and induce DNA double-strand breaks at specific target locations. The DNA double-strand breaks are repaired by homologous recombination (HR) in the presence of the PKLR gene repair donor sequence.

Description

[Technical Field]

[0001] This invention relates to the treatment of pyruvate kinase deficiency (PKD) using a clustered, regularly arranged short palindromic sequence repeat (CRISPR) system. This technology offers the possibility of designing improved single guide RNAs (sgRNAs), particularly improved crRNAs, that can associate with tracrRNA or linkers and be incorporated into CRISPR-associated protein (Cas9) to recognize and induce DNA double-strand breaks at specific target sites. DNA double-strand breaks are repaired by homologous recombination (HR) in the presence of a donor sequence for PKLR gene repair. [Background technology]

[0002] Pyruvate kinase deficiency (PKD) is the most common hereditary erythrocyte deficiency, causing chronic nonspherocytic hemolytic anemia. The prevalence of PKD in the Caucasian population is estimated at 1–9 cases per 100,000 people. PKD is an autosomal recessive genetic disorder caused by mutations in the PKLR gene. This gene encodes two different transcriptional variants, RPK and LPK, which are expressed in erythrocytes and the liver, respectively. To date, more than 200 different PKLR gene mutations have been associated with PKD. Treatment options for PKD are symptomatic, including regular erythrocyte transfusions, splenectomy, and iron chelation therapy. To date, allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for severely ill patients. Autologous HSCT, using genetically corrected cells, also known as hematopoietic stem cell and progenitor cell (HSPC) gene therapy, is used to treat many hematopoietic cell genetic disorders. CIEMAT has recently developed a lentiviral vector to correct PKD, which has received orphan drug designation (EU / 3 / 14 / 1330; FDA#DRU-2016-5168). This lentiviral-mediated gene therapy approach is expected to provide permanent and curative clinical benefits with a single treatment.

[0003] In recent years, gene editing has emerged as a promising gene therapy approach for hematological diseases due to its ability to precisely correct gene mutations. The generation of double-strand breaks (DSBs) using modified endonucleases that cleave specific genomic sites that mobilize DNA repair mechanisms, when combined with the introduction of desired DNA sequences to these specific sites, has increased gene editing efficiency to a percentage that can be considered clinically applicable. The CRISPR-Cas9 system is one of the modified endonucleases described to date. A specific sgRNA that recognizes a single specific site in the genome, combined with the Cas9 protein that generates DSBs at that specific site, is also called a ribonucleoprotein complex (RNP). The introduction of this into cells, when combined with adeno-associated virus (AAV) for donor template delivery, has demonstrated to be the most efficient system for approaching the field of gene editing to treat patients with hereditary hematopoietic disorders (Non-Patent Literature 1, Non-Patent Literature 2, Non-Patent Literature 3, Non-Patent Literature 4). CIEMAT set up this approach for the correction of PKD and found that by combining specific RNPs and AAVs, up to 40% of human hematopoietic progenitor cells were gene-edited at the therapeutic RPK (R-type pyruvate kinase) locus. The results suggest that the clinical use of gene editing therapy has a very high potential to correct PKD in a short period of time.

[0004] One of the current bottlenecks in using gene editing to treat genetic disorders is the generation of double-stroke bonds (DSBs) at locations other than the selected on-target site, which can lead to unwanted genetic modifications with unexpected clinical effects. This side effect of the CRISPR-Cas9 system resulting from the generation of unwanted DSBs is called an off-target effect. Off-target effects generated by specific sgRNAs can hinder its clinical application because hematopoietic stem cell gene therapy involves genetic modification of millions of HSPCs, and therefore the number of cells with unwanted genome changes can be enormous. Even one unwanted modification in one of the transplanted HSPCs could lead to uncontrolled clonal proliferation, potentially derailing the therapy and posing a risk to the patient's health. As previously described in both the natural context and the context of hematopoietic stem cell gene therapy, alterations to a single HSPC genome or the random incorporation of a viral vector into the host genome can also trigger leukemic processes (Non-Patent Literature 5). Therefore, a key challenge with regard to the use of CRISPR systems is the need to identify and minimize potentially harmful off-target mutations induced by these nucleases. Although the use of high-fidelity Cas9 significantly reduced off-target activity (Non-Patent Literature 6), the off-target effects of therapeutic RNPs need to be carefully analyzed before using gene editing for patient treatment.

[0005] High-sensitivity analysis of potential off-target effects is essential for any clinical application, because even low-frequency events can potentially lead to adverse outcomes. Methods involving computer simulation prediction of potential off-target effects are most frequently used. However, these approaches fail to consider the genomic location of potential off-target effects, nor the cell type of interest. Therefore, their ability to identify off-target sites for specific sgRNAs is very limited. The ideal method for analyzing the side effects of gene editing technologies should be genome-wide, unbiased, and highly sensitive in identifying off-target sites—that is, capable of detecting even low-frequency mutations in very large cell populations. GUIDE-seq is based on efficient incorporation of double-stranded oligodeoxynucleotide (dsODN) tags, followed by tag-specific amplification and high-processing sequencing. GUIDE-seq is highly sensitive and can detect off-target sites mutagenerated by the tested sgRNA within a cell population. Some of the advantages of GUIDE-seq include its experimental simplicity, efficiency, and accuracy, as well as its ability to detect nuclease-induced double-sequence break (DSB) repair outcomes in a physiologically relevant cellular context. CIEMAT has evaluated the off-target effects of relevant sgRNAs through GUIDE-seq within HSPC itself, paying close attention to the most stringent criteria for selecting gene-editing-based therapeutic tools. This type of genome-wide, unbiased analysis of the side effects of gene-editing platforms will ensure the safety of the clinical use of gene editing within HSPC. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Dever, Bak et al. 2016 [Non-Patent Document 2] Bak, Dever et al. 2018 [Non-Patent Document 3] Charlesworth, Camarena et al. 2018 [Non-Patent Document 4] Pavel-Dinu, Wiebking et al. 2019 [Non-Patent Document 5] Greaves and Maley 2012 [Non-Patent Document 6] Vakulskas, Dever et al. 2018 [Brief explanation of the drawing]

[0007]

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Mode for Carrying Out the Invention

[0008] Description of the Present Invention Definitions As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0009] As used herein, the singular forms (The terms "a," "an," or "the") that do not specify a quantity include embodiments having only one member as well as embodiments having two or more members. For example, the singular forms that do not specify a quantity include multiple objects unless the context clearly indicates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, and a reference to "the agent" includes a reference to one or more agents known to those skilled in the art, and so on. <000009]]

[0010] The term "gene" refers to a combination of polynucleotide elements that, when operably linked together, either in a natural or recombinant manner, provide some product or function. The term "gene" should be interpreted broadly and may encompass the mRNA, cDNA, genomic DNA forms of genes.

[0011] The term "homologous-directed repair" or "HDR" refers to an intracellular mechanism that correctly and accurately repairs double-strand DNA breaks using homologous templates to guide the repair process. The underlying mechanism of HDR is homologous recombination (HR).

[0012] The term "homologous recombination" or "HR" refers to a genetic process in which nucleotide sequences are exchanged between two similar molecules of DNA. Homologous recombination (HR) is used by cells to properly repair harmful breaks that occur on both strands of DNA (known as double-strand breaks or other breaks that produce overhang sequences).

[0013] The term "single guide RNA" or "sgRNA" refers to DNA targeting RNA containing a guide sequence that targets a Cas nuclease to target genomic DNA and a scaffold sequence (e.g., tracrRNA) that interacts with the Cas nuclease. Preferably, the sgRNA contains or consists solely of Sequence ID No. 1.

[0014] The terms "Cas polypeptide" or "Cas nuclease" refer to clustered, regularly arranged, short palindromic repeat-associated polypeptides or nucleases that cleave DNA at sites identified by a 20-nucleotide guide sequence contained within a crRNA molecule, generating blunt ends in double-strand breaks. Cas nucleases require both crRNA and tracrRNA for site-specific DNA recognition and cleavage. The crRNA associates with the tracrRNA via a partially complementary region or via a linker, leading the Cas nuclease to a region homologous to the crRNA in the target DNA called a "protospacer."

[0015] The term "HiFi-Cas9" is interpreted herein as a high-fidelity Cas9 variant that has on-target activity similar to wild-type Cas9 but reduced off-target activity, and is delivered as a ribonucleoprotein complex that enables efficient gene editing. HiFi-Cas9 was described by Valklskas et al. (Non-Patent Literature 6).

[0016] The term "ribonucleoprotein complex" or "RNP complex" refers to a complex containing sgRNA and Cas polypeptide.

[0017] The terms "adeno-associated virus vector delivery donor template" or "donor template-containing adeno-associated virus vector" refer to adeno-associated virus particles capable of delivering recombinant donor templates for CRISPR-based gene editing via homology-directed repair in target cells, such as primary cells.

[0018] The term "recombinant donor template" sometimes refers to the nucleic acid strand, such as the donor DNA strand, at the time of homologous recombination intrusion, which is initiated by the DNA damage repair mechanism resulting from a double-strand break. The donor polynucleotide acts as template material that directs the repair of the damaged DNA region.

[0019] In the context of two or more nucleic acids or polypeptides, the terms “sequence identity” or “percent identity” refer to two or more sequences or subsequences that are the same (“identical”) or have a specific percentage of amino acid residues or nucleotides that are identical when compared and aligned to maximize match with a second molecule (“percent identity”), and are measured using a sequence comparison algorithm (e.g., BLAST alignment, or any other algorithm known to those skilled in the art) or, instead, by visual inspection.

[0020] The term "homologous" refers to two or more amino acid sequences that are derived naturally or artificially from a common ancestral protein or amino acid sequence. Similarly, nucleotide sequences are homologous if they are derived naturally or artificially from a common ancestral nucleic acid.

[0021] The term "primary cells" refers to cells isolated directly from a multicellular organism. Primary cells typically undergo little population doubling and therefore better represent the major functional components of the tissue from which they originate, compared to successive (tumor or artificially immortalized) cell lines. In some cases, primary cells are isolated and immediately used. In other cases, primary cells cannot divide indefinitely and therefore cannot be cultured in vitro for extended periods.

[0022] The terms "genetically modified primary cells" or "genome-edited primary cells" sometimes refer to primary cells in which heterologous nucleic acids have been introduced into their endogenous genomic DNA.

[0023] The term "pharmaceutical composition" refers to a composition that is physiologically and pharmacologically acceptable. In some examples, a composition may contain an active ingredient for buffering and storage during preservation, and may also contain appropriate buffers and carriers for delivery depending on the route of administration.

[0024] The term "pharmaceutically acceptable carrier" refers to a substance that assists in the administration of an active agent (e.g., Cas nuclease, modified single guide RNA, genetically modified primary cells, etc.) to a cell, organism, or target. A "pharmaceutically acceptable carrier" refers to a carrier or excipient that can be included in a composition or formulation and does not cause a significantly harmful toxicological effect to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, lactated Ringer's solution, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, and colorants. Those skilled in the art will recognize that other pharmaceutical carriers may be useful in the present invention.

[0025] The terms “administer” or “dosage” refer to the process by which the active substances, compositions, dosage forms and / or combinations disclosed herein are delivered to a subject for therapeutic or preventive purposes. The compositions, dosage forms and / or combinations disclosed herein are administered in accordance with good medical practice, taking into account the subject’s clinical condition, site and method of administration, dosage, the subject’s age, sex, weight and other factors known to the physician. For example, the terms “administer,” “dosage,” or “dosage” include providing, giving, administering and / or prescribing the active substances, compositions, dosage forms and / or combinations disclosed herein by a clinician or other clinical professional.

[0026] The term "treat" refers to an approach to obtain beneficial or desired outcomes, including but not limited to therapeutic and / or preventive benefits. Therapeutic benefits mean any therapeutically related improvement or effect on one or more diseases, conditions, or symptoms being treated. In the case of preventive benefits, a composition may be administered to subjects at risk of developing a particular disease, condition, or symptom, or to subjects complaining of one or more physiological symptoms of a disease, even if the disease, condition, or symptoms have not yet manifested.

[0027] The terms “subject,” “patient,” and “individual” are used herein interchangeably to include humans or animals. For example, animal subjects may be mammals, primates (e.g., monkeys), domestic animals (e.g., horses, cattle, sheep, pigs, or goats), companion animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, guinea pigs, birds), animals of veterinary importance, or animals of economic importance.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which this art belongs. While exemplary methods, apparatus, and materials are described herein, any methods and materials similar or equivalent to those expressly described herein may be used in the practice or testing of this art. For example, the reagents described herein are merely illustrative, and such equivalents are known in the art. Unless otherwise specified, the practice of this art may employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the scope of the art of those skilled in the art.For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Ausubel et al. eds. (2007) Current Protocols in Molecular Biology series, Methods in Enzymology (Academic Press, Inc., NY) series, MacPherson et al. (1991) PCR I: A Practical Approach (IRL Press at Oxford University Press), MacPherson et al. al. (1995) PCR 2: A Practical Approach, Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual, Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition, Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory), and Makrides ed. (2003) Gene Transfer and Expression in Please refer to Mammalian Cells (Cold Spring Harbor Laboratory).

[0029] explanation As already stated in the background of this invention, in recent years, gene editing has emerged as a promising gene therapy approach for hematological diseases due to its ability to precisely correct gene mutations. RNPs, when used in combination with adeno-associated viruses (AAVs) for donor template delivery, are approaching the field of gene editing for the treatment of various diseases (Non-Patent Literature 1, Non-Patent Literature 2, Non-Patent Literature 3, Non-Patent Literature 4). In this sense, the inventors have set this approach toward the correction of PKD and have found that up to 40% of human hematopoietic progenitor cells were gene-edited at the therapeutic RPK (R-type pyruvate kinase) gene locus through the combination of specific RNPs and AAVs. These results suggest that the clinical use of gene editing therapy has the potential to correct PKD, however, the most concerning issue for the clinical application of this novel gene editing technology is the off-target effects caused by RNPs. Although off-target activity has been reduced by using high-fidelity Cas9, it is still necessary to significantly reduce off-target effects before using gene editing to treat patients.

[0030] For this purpose, and to avoid off-target effects, double-strand breaks (DSBs) must be located as close as possible to the site where the exogenous DNA is intended to be incorporated. The sgRNA, more specifically the crRNA, determines this location. In this sense, the sgRNA, more specifically the crRNA, must be carefully selected to maximize on-target breaks and minimize off-target effects. Homology arms must be located around the DSB site. These must be designed, according to the selected sgRNA, to maintain function and avoid additional sequence modifications.

[0031] In this invention, various single guide RNAs for introducing DSBs into genomic sites of interest were designed using various web tools available for that purpose (see Example 1). The effectiveness of creating DSBs from the various crRNAs obtained (SEQ ID NOs: 1 to 10) was tested and evaluated by Surveyor assay, TIDE, and / or GUIDE-Seq and rhAmp-Seq. The results showed that only one of the selected crRNAs generated very high-frequency indels at the on-target site in K562 cells, and human CB-CD34 + This particular crRNA generated indels at a very high frequency at the on-target site of the cell, and while it exhibited several off-target effects when transfused into HEK293-Cas9 cells and Jurcut cells, these were negligible when HiFi-Cas9 RNPs were used. Such crRNAs correspond to crRNA SG1 of SEQ ID NO: 1 (as DNA) or SEQ ID NO: 11 (as RNA). It should be noted that such crRNAs (SG1) were due to errors in selecting the appropriate ATG start codon for the RPK transcript variant, as shown in Example 1. In this sense, the fact that such crRNAs SG1 showed a clearly improved effect compared to the various crRNAs tested was surprising. In fact, such crRNA SG1 showed a clear improvement in efficacy when compared to various crRNAs identified around a hidden ATG located 30 bp upstream of the RPK start codon, and also when compared to those designed between the hidden ATG and the appropriate ATG RPK start site (SG5-SG8) for the purpose of correcting the aforementioned design process or errors, because, in order to avoid off-target effects, the double-strand breaks (DSBs) must be located as close as possible to the site where the exogenous DNA is to be incorporated. Based on these results, we propose herein to provide a novel RNP complex for use in gene editing therapy to correct PKD, using the crRNA of SEQ ID NO: 1 or SEQ ID NO: 11, for the purpose of eliminating or reducing off-target effects caused by RNPs.

[0032] In this sense, in contrast to other methods, the RNP complex provided herein significantly reduces the percentage of off-target effects. Overall, the present invention provides evidence that the RNP complex, when used in combination with the adeno-associated virus (AAV) for delivery of the coRPK donor sequence of the present invention, is particularly well-suited as an ex vivo effective genome editing tool capable of achieving gene correction in a variety of cell types, and provides a source for developing different cell therapies for pyruvate kinase deficiency (PKD).

[0033] Therefore, in a first aspect of the present invention, the inventors provide herein a modified crRNA comprising SEQ ID NO: 1 or 11, or comprising SEQ ID NO: 1 or 11 alone. In other examples, the modified crRNA of the present invention is a variant of SEQ ID NO: 1 or SEQ ID NO: 1 that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 11, for example, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 1 (hereafter, SEQ ID NO: 1, SEQ ID NO: 11, and any variant thereof will be referred to as the modified crRNA of the present invention).

[0034] Preferably, the modified crRNA of the present invention is associated with or bound to a tracrRNA nucleotide sequence or to a linker that interacts with a CRISPR-related protein (Cas) polypeptide (in this specification, the modified crRNA of the present invention associated with tracrRNA will be referred to as the modified single guide RNA (sgRNA) of the present invention). Preferably, the modified sgRNA of the present invention includes SEQ ID NO: 12, or a variant of SEQ ID NO: 12 having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 12, for example, a variant of SEQ ID NO: 12 having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12, or consists only of SEQ ID NO: 12 or a variant of SEQ ID NO: 12.

[0035] A second aspect of the present invention relates to a ribonucleoprotein (RNP) comprising a modified crRNA or sgRNA of the present invention and a CRISPR-related protein (Cas) polypeptide. For example, the modified sgRNA and the Cas polypeptide can be mixed together in a container to form the RNP complex of the present invention, and the RNP complex can then be introduced into primary cells.

[0036] In another embodiment, the present invention shows a “total RNA” CRISPR system comprising mRNA encoding a Cas polypeptide and a modified sgRNA of the present invention.

[0037] A third aspect of the present invention is a vector comprising a coRPK cDNA sequence including homology arms (LHA and RHA), a coRPK sequence, and a dedicated termination sequence for protein expression in eukaryotic cells, such as a bGH poly(A) sequence. In this vector, preferably, LHA is SEQ ID NO: 13, RHA is SEQ ID NO: 14, the coRPK sequence is SEQ ID NO: 16, and the bGH poly(A) sequence is SEQ ID NO: 18. Preferably, the coRPK sequence of the present invention comprises SEQ ID NO: 16, or a variant of SEQ ID NO: 16 having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 16, for example, a variant of SEQ ID NO: 16 having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16, or consists only of SEQ ID NO: 16 or a variant of SEQ ID NO: 16. Preferably, the LHA sequence of the present invention includes SEQ ID NO: 13, or a variant of SEQ ID NO: 13 having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 13, for example, a variant of SEQ ID NO: 13 having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13, or consists only of SEQ ID NO: 13 or a variant of SEQ ID NO: 13. Preferably, the RHA sequence of the present invention includes SEQ ID NO: 14, or a variant of SEQ ID NO: 14 having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 14, for example, a variant of SEQ ID NO: 14 having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14, or consists only of SEQ ID NO: 14 or a variant of SEQ ID NO: 14. Preferably, the bGH poly(A) sequence of the present invention includes SEQ ID NO: 18, or a variant of SEQ ID NO: 18 having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 18, for example, a variant of SEQ ID NO: 18 having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18, or consists only of SEQ ID NO: 18 or a variant of SEQ ID NO: 18.

[0038] In some examples, the vector comprising the coRPK cDNA sequence of the present invention further comprises a 5'UTR sequence, preferably such a sequence is SEQ ID NO: 15. More preferably, the vector comprising the coRPK cDNA sequence comprises SEQ ID NO: 15, or a variant of SEQ ID NO: 15 having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 15, for example, having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15.

[0039] In further examples, a vector containing coRPK cDNA may include any of SEQ ID NOs. 19 to 22, or variants of SEQ ID NOs. 19 to 22 having at least 95% sequence identity with any of SEQ ID NOs. 19 to 22, for example, variants of SEQ ID NOs. 19 to 22 having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NOs. 19 to 22 or only variants of SEQ ID NOs. 19 to 22 may also include a vector containing, or a vector containing only variants of SEQ ID NOs. 19 to 22.

[0040] A fourth aspect of the present invention describes a system comprising the RNP complex of the present invention or the total RNA CRISPR system of the present invention and adeno-associated virus particles or homologous donor AAVs capable of delivering recombinant donor templates for CRISPR gene editing via homology-directed repair in target cells, e.g., primary cells. In some examples, the adeno-associated virus or homologous donor AAV skeleton, e.g., (AAV-6) or (AAV-1) or any other possible AAV serotype or serotype chimera, has at least about 90% sequence identity with the AAV skeleton. In the present invention, the AAV skeleton is interpreted as adeno-associated virus particles or homologous donor AAVs that do not contain the recombinant donor templates of the present invention for CRISPR gene editing via homology-directed repair in target cells. In other examples, the AAV skeleton is a variant of AAV6 having at least 95% sequence identity with wild-type AAV6 or SEQ ID NO: 23, for example, having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23. In some embodiments, a polynucleotide vector encoding one or more of the various components of the AAV skeleton, e.g., (AAV-6) or (AAV-1) or any other possible AAV serotype or serotype chimera, is operably ligated to an inductive promoter, a repressive promoter, or a constitutive promoter. The regulatory sequences operably ligated to the components may include activator-binding sequences, enhancers, introns, polyadenylation-recognition sequences, promoters, repressor-binding sequences, stem-loop structures, translation initiation sequences, translation reader sequences, transcription termination sequences, translation termination sequences, primer-binding sites, etc. Commonly used promoters include the constitutive mammalian promoters CMV, EF1a, SV40, PGK1 (mouse or human), Ubc, CAG, CaMKIIa, and beta-Act, as well as others known in the art (Khan, KH (2013) "Gene Expression in Mammalian Cells and its Applications", Advanced Pharmaceutical Bulletin 3(2), 257-263).Furthermore, mammalian RNA polymerase III promoters, including H1 and U6, can also be used.

[0041] In some embodiments, the adeno-associated virus particle or homologous donor comprises an AAV skeleton and the coRPK cDNA sequence of the present invention delivered to primary cells. More preferably, the adeno-associated virus particle or homologous donor AAV comprises any of SEQ ID NOs. 19 to 22, or variants of SEQ ID NOs. 19 to 22 having at least 95% sequence identity with any of SEQ ID NOs. 19 to 22, for example, variants of SEQ ID NOs. 19 to 22 having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NOs. 19 to 22.

[0042] More preferably, the adeno-associated virus particle or homologous donor AAV comprising a vector containing the coRPK cDNA sequence of the present invention is selected from any of SEQ ID NOs. 24 to 27, or from variants of SEQ ID NOs. 24 to 27 having at least 95% sequence identity with any of SEQ ID NOs. 24 to 27, for example, variants of SEQ ID NOs. 24 to 27 having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of SEQ ID NOs. 24 to 27.

[0043] Furthermore, in a fifth aspect of the present invention, the inventors provide a method for inducing stable gene modification in primary cells, preferably hematopoietic stem cells and progenitor cells (HSPCs), or embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), or any other cell type capable of differentiating into HSPCs or erythrocytes, via homologous recombination, comprising a target nucleic acid comprising a PKLR gene containing one or more mutations in the PKLR gene and a nucleotide sequence complementary to SEQ ID NO: 1 or 11, wherein the method is described below: (a) Introducing into primary cells a composition comprising the modified crRNA or sgRNA of the present invention associated with a CRISPR-related protein (Cas) polypeptide, i.e., the "total RNA" CRISPR system of the present invention, and simultaneously or sequentially thereafter, (b) Introducing adeno-associated virus particles or homologous donor AAV containing the coRPK cDNA sequence of the present invention corresponding to the target nucleic acid to be subjected to homologous recombination into primary cells, Includes, In this method, stable gene modification of the target nucleic acid includes compensation for pathogenic mutations in the PKLR gene (target nucleic acid) by introducing homologous donor AAVs containing a corrective donor template, such as (AAV-6) or (AAV-1), or any other possible AAV serotype or serotype chimera, or vector.

[0044] The above-described gene modification strategies in primary cells, preferably hematopoietic stem cells and progenitor cells (HSPCs), or embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), or any other cell type capable of differentiating into HSPCs or erythrocytes, are performed for the purpose of treating subjects who have or suffer from pyruvate kinase deficiency (PKD). Pyruvate kinase deficiency (PKD) is a hereditary autosomal recessive genetic disorder caused by mutations in the PKLR gene and is the leading cause of chronic nonspherocytic hemolytic anemia. It is estimated that 1 in 20,000 people worldwide suffer from PKD, and approximately 17% of them still lack a curative treatment. The PKLR gene encodes erythrocyte pyruvate kinase protein (RPK), which is involved in the final step of anaerobic glycolysis in erythrocytes. These PKD-causing mutations lead to a general or partial decrease in RPK activity and a subsequent decrease in ATP levels, which contributes to red blood cell hemolysis and resulting anemia. The disease becomes clinically relevant when protein activity decreases to less than 25% of normal red blood cell activity.

[0045] Therefore, in a further embodiment of the fifth aspect of the present invention, the homology donor AAV comprises a coRPK cDNA sequence, for example, SEQ ID NO: 16, and more preferably, the donor template comprises a coRPK cDNA sequence and two homology portions of a target nucleic acid, for example, SEQ ID NOs: 13 and 14.

[0046] In some embodiments, primary cells are selected from the group consisting of primary HSPCs, or embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), or any other cell type capable of differentiating into HSPCs or erythrocytes, and any combination thereof. In some embodiments, primary cells are isolated from a mammal before introducing the modified crRNA or sgRNA of the present invention, i.e., the “total RNA” CRISPR system of the present invention, and a homologous donor AAV vector into the primary cells. For example, primary cells can be collected from a human subject. In some examples, primary cells or their offspring are returned to a mammal after introducing the modified crRNA or sgRNA of the present invention, i.e., the “total RNA” CRISPR system of the present invention, and a homologous donor AAV vector into the primary cells. In other words, the genetically modified primary cells undergo autologous transplantation. In other examples, the genetically modified cells undergo allogeneic transplantation. For example, unstable genetically modified cells are isolated from a donor, and then the genetically modified donor cells are transplanted into a recipient different from the donor.

[0047] Primary cells can constitute a population of primary cells. In some cases, a population of primary cells includes heterogeneous populations of primary cells. In other cases, a population of primary cells includes homogeneous populations of primary cells.

[0048] In further examples, a homologous donor AAV skeleton, e.g., (AAV-6) or (AAV-1), has at least about 90% sequence identity with the AAV skeleton. In other examples, the homologous donor AAV skeleton is a variant of the AAV6 skeleton having at least 95% sequence identity with wild-type AAV6 or SEQ ID NO: 23, e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23. In some embodiments, a vector of polynucleotides encoding one or more of the various components of the AAV skeleton, e.g., (AAV-6) or (AAV-1), is operably ligated to an inducible promoter, a repressive promoter, or a constitutive promoter. Furthermore, the regulatory sequences operably linked to the components may include activator-binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor-binding sequences, stem-loop structures, translation initiation sequences, translation reader sequences, transcription termination sequences, translation termination sequences, primer binding sites, etc. Commonly used promoters include the constitutive mammalian promoters CMV, EF1a, SV40, PGK1 (mouse or human), Ubc, CAG, CaMKIIa, and beta-Act, as well as others known in the art (Khan, KH (2013) "Gene Expression in Mammalian Cells and its Applications," Advanced Pharmaceutical Bulletin 3(2), 257-263). In addition, mammalian RNA polymerase III promoters, including H1 and U6, may also be used.

[0049] In some embodiments, homologous donor AAV scaffolds can preferentially direct the expression of nucleic acids in specific cell types (e.g., expressing polynucleotides using tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art and include, but are not limited to, albumin promoters, lymphoid-specific promoters, neuron-specific promoters (e.g., neurofilament promoters), pancreas-specific promoters, mammary gland-specific promoters (e.g., whey promoters), and in particular T cell receptor and immunoglobulin promoters. Developmentally regulated promoters, such as the mouse hox promoter and alpha-fetoprotein promoter, are also included.

[0050] Methods for introducing AAV, such as (AAV-6) or (AAV-1), expression vectors into host cells are known in the art and are typically selected based on the type of host cell.

[0051] In several embodiments, stable genetic modification of the target nucleic acid is induced in more than 30% of the primary cell population, for example, about 35%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% of the primary cell population. In other embodiments, stable genetic modification of the target nucleic acid is induced in more than 80% of the primary cell population, for example, in about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% of the primary cell population.

[0052] In some embodiments, the sequences of the fifth aspect of the present invention may include modified nucleotides, such as modifications at a ribose group, a phosphate group, a nucleic acid base, or a combination thereof. In some examples, modifications at the ribose group include modifications at the 2' position of the ribose group. In some cases, modifications at the 2' position of the ribose group are selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, 2'-O-(2-methoxyethyl), and combinations thereof. In other examples, modifications at the phosphate group include phosphorothioate modifications. In other embodiments, the modified nucleotides are selected from the group consisting of 2'-O-methyl (M) nucleotides, 2'-O-methyl 3'-phosphorothioate (MS) nucleotides, 2'-O-methyl 3'-thioPACE (MSP) nucleotides, and combinations thereof.

[0053] Preferably, in all aspects and embodiments of the present invention, the Cas polypeptide is a Cas9 polypeptide or a high-fidelity or highly specific Cas9 polypeptide variant. In certain embodiments, the modified sgRNA and Cas polypeptide of the present invention are introduced into primary cells simultaneously. In other embodiments, the modified sgRNA and Cas polypeptide are introduced into primary cells sequentially. In some cases, the modified sgRNA is introduced first, followed by the Cas polypeptide. In other cases, the Cas polypeptide is introduced first, followed by the modified sgRNA of the present invention.

[0054] In some embodiments, the Cas polypeptide described herein may be an mRNA encoding a Cas polypeptide, and this Cas mRNA is introduced into primary cells as a “total RNA” CRISPR system together with the modified gRNA of the present invention. In certain examples, the modified gRNA and Cas mRNA are introduced into primary cells simultaneously. In other examples, the modified gRNA and Cas mRNA are introduced into primary cells sequentially. In some cases, the modified gRNA is introduced first, followed by the Cas mRNA. In other cases, the Cas mRNA is introduced first, followed by the modified gRNA of the present invention.

[0055] In some embodiments, the RNP complex and homologous donor AAV, such as (AAV-6) or (AAV-1), are introduced into primary cells simultaneously. In other embodiments, the RNP complex and homologous donor AAV vector are introduced into primary cells sequentially. In some examples, the RNP complex is introduced into primary cells before the homologous donor AAV vector. In other examples, the homologous donor AAV vector is introduced into primary cells before the RNP complex. For example, the RNP complex can be introduced into primary cells approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90, 120, 150, 180, 210 or 240 minutes, or more, before the homologous donor AAV vector, and vice versa. In certain embodiments, the RNP complex is introduced into primary cells approximately 15 minutes (e.g., approximately 10 to 20 minutes) before the homologous donor AAV vector.

[0056] In some embodiments, the "total RNA" CRISPR system and homologous donor AAV vector are introduced simultaneously into primary cells. In other embodiments, the "total RNA" CRISPR system and homologous donor AAV vector are introduced sequentially into primary cells. In some examples, the "total RNA" CRISPR system is introduced into primary cells before the homologous donor AAV vector. In other examples, the homologous donor AAV vector is introduced into primary cells before the "total RNA" CRISPR system. For example, the "total RNA" CRISPR system can be introduced into primary cells approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90, 120, 150, 180, 210 or 240 minutes, or more, before the homologous donor AAV vector, and vice versa. In a particular embodiment, the "total RNA" CRISPR system is introduced into primary cells approximately 15 minutes (e.g., approximately 10 to 20 minutes) before the homologous donor AAV vector.

[0057] In some embodiments, any of the methods described herein may also include purifying primary cells having a stable genetic modification of the target nucleic acid using a marker. In some cases, the composition isolated by the purification step contains at least about 80%, for example, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more primary cells having a stable genetic modification of the target nucleic acid.

[0058] In some embodiments, the step of introducing the modified gRNA and Cas polypeptide of the present invention into primary cells includes electroporation of the primary cells with the modified gRNA and Cas polypeptide. In some embodiments, the step of introducing a homologous donor AAV, for example (AAV-6) or (AAV-1), vector into primary cells includes transduction of the primary cells.

[0059] In other embodiments, provided herein are genetically modified primary cells produced by any of the methods described herein. In some embodiments, the genetically modified primary cells are selected from the group consisting of HSPCs, or embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), or any other cell type capable of differentiating into HSPCs or erythrocytes, or any combination thereof.

[0060] In yet another embodiment, provided herein are pharmaceutical compositions comprising any of the genetically modified primary cells described herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises one type of genetically modified primary cell. In another embodiment, the pharmaceutical composition comprises two or more different types of genetically modified primary cells, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of genetically modified primary cells.

[0061] In a further embodiment, provided herein is the in vitro use of a kit comprising adeno-associated virus particles or homologous donor AAV, comprising (a) a modified crRNA or sgRNA of the present invention associated with a CRISPR-associated protein (Cas) polypeptide, i.e., the “total RNA” CRISPR system of the present invention, and / or (b) a coRPK cDNA sequence of the present invention corresponding to a target nucleic acid to be subjected to homologous recombination.

[0062] In some cases, the kit also includes reagents for collecting or isolating primary cells from the subject. The subject may be a mammalian subject, such as a human subject.

[0063] In a further embodiment, the foregoing provides a method for preventing or treating PKD in a subject requiring prevention or treatment of PKD, the method comprising administering any of the genetically modified primary cells described herein or any of the pharmaceutical compositions described herein to a subject for preventing the disease or improving one or more symptoms of the disease.

[0064] In some embodiments, the administration step includes a delivery route selected from the group consisting of intravenous, intraperitoneal, intraosseous, or a combination thereof.

[0065] In certain embodiments, the genetically modified primary cells or pharmaceutical composition of the present invention are administered to a subject in an amount sufficient to correct a mutation in a disease-related target nucleic acid. In some examples, the mutation is corrected by replacing the mutated allele in the target nucleic acid with the wild-type allele.

[0066] Other objects and advantages of the present invention will become apparent to those skilled in the art from the summary of the following detailed description, which proceeds with reference to the following exemplary drawings and the appended claims.

[0067] As a result, the applicants herein demonstrate the use of a CRISPR system for reliably repairing PKD mutations. The applicants target the site surrounding the mutation site. DNA repair of PKD disease mutations using a CRISPR / Cas9 system represents a novel and original therapeutic approach. The present invention offers the possibility of acting at the DNA level using nucleases engineered to inactivate or repair pathogenic mutations.

[0068] The following embodiments are merely illustrative examples of the present invention and do not limit the scope of the invention. [Examples]

[0069] This embodiment illustrates various components that integrate the procedures necessary to expand HSPCs ex vivo for 16, 24, or 48 hours in the presence of hematopoietic cytokines, i) introducing a system that induces DNA double-strand breaks (DSBs), such as the CRISPR / Cas9 system, upstream of the transcription start site of the RPK transcript variant of the PKLR gene, and ii) introducing a donor matrix containing coRPK cDNA adjacent to homology arms (left [LHA] and right [RHA]). The LHA and RHA are identical to the genomic sequences into which the exogenous sequences will be inserted. The coRPK cDNA used herein is preceded by a 5'UTR RPK sequence portion and expresses a therapeutic cassette under endogenous regulation. CRISPR / Cas9 is introduced by electroporation to facilitate DNA nucleus access, and the donor matrix is ​​introduced by adeno-associated virus vector serotype 6 (AAV-coRPK). To assemble ribonucleoproteins (RNPs), Cas9 protein is mixed with sgRNA. Electroporation is used to nuclear-transfer (nuclefection) the RNPs into cells. Cells are pre-stimulated and then resuspended in electroporation solution. The RNP complex is added to the cell suspension, and the cells are electroporated. After the electrical pulse, the HSPCs are incubated at 37°C for 10 minutes. Then, pre-warmed medium is added, and the cells are transferred to culture plates. Nucleofected cells are immediately transduced with the corresponding AAV at various concentrations.

[0070] These cells, i.e., hematopoietic stem cells (HSCs), are obtained from the patient in this manner, manipulated in vitro, and then frozen. Once the product is characterized and verified as correct, the cells are thawed and injected into the patient who has been pre-treated with chemotherapy to allow for the engraftment of the corrective cells to be injected.

[0071] Example 1. Design of the CRISPR / Cas9 system of the present invention. sgRNA: 1.sgRNA design: Following previous reports, the sgRNA that introduces the DSB and promotes knock-in integration at the beginning of the gene must be designed as close as possible to the start codon of the gene's RPK transcript variant. crRNA design was performed using various web tools available for this purpose, such as the Dr. Zhang Lab tool (https: / / zlab.bio / guide-design-resources) or the IDT tool (https: / / eu.idtdna.com / site / order / designtool / index / CRISPR_SEQUENCE). Due to errors in selecting the appropriate ATG start codon for the RPK transcript variant, different crRNAs were identified around a hidden ATG located 30 bp upstream of the RPK start codon. These initially designed crRNAs were SG1-SG4. Further crRNAs (SG5-SG8) were designed between the hidden ATG and the appropriate ATG RPK start site to correct the aforementioned design process or errors. This is because, in order to avoid off-target effects, double-strand breaks (DSBs) must be located as close as possible to the site where the foreign DNA is intended to be incorporated.

[0072] [Table 1]

[0073] 2. Efficacy and safety of sgRNA: The effectiveness of DSB generation for the 10 different sgRNAs (SEQ ID NOs: 1-10) listed in Table 1 was evaluated using Surveyor assay, TIDE, and / or GUIDE-Seq and rhAmp-Seq.

[0074] For the Surveyor assay, genomic DNA was purified, PCR was performed, and the region around the start codon of the RPK transcript variant was amplified. The PCR product was then digested using Surveyor nuclease S according to the manufacturer's instructions. The digested products were separated and evaluated on a 10% Novex TBE gel. To measure cleavage, the gel image was analyzed by measuring the concentration of different bands.

[0075] Furthermore, the indel frequencies of SG1, SG3, SG5, SG6, and SG8 were compared with human CB-CD34. + Cellular analysis was performed using the TIDE assay. Genomic DNA was purified and PCR was performed to amplify the region around the start codon of the RPK transcript variant of the PKLR gene. Sanger sequencing analysis was then performed on the PCR products. Unedited cells were always used as a negative control for calculating indel frequencies with TIDE. Finally, the activity of the designed guide was evaluated by calculating indel frequencies using TIDE software (https: / / tide.deskgen.com / ).

[0076] Furthermore, the off-target activity of the three most promising sgRNAs (SG1, SG2, and SG3) and the newly designed sgRNAs (SG4, SG9, and SG10) was analyzed together according to the most rigorous standards currently available, GUIDE-seq and rhAmpSeq, and one of them was selected for clinical use. First, GUIDE-seq analysis was performed on HEK293T cell lines constitutively expressing WT-Cas9 to broadly identify off-targets considering the genomic context in vivo. Cells were transfused with different sgRNAs. After 5 days, cells were collected and genomic DNA was isolated. Off-target sites were identified using GUIDE-seq-tags with the IDT in-house guided analysis tool, and those appearing in more than 1% of readouts were also observed. In vivo off-targets were identified for these sgRNAs, and the proportion of on-target modifications in the overall gene editing was calculated (Table 3). However, when the same GUIDE-seq analysis was performed on jarcut cells electroporated using RNP-formatted SG1, the number of off-target effects dramatically decreased (Figure 4). Furthermore, using HiFi-Cas9 in the RNP complex reduced the off-target effects of SG1. To quantify on-target and off-target activity in regions not covered by GUIDE-seq, the indel frequencies of on-target and off-target processes were measured by rhAmpSeq. This assay contributed to a more accurate quantitative analysis of SG1 gene editing in cellular and genomic contexts. CB-CD34 +Cells were edited using SG1, SG9, or SG10. These showed reduced off-target contributions (Table 3), and were complexed with either WT-Cas9 or HiFi-Cas9 to form RNP complexes. Furthermore, a specific ssODN HDR template was added to a portion of the sample to identify the ability of each sgRNA to mediate HDR. Top hits from GUIDE-seq experiments (one on-target site, ON, and 48 off-target sites, OT) were amplified using the rhAmpSeq pool. The libraries were then run on the MiSeq system and analyzed using in-house analytical tools. The editing level at the cleavage sites was calculated by adding the percentage of NHEJ to the percentages of incomplete and complete HDR. As shown in Figure 5, SG1 was confirmed to have the highest gene editing activity at on-target sites. Moreover, the frequency of complete HDR did not change when HiFi-Cas9 was used to form the RNP complex. Therefore, using HiFi-Cas9 in the SG1 RNP complex did not interfere with HDR at the target site. Furthermore, hCD34 + The off-target effects of SG1 RNP were analyzed (Figure 6). When HiFi-Cas9 was used for SG1 RNP, all off-target modifications were reduced to less than 0.1, the detection limit of the technique. Therefore, hCD34 using the SG1 HiFi-Cas9 RNP complex + Cellular gene editing maintained a high level of on-target site modification without altering HDR, while reducing off-target effects to undetectable levels.

[0077] 2.1. Surveyor Assay The results obtained from the Surveyor assay are shown in Figure 2, in addition to Table 2 below.

[0078] [Table 2]

[0079] Surveyor assay results: SG1 (SEQ ID NO: 1) resulted in the highest indel frequency at the on-target site in K562 cells.

[0080] 2.2. TIDE Assay The results obtained from the TIDE assay are shown in Figure 3.

[0081] TIDE assay results: SG1 (Sequence ID 1) is human CB-CD34 + This resulted in the highest indel frequency at the on-target site in the cell.

[0082] 2.3. GUIDE-Seq and rhAmp-Seq: Identification of in vivo off-targets: GUIDE-seq analysis was performed on HEK293 cell lines constitutively expressing WT-Cas9 (HEK293-Cas9) to advance the identification of in vivo off-targets.

[0083] [Table 3]

[0084] result: SG1 (SEQ ID NO: 1) exhibited multiple off-target behaviors when transfected into HEK293-Cas9. The safety results for PKLR SG1 ATG in ribonucleoprotein (RNP) form are shown in Figures 4 and 6. hCD34 + The off-target effects originating from SG1 were reduced to less than 0.1% when using HiFi Cas9-RNP.

[0085] 3. Quantitative on-target modification and HDR frequency using rhAmp-Seq The results are shown in Figure 5. In the figure, the on-target frequency is CB-CD34. + In this case, the highest performance was observed when using SG1. Furthermore, the high HDR frequency of SG1 was not compromised when using HiFi Cas9-RNP.

[0086] Sequence ID 1: Protospacer SG1 sequence CTGCGGGACCATGGAATGAG

[0087] Sequence ID 11: crRNA SG1 sequence (as RNA) CUGCGGGACCAUGGAAUGAG

[0088] Sequence ID 12: sgRNA SG1 sequence (as RNA) CUGCGGGACCAUGGAAUGAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAUAAGGCUAGUCCG

[0089] Example 2. Design of coRPK-AAV The most efficient and safest sgRNA, which was particularly a product of the experimental process or errors, was selected, and the coRPK-AAV was designed from there. As described in the following sections, the coRPK-AAV is formed by two homology arms around the SG1 on-target site (left homology arm i.e., LHA, and right homology arm i.e., RHA), a sequence upstream of the ATG (5'UTR), coRPK cDNA, FLAG-Tag, and bovine growth hormone polyadenylation (bgh-polyA) signaling. This is shown in Figures 7 and 8. The coRPK-AAV was designed taking into account the SG1 cleavage site, which is located 38 bp upstream of the RPK start codon. The two homology arms were selected from sequences around the SG1 cleavage site. LHA encompasses a genomic region of 463 bp–39 bp upstream of the RPK start codon (total size 425 bp). The RHA encompasses the region from 30 bp upstream to 395 bp downstream of the RPK start codon (total size 425 bp). Between the LHA and the homologous genomic region of the RHA lies an 8 bp region of SG1 target sequences, which contains the SG1 cleavage site. This 8 bp gap between the two homologous arms was considered to prevent re-cleavage of SG1 after homology-directed repair has occurred. On the other hand, the sequence encompassing 37 bp upstream of the RPK start codon is part of the Kozac sequence of the RPK transcript variant, and this was cloned into the 5' of the coRPK cDNA of coRPK-AAV without the ATG and stop codon. A 1 bp gap exists between the LHA and the 5'UTR, which corresponds to the SG1 cleavage site and is included to reduce the risk of the therapeutic cassette being cleaved by SG1 after HDR has occurred.

[0090] 1. LHA (Left Homology Arm) (Sequence ID 13): As shown in Figure 9, the LHA encompasses 425 bp upstream of the SG1 cleavage site. CAGAGTGGTGAAGGCACTCTGCATTTCTTGGTTGAGACAGAGAAAAAAAGTGGTCAGAACTGGGTAACCCTCCCCCCACCATATTATCACAGTGATCCCTTTTGTCTTTCTTCAGGCTCCAGCCCCACCCTACAGCCCCTGCTCCCTGGATTCACTAGAGCTAACTTCAGTAAAGTACAAAGAAAATGGGGCCATATGACTGGCCAAAAAAAAAATATCTATTCACGTGGATGACCAGATAGTATGAATGGATTGAAAATTTATCAGGAAAAAAGGATGAGAGGAAATGCCAGGAGATGAGGGCAGAGAGCAGGCCGTTCTGGGGGAGGGATTCTGTGGGGACAGGGTGGCCTACTGGGTGTGCCCCTTTTCTCTTCTCTGTCTCCCTTAGATAAGACCAGCAGTTTTGTCATCCTCTCCCTCTC

[0091] 2. RHA (Right Homologous Arm) (SEQ ID NO: 14): As shown in FIG. 10, RHA includes 425 bp starting 11 bp away from the SG1 PAM sequence. GTCCCGCAGCCCCAGGCCCACACTGAAAGCATGTCGATCCAGGAGAACATATCATCCCTGCAGCTTCGGTCATGGGTCTCTAAGTCCCAAAGAGACTTAGCAAAGTCCATCCTGATTGGGGCTCCAGGAGGTAAGAAGGGGAGACAGAAGCCATGGAACATAGGAGGAAAATGAGGGTGAAAACTAGGAGCCAGGGTGGAGGGCATAAATGATCCACATCAGCCACTGGCTAGGTGGGTTTTGGAGAGGAACGTACGTTCTTCAGAGCCTCCCGTGTGTTAAATTATGGACCCTGGCCTGGGTCTTTTCCAGGCCCTATAGGCAGGCCAGAGCCACAGCATGTAAGCCACGGGGCACTCCCGTGGTTCCTGGACTCTGGCCCCTGGCATACAGGGCTTCCAATGGAACAGGAGACAGTGGTGACA

[0092] 3.5'UTR sequence (sequence number 15): A 40bp sequence, including the 4th nt of the SG1 protospacer up to the RPK start codon, was cloned downstream of the coRPK cDNA, excluding the start and stop codons. This sequence provides the WT RPK with the most appropriate 5'UTR for similar erythrocyte expression of coRPK, separate from the start codon, because the Kozac sequence of the RPK transcript variant is part of this sequence.

[0093] Note: A 1 bp gap exists between the LHA and the 5'UTR to prevent re-cleavage by SG1 after gene editing correction. This is because the SG1 protospacer is not completely reconstructed after gene editing. TTCCATGGTCCCGCAGCCCCAGGCCCACACTGAAAGCATG

[0094] 4. coRPK cDNA (SEQ ID NO: 16): The coRPK cDNA sequence is a modified version of LV coRPK (Garcia-Gomez et al. Mol Ther. 2016), obtained after codon optimization by GeneArt. In this specification, the inventors have made the following modifications: i) cloned this sequence without a start codon to use an endogenous PKLR sequence for the purpose of driving coRPK expression by an endogenous PKLR promoter and endogenous regulatory sequence; ii) cloned this sequence without a stop codon for the purpose of fusing it with a FLAG-Tag.

[0095] 5. FLAG-Tag (SEQ ID NO: 17): hCD34 from healthy donors + During the setting of gene editing conditions using cells, for the purpose of tracking the therapeutic RPK protein after gene editing, this sequence was added in-frame with coRPK without a stop codon to generate a fusion protein. However, FLAG-Taq does not exist in coRPK-AAV proposed for clinical use. This is because FLAG-Taq does not make any functional contribution to the correction of PKD. GACTACAAAGACGATGACGATAAATGA

[0096] 6. bGH poly(A) signal (SEQ ID NO: 18): The bovine growth hormone polyadenylation (bGH-polyA) signal is a dedicated termination sequence for protein expression in eukaryotic cells. CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0097] 7. LHA-5’UTR-coRPK-FLAG-bGH poly(A)-RHA (SEQ ID NO: 19) This is a donor sequence designed to correct PKD via HDR at the SG1 target site during the setup experiment. It includes LHA (sequence number 13, bold), 5'UTR (sequence number 15, italic), coRPK without start and stop codons (sequence number 16, underlined), FLAG-Taq (sequence number 17, bold and italic), and bGH poly(A) (sequence number 18, bold and underlined)RHA (sequence number 14, bold and italic), in the order mentioned. The functions of these element sequences are described below: The homology arm intervenes in the insertion of the coRPK cassette at the SG1 target site. The 5'UTR ensures proper regulation of coRPK expression. coRPK is a codon-optimized version of the RPK transcript, which encodes an RPK protein that corrects the PKD phenotype in red blood cells. FLAG-Taq is derived from healthy donor hCD34 + During the development of gene editing conditions using cells, FLAG-Taq was added to distinguish between WT RPK protein and the RPK encoded by coRPK, but it is not present in coRPK-AAV proposed for clinical use. This is because FLAG-Taq does not make any functional contribution to the correction of PKD. Finally, the bGH poly(A) signaling pathway promotes the translation of coRPK-FLAG into protein. CAGAGTGGTGAAGGCACTCTGCATTTCTTGGTTGAGACAGAGAAAAAAAGTGGTCAGAACTGGGTAACCCTCCCCCCACCATATTATCACAGTGATCCCTTTTGTCTTTCTTCAGGCTCCAGCCCCACCCTACAGCCCCTGCTCCCTGGATTCACTAGAGCTAACTTCAGTAAAGTACAAAGAAAATGGGGCCATATGACTGGCCAAAAAAAAAATATCTATTCACGTGGATGACCAGATAGTATGAATGGATTGAAAATTTATCAGGAAAAAAGGATGAGAGGAAATGCCAGGAGATGAGGGCAGAGAGCAGGCCGTTCTGGGGGAGGGATTCTGTGGGGACAGGGTGGCCTACTGGGTGTGCCCCTTTTCTCTTCTCTGTCTCCCTTAGATAAGACCAGCAGTTTTGTCATCCTCTCCCTCTCTTCCATGGTCCCGCAGCCCCAGGCCCACACTGAAAGCATG AGCATCCAGGAAAATATCAGCTCTCTGCAGCTGCGGTCCTGGGTGTCCAAGAGCCAGAGAGACCTGGCCAAGAGCATCCTGATCGGAGCCCCTGGCGGACCAGCCGGATACCTGAGAAGGGCTAGCGTGGCCCAGCTGACCCAGGAACTGGGCACCGCCTTTTTCCAGCAGCAGCAGCTGCCAGCCGCCATGGCCGACACCTTTCTGGAACACCTGTGCCTGCTGGACATCGACTCTGAGCCCGTGGCCGCCAGAAGCACCAGCATCATTGCCACCATCGGCCCTGCCAGCAGAAGCGTGGAGCGGCTGAAAGAGATGATCAAGGCCGGCATGAATATCGCCCGGCTGAACTTCTCCCACGGCAGCCACGAGTACCACGCAGAGAGCATTGCCAACGTCCGGGAGGCCGTGGAGAGCTTTGCCGGCAGCCCCCTGAGCTACAGACCCGTGGCCATTGCCCTGGACACCAAGGGCCCCGAGATCAGAACAGGAATTCTGCAGGGAGGGCCTGAGAGCGAGGTGGAGCTGGTGAAGGGCAGCCAAGTGCTGGTGACCGTGGACCCCGCCTTCAGAACCAGAGGCAACGCCAACACAGTGTGGGTGGACTACCCCAACATCGTGCGGGTGGTGCCTGTGGGCGGCAGAATCTACATCGACGACGGCCTGATCAGCCTGGTGGTGCAGAAGATCGGACCTGAGGGCCTGGTGACCCAGGTCGAGAATGGCGGCGTGCTGGGCAGCAGAAAGGGCGTGAATCTGCCAGGCGCCCAGGTGGACCTGCCTGGCCTGTCTGAGCAGGACGTGAGAGACCTGAGATTTGGCGTGGAGCACGGCGTGGACATCGTGTTCGCCAGCTTCGTGCGGAAGGCCTCTGATGTGGCCGCCGTGAGAGCCGCTCTGGGCCCTGAAGGCCACGGCATCAAGATCATCAGCAAGATCGAGAACCACGAGGGCGTGAAGCGGTTCGACGAGATCCTGGAAGTGTCCGACGGCATCATGGTGGCCAGAGGCGACCTGGGCATCGAGATCCCCGCCGAGAAGGTGTTCCTGGCCCAGAAAATGATGATCGGACGGTGCAACCTGGCCGGCAAACCTGTGGTGTGCGCCACCCAGATGCTGGAAAGCATGATCACCAAGCCCAGACCCACCAGAGCCGAGACAAGCGACGTGGCCAACGCCGTGCTGGATGGCGCTGACTGCATCATGCTGTCCGGCGAGACAGCCAAGGGCAACTTCCCCGTGGAGGCCGTGAAGATGCAGCACGCCATTGCCAGAGAAGCCGAGGCCGCCGTGTACCACCGGCAGCTGTTCGAGGAACTGCGGAGAGCCGCCCCTCTGAGCAGAGATCCCACCGAAGTGACCGCCATCGGAGCCGTGGAAGCCGCCTTCAAGTGCTGCGCCGCTGCAATCATCGTGCTGACCACCACAGGCAGAAGCGCCCAGCTGCTGTCCAGATACAGACCCAGAGCCGCCGTGATCGCCGTGACAAGATCCGCCCAGGCCGCTAGACAGGTCCACCTGTGCAGAGGCGTGTTCCCCCTGCTGTACCGGGAGCCTCCCGAGGCCATCTGGGCCGACGACGTGGACAGACGGGTGCAGTTCGGCATCGAGAGCGGCAAGCTGCGGGGCTTCCTGAGAGTGGGCGACCTGGTGATCGTGGTGACAGGCTGGCGGCCTGGCAGCGGCTACACCAACATCATGAGGGTGCTGTCCATCAGC GACTACAAAGACGATGACGATAAATGAACGCGTGAGTTACAAATAAAGCA CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG GTCCCGCAGCCCCAGGCCCACACTGAAAGCATGTCGATCCAGGAGAACATATCATCCCTGCAGCTTCGGTCATGGGTCTCTAAGTCCCAAAGAGACTTAGCAAAGTCCATCCTGATTGGGGCTCCAGGAGGTAAGAAGGGGAGACAGAAGCCATGGAACATAGGAGGAAAATGAGGGTGAAAACTAGGAGCCAGGGTGGAGGGCATAAATGATCCACATCAGCCACTGGCTAGGTGGGTTTTGGAGAGGAACGTACGTTCTTCAGAGCCTCCCGTGTGTTAAATTATGGACCCTGGCCTGGGTCTTTTCCAGGCCCTATAGGCAGGCCAGAGCCACAGCATGTAAGCCACGGGGCACTCCCGTGGTTCCTGGACTCTGGCCCCTGGCATACAGGGCTTCCAATGGAACAGGAGACAGTGGTGACA

[0098] 8. LHA-5'UTR-coRPK-bGH poly(A)-RHA (SEQ ID NO: 20) Sequence ID 20 is the donor matrix (Sequence ID 19) with the FLAG-Tag sequence removed. CAGAGTGGTGAAGGCACTCTGCATTTCTTGGTTGAGACAGAGAAAAAAAGTGGTCAGAACTGGGTAACCCTCCCCCCATATTATCACAGTGATCCCTTTTGTCTTTCTTCAGGCTCCAGCCCCACCCTACAGCCCCTGCTCCCTGGATTCACTAGAGCTAACTTCAGTAAAGTACAAAGAAAATGGGGCCATATGACTGGCCAAAAAAAAAATATCTATTCACGTGGAT GACCAGATAGTATGAATGGATTGAAAATTTATCAGGAAAAAAGGATGAGAGGAAATGCCAGGAGATGAGGGCAGAGAGCAGGCCGTTCTGGGGGAGGGATTCTGTGGGGACAGGGTGGCCTACTGGGTGTGCCCCTTTTCTCTTCTCTGTCTCCCTTAGATAAGACCAGCAGTTTTGTCATCCTCTCCCTCTCTTCCATGGTCCCGCAGCCCCAGGCCCACACTGAAAGCATG AGCATCCAGGAAAATATCAGCTCTCTGCAGCTGCGGTCCTGGGTGTCCAAGAGCCAGAGAGACCTGGCCAAGAGCATCCTGATCGGAGCCCCTGGCGGACCAGCCGGATACCTGAGAAGGGCTAGCGTGGCCCAGCTGACCCAGGAACTGGGCACCGCCTTTTTCCAGCAGCAGCAGCTGCCAGCCGCCATGGCCGACACCTTTCTGGAACACCTGTGCCTGCTGGACATCGACTCTGAGCCCGTGGCCGCCAGAAGCACCAGCATCATTGCCACCATCGGCCCTGCCAGCAGAAGCGTGGAGCGGCTGAAAGAGATGATCAAGGCCGGCATGAATATCGCCCGGCTGAACTTCTCCCACGGCAGCCACGAGTACCACGCAGAGAGCATTGCCAACGTCCGGGAGGCCGTGGAGAGCTTTGCCGGCAGCCCCCTGAGCTACAGACCCGTGGCCATTGCCCTGGACACCAAGGGCCCCGAGATCAGAACAGGAATTCTGCAGGGAGGGCCTGAGAGCGAGGTGGAGCTGGTGAAGGGCAGCCAAGTGCTGGTGACCGTGGACCCCGCCTTCAGAACCAGAGGCAACGCCAACACAGTGTGGGTGGACTACCCCAACATCGTGCGGGTGGTGCCTGTGGGCGGCAGAATCTACATCGACGACGGCCTGATCAGCCTGGTGGTGCAGAAGATCGGACCTGAGGGCCTGGTGACCCAGGTCGAGAATGGCGGCGTGCTGGGCAGCAGAAAGGGCGTGAATCTGCCAGGCGCCCAGGTGGACCTGCCTGGCCTGTCTGAGCAGGACGTGAGAGACCTGAGATTTGGCGTGGAGCACGGCGTGGACATCGTGTTCGCCAGCTTCGTGCGGAAGGCCTCTGATGTGGCCGCCGTGAGAGCCGCTCTGGGCCCTGAAGGCCACGGCATCAAGATCATCAGCAAGATCGAGAACCACGAGGGCGTGAAGCGGTTCGACGAGATCCTGGAAGTGTCCGACGGCATCATGGTGGCCAGAGGCGACCTGGGCATCGAGATCCCCGCCGAGAAGGTGTTCCTGGCCCAGAAAATGATGATCGGACGGTGCAACCTGGCCGGCAAACCTGTGGTGTGCGCCACCCAGATGCTGGAAAGCATGATCACCAAGCCCAGACCCACCAGAGCCGAGACAAGCGACGTGGCCAACGCCGTGCTGGATGGCGCTGACTGCATCATGCTGTCCGGCGAGACAGCCAAGGGCAACTTCCCCGTGGAGGCCGTGAAGATGCAGCACGCCATTGCCAGAGAAGCCGAGGCCGCCGTGTACCACCGGCAGCTGTTCGAGGAACTGCGGAGAGCCGCCCCTCTGAGCAGAGATCCCACCGAAGTGACCGCCATCGGAGCCGTGGAAGCCGCCTTCAAGTGCTGCGCCGCTGCAATCATCGTGCTGACCACCACAGGCAGAAGCGCCCAGCTGCTGTCCAGATACAGACCCAGAGCCGCCGTGATCGCCGTGACAAGATCCGCCCAGGCCGCTAGACAGGTCCACCTGTGCAGAGGCGTGTTCCCCCTGCTGTACCGGGAGCCTCCCGAGGCCATCTGGGCCGACGACGTGGACAGACGGGTGCAGTTCGGCATCGAGAGCGGCAAGCTGCGGGGCTTCCTGAGAGTGGGCGACCTGGTGATCGTGGTGACAGGCTGGCGGCCTGGCAGCGGCTACACCAACATCATGAGGGTGCTGTCCATCAGC TGAACGCGTGAGTTACAAATAAAGCA CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTCCCGCAGCCCCAGGCCCACACTGAAAGCATGTCGATCCAGGAGAACATATCATCCCTGCAGCTTCGGTCATGGGTCTCTAAGTCCCAAAGAGACTTAGCAAAGTCCATCCTGATTGGGGCTCCAGGAGGTAAGAAGGGGAGACAGAAGCCATGGAACATAGGAGGAAAATGAGGGTGAAAACTAGGAGCCAGGGTGGAGGGCATAAATGA TCCACATCAGCCACTGGCTAGGTGGGTTTTGGAGAGGAACGTACGTTCTTCAGAGCCTCCCGTGTGTTAAATTATGGACCCTGGCCTGGGTCTTTTCCAGGCCCTATAGGCAGGCCAGAGCCACAGCATGTAAGCCACGGGGCACTCCCGTGGTTCCTGGACTCTGGCCCCTGGCATACAGGGCTTCCAATGGAACAGGAGACAGTGGTGACA

[0099] 9. LHA-5'UTR-coRPK-bGH poly(A)-RHA (SEQ ID NO: 21) Sequence ID 21 is the reverse sequence of sequence ID 19.

[0100] The order from the coRPK treatment donor to the AAV is from 5' to 3': right homology arm (bold), bGH poly(A), FLAG-Tag (bold and italic), coRPK without start codon (underlined), 5'UTR (italic), and left homology arm (bold). TGTCACCACTGTCTCCTGTTCCATTGGAAGCCCTGTATGCCAGGGGCCAGAGTCCAGGAACCACGGGAGTGCCCCGTGGCTTACATGCTGTGGCTCTGGCCTGCCTATAGGGCCTGGAAAAGACCCAGGCCAGGGTCCATAATTTAACACACGGGAGGCTCTGAAGAACGTACGTTCCTCTCCAAAACCCACCTAGCCAGTGGCTGATGTGGATCATTTATGCCCTCCACCCTGGCTCCTAGTTTTCACCCTCATTTTCCTCCTATGTTCCATGGCTTCTGTCTCCCCTTCTTACCTCCTGGAGCCCCAATCAGGATGGACTTTGCTAAGTCTCTTTGGGACTTAGAGACCCATGACCGAAGCTGCAGGGATGATATGTTCTCCTGGATCGACATGCTTTCAGTGTGGGCCTGGGGCTGCGGGAC CCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGCAACTAGAAGGCACAG TGCTTTATTTGTAACTCACGCGTTCATTTATCGTCATCGTCTTTGTAGTC GCTGATGGACAGCACCCTCATGATGTTGGTGTAGCCGCTGCCAGGCCGCCAGCCTGTCACCACGATCACCAGGTCGCCCACTCTCAGGAAGCCCCGCAGCTTGCCGCTCTCGATGCCGAACTGCACCCGTCTGTCCACGTCGTCGGCCCAGATGGCCTCGGGAGGCTCCCGGTACAGCAGGGGGAACACGCCTCTGCACAGGTGGACCTGTCTAGCGGCCTGGGCGGATCTTGTCACGGCGATCACGGCGGCTCTGGGTCTGTATCTGGACAGCAGCTGGGCGCTTCTGCCTGTGGTGGTCAGCACGATGATTGCAGCGGCGCAGCACTTGAAGGCGGCTTCCACGGCTCCGATGGCGGTCACTTCGGTGGGATCTCTGCTCAGAGGGGCGGCTCTCCGCAGTTCCTCGAACAGCTGCCGGTGGTACACGGCGGCCTCGGCTTCTCTGGCAATGGCGTGCTGCATCTTCACGGCCTCCACGGGGAAGTTGCCCTTGGCTGTCTCGCCGGACAGCATGATGCAGTCAGCGCCATCCAGCACGGCGTTGGCCACGTCGCTTGTCTCGGCTCTGGTGGGTCTGGGCTTGGTGATCATGCTTTCCAGCATCTGGGTGGCGCACACCACAGGTTTGCCGGCCAGGTTGCACCGTCCGATCATCATTTTCTGGGCCAGGAACACCTTCTCGGCGGGGATCTCGATGCCCAGGTCGCCTCTGGCCACCATGATGCCGTCGGACACTTCCAGGATCTCGTCGAACCGCTTCACGCCCTCGTGGTTCTCGATCTTGCTGATGATCTTGATGCCGTGGCCTTCAGGGCCCAGAGCGGCTCTCACGGCGGCCACATCAGAGGCCTTCCGCACGAAGCTGGCGAACACGATGTCCACGCCGTGCTCCACGCCAAATCTCAGGTCTCTCACGTCCTGCTCAGACAGGCCAGGCAGGTCCACCTGGGCGCCTGGCAGATTCACGCCCTTTCTGCTGCCCAGCACGCCGCCATTCTCGACCTGGGTCACCAGGCCCTCAGGTCCGATCTTCTGCACCACCAGGCTGATCAGGCCGTCGTCGATGTAGATTCTGCCGCCCACAGGCACCACCCGCACGATGTTGGGGTAGTCCACCCACACTGTGTTGGCGTTGCCTCTGGTTCTGAAGGCGGGGTCCACGGTCACCAGCACTTGGCTGCCCTTCACCAGCTCCACCTCGCTCTCAGGCCCTCCCTGCAGAATTCCTGTTCTGATCTCGGGGCCCTTGGTGTCCAGGGCAATGGCCACGGGTCTGTAGCTCAGGGGGCTGCCGGCAAAGCTCTCCACGGCCTCCCGGACGTTGGCAATGCTCTCTGCGTGGTACTCGTGGCTGCCGTGGGAGAAGTTCAGCCGGGCGATATTCATGCCGGCCTTGATCATCTCTTTCAGCCGCTCCACGCTTCTGCTGGCAGGGCCGATGGTGGCAATGATGCTGGTGCTTCTGGCGGCCACGGGCTCAGAGTCGATGTCCAGCAGGCACAGGTGTTCCAGAAAGGTGTCGGCCATGGCGGCTGGCAGCTGCTGCTGCTGGAAAAAGGCGGTGCCCAGTTCCTGGGTCAGCTGGGCCACGCTAGCCCTTCTCAGGTATCCGGCTGGTCCGCCAGGGGCTCCGATCAGGATGCTCTTGGCCAGGTCTCTCTGGCTCTTGGACACCCAGGACCGCAGCTGCAGAGAGCTGATATTTTCCTGGATGCT CATGCTTTCAGTGTGGGCCTGGGGCTGCGGGACCATGGAAGAGAGGGAGAGGATGACAAAACTGCTGGTCTTATCTAAGGGAGACAGAGAAGAGAAAAGGGGCACACCCAGTAGGCCACCCTGTCCCCACAGAATCCCTCCCCCAGAACGGCCTGCTCTCTGCCCTCATCTCCTGGCATTTCCTCTCATCCTTTTTTCCTGATAAATTTTCAATCCATTCATACTATCTGGTCATCCACGTGAATAGATATTTTTTTTTTGGCCAGTCATATGGCCCCATTTTCTTTGTACTTTACTGAAGTTAGCTCTAGTGAATCCAGGGAGCAGGGGCTGTAGGGTGGGGCTGGAGCCTGAAGAAAGACAAAAGGGATCACTGTGATAATATGGTGGGGGGAGGGTTACCCAGTTCTGACCACTTTTTTTCTCTGTCTCAACCAAGAAATGCAGAGTGCCTTCACCACTCTG

[0101] 10. LHA-5'UTR-coRPK-bGH poly(A)-RHA (SEQ ID NO: 22) Sequence ID 22 is the reverse sequence of sequence ID 20.

[0102] The order from the coRPK treatment donor to the AAV is from 5' to 3': right homology arm (bold), bGH poly(A), coRPK without start codon (underlined), 5'UTR (italic), and left homology arm (bold). TGTCACCACTGTCTCCTGTTCCATTGGAAGCCCTGTATGCCAGGGGCCAGAGTCCAGGAACCACGGGAGTGCCCCGTGGCTTACATGCTGTGGCTCTGGCCTGCCTATAGGGCCTGGAAAAGACCCAGGCCAGGGTCCATAATTTAACACACGGGAGGCTCTGAAGAACGTACGTTCCTCTCCAAAACCCACCTAGCCAGTGGCTGATGTGG ATCATTTATGCCCTCCACCCTGGCTCCTAGTTTTCACCCTCATTTTCCTCCTATGTTCCATGGCTTCTGTCTCCCCTTCTTACCTCCTGGAGCCCCAATCAGGATGGACTTTGCTAAGTCTCTTTGGGACTTAGAGACCCATGACCGAAGCTGCAGGGATGATATGTTCTCCTGGATCGACATGCTTTCAGTGTGGGCCTGGGGCTGCGGGAC CCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGCAACTAGAAGGCACAG TGCTTTATTTGTAACTCACGCGTTCA GCTGATGGACAGCACCCTCATGATGTTGGTGTAGCCGCTGCCAGGCCGCCAGCCTGTCACCACGATCACCAGGTCGCCCACTCTCAGGAAGCCCCGCAGCTTGCCGCTCTCGATGCCGAACTGCACCCGTCTGTCCACGTCGTCGGCCCAGATGGCCTCGGGAGGCTCCCGGTACAGCAGGGGGAACACGCCTCTGCACAGGTGGACCTGTCTAGCGGCCTGGGCGGATCTTGTCACGGCGATCACGGCGGCTCTGGGTCTGTATCTGGACAGCAGCTGGGCGCTTCTGCCTGTGGTGGTCAGCACGATGATTGCAGCGGCGCAGCACTTGAAGGCGGCTTCCACGGCTCCGATGGCGGTCACTTCGGTGGGATCTCTGCTCAGAGGGGCGGCTCTCCGCAGTTCCTCGAACAGCTGCCGGTGGTACACGGCGGCCTCGGCTTCTCTGGCAATGGCGTGCTGCATCTTCACGGCCTCCACGGGGAAGTTGCCCTTGGCTGTCTCGCCGGACAGCATGATGCAGTCAGCGCCATCCAGCACGGCGTTGGCCACGTCGCTTGTCTCGGCTCTGGTGGGTCTGGGCTTGGTGATCATGCTTTCCAGCATCTGGGTGGCGCACACCACAGGTTTGCCGGCCAGGTTGCACCGTCCGATCATCATTTTCTGGGCCAGGAACACCTTCTCGGCGGGGATCTCGATGCCCAGGTCGCCTCTGGCCACCATGATGCCGTCGGACACTTCCAGGATCTCGTCGAACCGCTTCACGCCCTCGTGGTTCTCGATCTTGCTGATGATCTTGATGCCGTGGCCTTCAGGGCCCAGAGCGGCTCTCACGGCGGCCACATCAGAGGCCTTCCGCACGAAGCTGGCGAACACGATGTCCACGCCGTGCTCCACGCCAAATCTCAGGTCTCTCACGTCCTGCTCAGACAGGCCAGGCAGGTCCACCTGGGCGCCTGGCAGATTCACGCCCTTTCTGCTGCCCAGCACGCCGCCATTCTCGACCTGGGTCACCAGGCCCTCAGGTCCGATCTTCTGCACCACCAGGCTGATCAGGCCGTCGTCGATGTAGATTCTGCCGCCCACAGGCACCACCCGCACGATGTTGGGGTAGTCCACCCACACTGTGTTGGCGTTGCCTCTGGTTCTGAAGGCGGGGTCCACGGTCACCAGCACTTGGCTGCCCTTCACCAGCTCCACCTCGCTCTCAGGCCCTCCCTGCAGAATTCCTGTTCTGATCTCGGGGCCCTTGGTGTCCAGGGCAATGGCCACGGGTCTGTAGCTCAGGGGGCTGCCGGCAAAGCTCTCCACGGCCTCCCGGACGTTGGCAATGCTCTCTGCGTGGTACTCGTGGCTGCCGTGGGAGAAGTTCAGCCGGGCGATATTCATGCCGGCCTTGATCATCTCTTTCAGCCGCTCCACGCTTCTGCTGGCAGGGCCGATGGTGGCAATGATGCTGGTGCTTCTGGCGGCCACGGGCTCAGAGTCGATGTCCAGCAGGCACAGGTGTTCCAGAAAGGTGTCGGCCATGGCGGCTGGCAGCTGCTGCTGCTGGAAAAAGGCGGTGCCCAGTTCCTGGGTCAGCTGGGCCACGCTAGCCCTTCTCAGGTATCCGGCTGGTCCGCCAGGGGCTCCGATCAGGATGCTCTTGGCCAGGTCTCTCTGGCTCTTGGACACCCAGGACCGCAGCTGCAGAGAGCTGATATTTTCCTGGATGCTCATGCTTTCAGTGTGGGCCTGGGGCTGCGGGACCATGGAAGAGAGGGAGAGGATGACAAAACTGCTGGTCTTATCTAAGGGAGACAGAGAAGAGAAAAGGGGCACACCCAGTAGGCCACCCTGTCCCCACAGAATCCCTCCCCCAGAACGGCCTGCTCTCTGCCCTCATCTCCTGGCATTTCCTCTCATCCTTTTTTCCTGATAAATTTTCAATCCATTCATACTATCTGGT CATCCACGTGAATAGATATTTTTTTTTTGGCCAGTCATATGGCCCCATTTTCTTTGTACTTTACTGAAGTTAGCTCTAGTGAATCCAGGGAGCAGGGGCTGTAGGGTGGGGCTGGAGCCTGAAGAAAGACAAAAGGGATCACTGTGATAATATGGTGGGGGGAGGGTTACCCAGTTCTGACCACTTTTTTTCTCTGTCTCAACCAAGAAATGCAGAGTGCCTTCACCACTCTG

[0103] 11. AAV skeleton (Sequence ID 23): A transfer plasmid containing ITRs (Internal Terminal Repeats) derived from an AAV2 ITR-containing pAAV-MCS plasmid (Agilent Technologies) was used. The reverse LHA-5'UTR-coRPK-FLAG-bGH poly(A)-RHA (SEQ ID NO: 20) was cloned into the AAV backbone via NotI restriction site cloning.

[0104] 12. coRPK AAV (SEQ ID NO: 24) to be used in preclinical trials: A sequence containing a coRPK therapeutic donor (SEQ ID NO: 19) inserted into the AAV skeleton (SEQ ID NO: 23). CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCCAGAGTGGTGAA GGCACTCTGCATTTCTTGGTTGAGACAGAGAAAAAAAGTGGTCAGAACTGGGTAACCCTCCCCCCACCATATTATCACAGTGATCCCTTTTGTCTTTCTTCAGGCTCCAGCCCCACCCTACAGCCCCTGCTCCCTGGATTCACTAGAGCTA ACTTCAGTAAAGTACAAAGAAAATGGGGCCATATGACTGGCCAAAAAAAAAATATCTATTCACGTGGATGACCAGATAGTATGAATGGATTGAAAATTTATCAGGAAAAAAGGATGAGAGGAAATGCCAGGAGATGAGGGCAGAGAGCAGG CCGTTCTGGGGGAGGGATTCTGTGGGGACAGGGTGGCCTACTGGGTGTGCCCCTTTTCTCTTCTCTGTCTCCCTTAGATAAGACCAGCAGTTTTGTCATCCTCTCCCTCTCTTCCATGGTCCCGCAGCCCCAGGCCCACACTGAAAGCATG AGCATCCAGGAAAATATCAGCTCTCTGCAGCTGCGGTCCTGGGTGTCCAAGAGCCAGAGAGACCTGGCCAAGAGCATCCTGATCGGAGCCCCTGGCGGACCAGCCGGATACCTGAGAAGGGCTAGCGTGGCCCAGCTGACCCAGGAACTGGGCACCGCCTTTTTCCAGCAGCAGCAGCTGCCAGCCGCCATGGCCGACACCTTTCTGGAACACCTGTGCCTGCTGGACATCGACTCTGAGCCCGTGGCCGCCAGAAGCACCAGCATCATTGCCACCATCGGCCCTGCCAGCAGAAGCGTGGAGCGGCTGAAAGAGATGATCAAGGCCGGCATGAATATCGCCCGGCTGAACTTCTCCCACGGCAGCCACGAGTACCACGCAGAGAGCATTGCCAACGTCCGGGAGGCCGTGGAGAGCTTTGCCGGCAGCCCCCTGAGCTACAGACCCGTGGCCATTGCCCTGGACACCAAGGGCCCCGAGATCAGAACAGGAATTCTGCAGGGAGGGCCTGAGAGCGAGGTGGAGCTGGTGAAGGGCAGCCAAGTGCTGGTGACCGTGGACCCCGCCTTCAGAACCAGAGGCAACGCCAACACAGTGTGGGTGGACTACCCCAACATCGTGCGGGTGGTGCCTGTGGGCGGCAGAATCTACATCGACGACGGCCTGATCAGCCTGGTGGTGCAGAAGATCGGACCTGAGGGCCTGGTGACCCAGGTCGAGAATGGCGGCGTGCTGGGCAGCAGAAAGGGCGTGAATCTGCCAGGCGCCCAGGTGGACCTGCCTGGCCTGTCTGAGCAGGACGTGAGAGACCTGAGATTTGGCGTGGAGCACGGCGTGGACATCGTGTTCGCCAGCTTCGTGCGGAAGGCCTCTGATGTGGCCGCCGTGAGAGCCGCTCTGGGCCCTGAAGGCCACGGCATCAAGATCATCAGCAAGATCGAGAACCACGAGGGCGTGAAGCGGTTCGACGAGATCCTGGAAGTGTCCGACGGCATCATGGTGGCCAGAGGCGACCTGGGCATCGAGATCCCCGCCGAGAAGGTGTTCCTGGCCCAGAAAATGATGATCGGACGGTGCAACCTGGCCGGCAAACCTGTGGTGTGCGCCACCCAGATGCTGGAAAGCATGATCACCAAGCCCAGACCCACCAGAGCCGAGACAAGCGACGTGGCCAACGCCGTGCTGGATGGCGCTGACTGCATCATGCTGTCCGGCGAGACAGCCAAGGGCAACTTCCCCGTGGAGGCCGTGAAGATGCAGCACGCCATTGCCAGAGAAGCCGAGGCCGCCGTGTACCACCGGCAGCTGTTCGAGGAACTGCGGAGAGCCGCCCCTCTGAGCAGAGATCCCACCGAAGTGACCGCCATCGGAGCCGTGGAAGCCGCCTTCAAGTGCTGCGCCGCTGCAATCATCGTGCTGACCACCACAGGCAGAAGCGCCCAGCTGCTGTCCAGATACAGACCCAGAGCCGCCGTGATCGCCGTGACAAGATCCGCCCAGGCCGCTAGACAGGTCCACCTGTGCAGAGGCGTGTTCCCCCTGCTGTACCGGGAGCCTCCCGAGGCCATCTGGGCCGACGACGTGGACAGACGGGTGCAGTTCGGCATCGAGAGCGGCAAGCTGCGGGGCTTCCTGAGAGTGGGCGACCTGGTGATCGTGGTGACAGGCTGGCGGCCTGGCAGCGGCTACACCAACATCATGAGGGTGCTGTCCATCAGC GACTACAAAGACGATGACGATAAATGAACGCGTGAGTTACAAATAAAGCA CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0105] 13. coRPK AAV (SEQ ID NO: 25) to be used in preclinical trials: A sequence containing a coRPK therapeutic donor (SEQ ID NO: 20) inserted into the AAV skeleton (SEQ ID NO: 23). CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCCAGAGTGGTGAA GGCACTCTGCATTTCTTGGTTGAGACAGAGAAAAAAAGTGGTCAGAACTGGGTAACCCTCCCCCCACCATATTATCACAGTGATCCCTTTTGTCTTTCTTCAGGCTCCAGCCCCACCCTACAGCCCCTGCTCCCTGGATTCACTAGAGCTA ACTTCAGTAAAGTACAAAGAAAATGGGGCCATATGACTGGCCAAAAAAAAAATATCTATTCACGTGGATGACCAGATAGTATGAATGGATTGAAAATTTATCAGGAAAAAAGGATGAGAGGAAATGCCAGGAGATGAGGGCAGAGAGCAGG CCGTTCTGGGGGAGGGATTCTGTGGGGACAGGGTGGCCTACTGGGTGTGCCCCTTTTCTCTTCTCTGTCTCCCTTAGATAAGACCAGCAGTTTTGTCATCCTCTCCCTCTCTTCCATGGTCCCGCAGCCCCAGGCCCACACTGAAAGCATG AGCATCCAGGAAAATATCAGCTCTCTGCAGCTGCGGTCCTGGGTGTCCAAGAGCCAGAGAGACCTGGCCAAGAGCATCCTGATCGGAGCCCCTGGCGGACCAGCCGGATACCTGAGAAGGGCTAGCGTGGCCCAGCTGACCCAGGAACTGGGCACCGCCTTTTTCCAGCAGCAGCAGCTGCCAGCCGCCATGGCCGACACCTTTCTGGAACACCTGTGCCTGCTGGACATCGACTCTGAGCCCGTGGCCGCCAGAAGCACCAGCATCATTGCCACCATCGGCCCTGCCAGCAGAAGCGTGGAGCGGCTGAAAGAGATGATCAAGGCCGGCATGAATATCGCCCGGCTGAACTTCTCCCACGGCAGCCACGAGTACCACGCAGAGAGCATTGCCAACGTCCGGGAGGCCGTGGAGAGCTTTGCCGGCAGCCCCCTGAGCTACAGACCCGTGGCCATTGCCCTGGACACCAAGGGCCCCGAGATCAGAACAGGAATTCTGCAGGGAGGGCCTGAGAGCGAGGTGGAGCTGGTGAAGGGCAGCCAAGTGCTGGTGACCGTGGACCCCGCCTTCAGAACCAGAGGCAACGCCAACACAGTGTGGGTGGACTACCCCAACATCGTGCGGGTGGTGCCTGTGGGCGGCAGAATCTACATCGACGACGGCCTGATCAGCCTGGTGGTGCAGAAGATCGGACCTGAGGGCCTGGTGACCCAGGTCGAGAATGGCGGCGTGCTGGGCAGCAGAAAGGGCGTGAATCTGCCAGGCGCCCAGGTGGACCTGCCTGGCCTGTCTGAGCAGGACGTGAGAGACCTGAGATTTGGCGTGGAGCACGGCGTGGACATCGTGTTCGCCAGCTTCGTGCGGAAGGCCTCTGATGTGGCCGCCGTGAGAGCCGCTCTGGGCCCTGAAGGCCACGGCATCAAGATCATCAGCAAGATCGAGAACCACGAGGGCGTGAAGCGGTTCGACGAGATCCTGGAAGTGTCCGACGGCATCATGGTGGCCAGAGGCGACCTGGGCATCGAGATCCCCGCCGAGAAGGTGTTCCTGGCCCAGAAAATGATGATCGGACGGTGCAACCTGGCCGGCAAACCTGTGGTGTGCGCCACCCAGATGCTGGAAAGCATGATCACCAAGCCCAGACCCACCAGAGCCGAGACAAGCGACGTGGCCAACGCCGTGCTGGATGGCGCTGACTGCATCATGCTGTCCGGCGAGACAGCCAAGGGCAACTTCCCCGTGGAGGCCGTGAAGATGCAGCACGCCATTGCCAGAGAAGCCGAGGCCGCCGTGTACCACCGGCAGCTGTTCGAGGAACTGCGGAGAGCCGCCCCTCTGAGCAGAGATCCCACCGAAGTGACCGCCATCGGAGCCGTGGAAGCCGCCTTCAAGTGCTGCGCCGCTGCAATCATCGTGCTGACCACCACAGGCAGAAGCGCCCAGCTGCTGTCCAGATACAGACCCAGAGCCGCCGTGATCGCCGTGACAAGATCCGCCCAGGCCGCTAGACAGGTCCACCTGTGCAGAGGCGTGTTCCCCCTGCTGTACCGGGAGCCTCCCGAGGCCATCTGGGCCGACGACGTGGACAGACGGGTGCAGTTCGGCATCGAGAGCGGCAAGCTGCGGGGCTTCCTGAGAGTGGGCGACCTGGTGATCGTGGTGACAGGCTGGCGGCCTGGCAGCGGCTACACCAACATCATGAGGGTGCTGTCCATCAGC TGAACGCGTGAGTTACAAATAAAGCA CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0106] 14. coRPK AAV (SEQ ID NO: 26) to be used in preclinical trials: A sequence containing a coRPK therapeutic donor (SEQ ID NO: 21) inserted into the AAV skeleton (SEQ ID NO: 23). CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCTG TCACCACTGTCTCCTGTTCCATTGGAAGCCCTGTATGCCAGGGGCCAGAGTCCAGGAACCACGGGAGTGCCCCGTGGCTTACATGCTGTGGCTCTGGCCTGCCTATAGGGCCTGGAAAAGACCCAGGCCAGGGTCCATAAT TTAACACACGGGAGGCTCTGAAGAACGTACGTTCCTCTCCAAAACCCACCTAGCCAGTGGCTGATGTGGATCATTTATGCCCTCCACCCTGGCTCCTAGTTTTCACCCTCATTTTCCTCCTATGTTCCATGGCTTCTGTCT CCCCTTCTTACCTCCTGGAGCCCCAATCAGGATGGACTTTGCTAAGTCTCTTTGGGACTTAGAGACCCATGACCGAAGCTGCAGGGATGATATGTTCTCCTGGATCGACATGCTTTCAGTGTGGGCCTGGGGCTGCGGGAC CCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGCAACTAGAAGGCACAG TGCTTTATTTGTAACTCACGCGTTCATTTATCGTCATCGTCTTTGTAGTC GCTGATGGACAGCACCCTCATGATGTTGGTGTAGCCGCTGCCAGGCCGCCAGCCTGTCACCACGATCACCAGGTCGCCCACTCTCAGGAAGCCCCGCAGCTTGCCGCTCTCGATGCCGAACTGCACCCGTCTGTCCACGTCGTCGGCCCAGATGGCCTCGGGAGGCTCCCGGTACAGCAGGGGGAACACGCCTCTGCACAGGTGGACCTGTCTAGCGGCCTGGGCGGATCTTGTCACGGCGATCACGGCGGCTCTGGGTCTGTATCTGGACAGCAGCTGGGCGCTTCTGCCTGTGGTGGTCAGCACGATGATTGCAGCGGCGCAGCACTTGAAGGCGGCTTCCACGGCTCCGATGGCGGTCACTTCGGTGGGATCTCTGCTCAGAGGGGCGGCTCTCCGCAGTTCCTCGAACAGCTGCCGGTGGTACACGGCGGCCTCGGCTTCTCTGGCAATGGCGTGCTGCATCTTCACGGCCTCCACGGGGAAGTTGCCCTTGGCTGTCTCGCCGGACAGCATGATGCAGTCAGCGCCATCCAGCACGGCGTTGGCCACGTCGCTTGTCTCGGCTCTGGTGGGTCTGGGCTTGGTGATCATGCTTTCCAGCATCTGGGTGGCGCACACCACAGGTTTGCCGGCCAGGTTGCACCGTCCGATCATCATTTTCTGGGCCAGGAACACCTTCTCGGCGGGGATCTCGATGCCCAGGTCGCCTCTGGCCACCATGATGCCGTCGGACACTTCCAGGATCTCGTCGAACCGCTTCACGCCCTCGTGGTTCTCGATCTTGCTGATGATCTTGATGCCGTGGCCTTCAGGGCCCAGAGCGGCTCTCACGGCGGCCACATCAGAGGCCTTCCGCACGAAGCTGGCGAACACGATGTCCACGCCGTGCTCCACGCCAAATCTCAGGTCTCTCACGTCCTGCTCAGACAGGCCAGGCAGGTCCACCTGGGCGCCTGGCAGATTCACGCCCTTTCTGCTGCCCAGCACGCCGCCATTCTCGACCTGGGTCACCAGGCCCTCAGGTCCGATCTTCTGCACCACCAGGCTGATCAGGCCGTCGTCGATGTAGATTCTGCCGCCCACAGGCACCACCCGCACGATGTTGGGGTAGTCCACCCACACTGTGTTGGCGTTGCCTCTGGTTCTGAAGGCGGGGTCCACGGTCACCAGCACTTGGCTGCCCTTCACCAGCTCCACCTCGCTCTCAGGCCCTCCCTGCAGAATTCCTGTTCTGATCTCGGGGCCCTTGGTGTCCAGGGCAATGGCCACGGGTCTGTAGCTCAGGGGGCTGCCGGCAAAGCTCTCCACGGCCTCCCGGACGTTGGCAATGCTCTCTGCGTGGTACTCGTGGCTGCCGTGGGAGAAGTTCAGCCGGGCGATATTCATGCCGGCCTTGATCATCTCTTTCAGCCGCTCCACGCTTCTGCTGGCAGGGCCGATGGTGGCAATGATGCTGGTGCTTCTGGCGGCCACGGGCTCAGAGTCGATGTCCAGCAGGCACAGGTGTTCCAGAAAGGTGTCGGCCATGGCGGCTGGCAGCTGCTGCTGCTGGAAAAAGGCGGTGCCCAGTTCCTGGGTCAGCTGGGCCACGCTAGCCCTTCTCAGGTATCCGGCTGGTCCGCCAGGGGCTCCGATCAGGATGCTCTTGGCCAGGTCTCTCTGGCTCTTGGACACCCAGGACCGCAGCTGCAGAGAGCTGATATTTTCCTGGATGCT

[0107] 15. coRPK AAV for clinical use (SEQ ID NO: 27): A sequence containing a coRPK therapeutic donor (SEQ ID NO: 22) inserted into the AAV skeleton (SEQ ID NO: 23). CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCTG TCACCACTGTCTCCTGTTCCATTGGAAGCCCTGTATGCCAGGGGCCAGAGTCCAGGAACCACGGGAGTGCCCCGTGGCTTACATGCTGTGGCTCTGGCCTGCCTATAGGGCCTGGAAAAGACCCAGGCCAGGGTCCATAAT TTAACACACGGGAGGCTCTGAAGAACGTACGTTCCTCTCCAAAACCCACCTAGCCAGTGGCTGATGTGGATCATTTATGCCCTCCACCCTGGCTCCTAGTTTTCACCCTCATTTTCCTCCTATGTTCCATGGCTTCTGTCT CCCCTTCTTACCTCCTGGAGCCCCAATCAGGATGGACTTTGCTAAGTCTCTTTGGGACTTAGAGACCCATGACCGAAGCTGCAGGGATGATATGTTCTCCTGGATCGACATGCTTTCAGTGTGGGCCTGGGGCTGCGGGAC CCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGCAACTAGAAGGCACAG TGCTTTATTTGTAACTCACGCGTTCA GCTGATGGACAGCACCCTCATGATGTTGGTGTAGCCGCTGCCAGGCCGCCAGCCTGTCACCACGATCACCAGGTCGCCCACTCTCAGGAAGCCCCGCAGCTTGCCGCTCTCGATGCCGAACTGCACCCGTCTGTCCACGTCGTCGGCCCAGATGGCCTCGGGAGGCTCCCGGTACAGCAGGGGGAACACGCCTCTGCACAGGTGGACCTGTCTAGCGGCCTGGGCGGATCTTGTCACGGCGATCACGGCGGCTCTGGGTCTGTATCTGGACAGCAGCTGGGCGCTTCTGCCTGTGGTGGTCAGCACGATGATTGCAGCGGCGCAGCACTTGAAGGCGGCTTCCACGGCTCCGATGGCGGTCACTTCGGTGGGATCTCTGCTCAGAGGGGCGGCTCTCCGCAGTTCCTCGAACAGCTGCCGGTGGTACACGGCGGCCTCGGCTTCTCTGGCAATGGCGTGCTGCATCTTCACGGCCTCCACGGGGAAGTTGCCCTTGGCTGTCTCGCCGGACAGCATGATGCAGTCAGCGCCATCCAGCACGGCGTTGGCCACGTCGCTTGTCTCGGCTCTGGTGGGTCTGGGCTTGGTGATCATGCTTTCCAGCATCTGGGTGGCGCACACCACAGGTTTGCCGGCCAGGTTGCACCGTCCGATCATCATTTTCTGGGCCAGGAACACCTTCTCGGCGGGGATCTCGATGCCCAGGTCGCCTCTGGCCACCATGATGCCGTCGGACACTTCCAGGATCTCGTCGAACCGCTTCACGCCCTCGTGGTTCTCGATCTTGCTGATGATCTTGATGCCGTGGCCTTCAGGGCCCAGAGCGGCTCTCACGGCGGCCACATCAGAGGCCTTCCGCACGAAGCTGGCGAACACGATGTCCACGCCGTGCTCCACGCCAAATCTCAGGTCTCTCACGTCCTGCTCAGACAGGCCAGGCAGGTCCACCTGGGCGCCTGGCAGATTCACGCCCTTTCTGCTGCCCAGCACGCCGCCATTCTCGACCTGGGTCACCAGGCCCTCAGGTCCGATCTTCTGCACCACCAGGCTGATCAGGCCGTCGTCGATGTAGATTCTGCCGCCCACAGGCACCACCCGCACGATGTTGGGGTAGTCCACCCACACTGTGTTGGCGTTGCCTCTGGTTCTGAAGGCGGGGTCCACGGTCACCAGCACTTGGCTGCCCTTCACCAGCTCCACCTCGCTCTCAGGCCCTCCCTGCAGAATTCCTGTTCTGATCTCGGGGCCCTTGGTGTCCAGGGCAATGGCCACGGGTCTGTAGCTCAGGGGGCTGCCGGCAAAGCTCTCCACGGCCTCCCGGACGTTGGCAATGCTCTCTGCGTGGTACTCGTGGCTGCCGTGGGAGAAGTTCAGCCGGGCGATATTCATGCCGGCCTTGATCATCTCTTTCAGCCGCTCCACGCTTCTGCTGGCAGGGCCGATGGTGGCAATGATGCTGGTGCTTCTGGCGGCCACGGGCTCAGAGTCGATGTCCAGCAGGCACAGGTGTTCCAGAAAGGTGTCGGCCATGGCGGCTGGCAGCTGCTGCTGCTGGAAAAAGGCGGTGCCCAGTTCCTGGGTCAGCTGGGCCACGCTAGCCCTTCTCAGGTATCCGGCTGGTCCGCCAGGGGCTCCGATCAGGATGCTCTTGGCCAGGTCTCTCTGGCTCTTGGACACCCAGGACCGCAGCTGCAGAGAGCTGATATTTTCCTGGATGCT

[0108] Example 3. PKLR correction in PKD-HSPC To evaluate the therapeutic potential of the inventors' gene editing system, human HSPC and HD-CD34 derived from four PKD patients with mutations in the PKLR gene were used. + Cells were pre-stimulated for 48 hours. Then, nuclear translocation and transduction were performed on the cells using rAAV. 24 hours after the gene editing procedure, the cells were collected and transferred to erythrocyte differentiation medium. Throughout all experiments, the erythrocyte differentiation process was evaluated by FACS, and no difference in maturation profiles was observed between healthy samples and gene-edited donor samples. On day 14, cells were collected and genomic and functional analyses were performed. First, in a sample from one patient (PKD2), vector insertion was evaluated by specific PCR of the 3' and 5' junctions. As observed in Figure 15C, specific bands were detected in the patient's sample. Furthermore, functional analysis was performed based on the quantification of erythrocyte ATP. Unedited PKD cells or PKD cells edited with GFP-AAV produced low levels of ATP. However, erythrocytes derived from PKD-HSPCs gene-edited with RNP and coRPK-AAV were able to restore ATP levels close to those of HD cells (Figure 15D). In summary, our data demonstrates that gene editing of the PKLR gene can restore the in vitro function of patients' red blood cells.

Claims

1. An isolated crRNA that targets the PKLR gene and has a sequence containing Sequence ID No.

11.

2. The isolated crRNA according to claim 1, wherein the sequence consists solely of sequence number 11.

3. The isolated crRNA according to claim 1 or 2, wherein the sequence is chemically bound to a tracrRNA nucleotide sequence that interacts with a CRISPR-related protein (Cas) polypeptide.

4. A single guide sgRNA targeting the PKLR gene, comprising the crRNA sequence and tracrRNA sequence described in claim 3.

5. The sgRNA is the sgRNA according to claim 4, wherein the sgRNA includes sequence number 12.

6. A ribonucleoprotein (RNP) complex targeting the PKLR gene, comprising isolated crRNA according to claim 1 or 2 or sgRNA according to claim 5, and a CRISPR-related protein (Cas) polypeptide.

7. A CRISPR system targeting the PKLR gene, comprising mRNA encoding a Cas polypeptide and the sgRNA described in claim 4 or 5.

8. The RNP complex according to claim 6 or the CRISPR system according to claim 7, wherein the Cas polypeptide is a Cas9 polypeptide.

9. The RNP complex according to claim 6 or the CRISPR system according to claim 7, wherein the Cas polypeptide is a high-fidelity or specificity-enhanced Cas9 polypeptide variant.

10. A system comprising the RNP complex described in claim 6 or the CRISPR system described in claim 7, and adeno-associated virus particles or homologous donor AAV vectors capable of delivering recombinant donor templates for CRISPR system gene editing via homology-directed repair in target cells, e.g., primary cells.

11. The system according to claim 10, wherein the adeno-associated virus particle or homologous donor AAV vector comprises a DNA sequence comprising homologous arms LHA and RHA, a corrected donor template encoding the PKLR gene, and a dedicated termination sequence for protein expression in eukaryotic cells, such as a bGH poly(A) sequence.

12. The system according to claim 10, wherein the LHA is sequence number 13, the RHA is sequence number 14, the corrected donor template encoding the PKLR gene is sequence number 16, and the bGH poly(A) sequence is sequence number 18, or a variant of any of these sequences having at least 95% sequence identity with any of these sequences.

13. The system according to claim 11 or 12, wherein the adeno-associated virus particle or homologous donor AAV vector further comprises a 5' UTR sequence.

14. A method for introducing a stable gene modification into primary cells via homologous recombination, wherein the primary cells include a target nucleic acid containing a PKLR gene with one or more mutations in the PKLR gene, and a nucleotide sequence complementary to SEQ ID NO: 1 or 21, and the method is as follows: a. Introducing the RNP complex described in claim 6 or the CRISPR system described in claim 7 into the primary cells, and simultaneously or sequentially thereafter, b. Introducing adeno-associated virus particles or homologous donor AAV as defined in any one of claims 10 to 13 into the primary cells, Includes, A method for stable gene modification of the target nucleic acid, comprising compensating for a pathogenic mutation in the PKLR gene (target nucleic acid) by introducing the homologous donor AAV vector containing the corrected donor template.

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