Gene editing for the treatment of epidermolysis bullosa

Abstract

The present invention relates to the treatment of epidermolysis bullosa, particularly the recessive dystrophic subtype (RDEB), using the clustered regularly interspaced short palindromic repeats (CRISPR) system. This technology offers the possibility of designing single guide RNAs (sgRNAs) integrated with 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 donor sequences for epidermolysis bullosa gene repair. In the context of epidermolysis bullosa, this allows for the repair of the disease-causing mutation(s).

Description

[Technical Field]

[0001] The present invention relates to the treatment of epidermolysis bullosa, particularly the recessive dystrophic subtype (RDEB), using the clustered regularly interspaced short palindromic repeats (CRISPR) system. This technology offers the possibility of designing single guide RNAs (sgRNAs) integrated with 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 donor sequences for epidermolysis bullosa gene repair. In the context of epidermolysis bullosa, this allows for the repair of the disease-causing mutation(s). [Background technology]

[0002] Epidermolysis bullosa is a group of rare genetic disorders characterized by severe skin fragility. The recessive dystrophic subtype, RDEB, is the most severe phenotype of the disease, causing skin and mucous blistering and a high risk of pseudosyndactyly and metastatic squamous cell carcinoma. Mutations along the COL7A1 gene, which expresses collagen VII (C7), are present in a high proportion of these patients, establishing this gene as a target for precision medicine therapy in RDEB.

[0003] Over the past few years, various site-specific nucleases capable of generating double-strand breaks in DNA, such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas9, have been explored as tools for genome editing. Genome editing-based approaches exploit the cells' natural DNA repair mechanisms triggered by nuclease-induced double-strand breaks (ds-breaks) to either introduce indels into gene sequences (NHEJ) or precisely correct them with a donor template (HDR). Although the NHEJ repair pathway is more frequent than HR, recent tool developments have improved the efficiency of this donor-based correction in different cell types.

[0004] Keratinocytes and fibroblasts have been primarily highlighted as cellular targets for gene therapy correction of EB. In 2013, Non-Patent Document 1 demonstrated 2% HDR correction in RDEB patient-derived fibroblasts using TALENs and oligonucleotide donors (ODNs). Subsequently, Non-Patent Document 2 developed HDR-based correction using meganucleases and achieved 4% COL7A1 correction. Non-Patent Document 3 also demonstrated genetic and functional correction in patient-derived RDEB keratinocyte cell lines using minicircle-based CRISPR / Cas9 for HDR. Recently, Non-Patent Document 4 demonstrated precise correction of exon 2-containing mutant RDEB primary cells, achieving a nearly 30% indel generation frequency in RDEB keratinocytes and fibroblasts with integration-deficient lentivirus-guided delivery. After donor template delivery, this achieved 11% and 15.7% corrected COL7A1 mRNA expression in keratinocytes and fibroblasts, respectively, without antibiotic selection.In another type of EB, Benaty et al. used HR to restore LAMB3 expression in situ in junctional epidermolysis bullosa (JEB)-immortalized keratinocytes using an adenovector carrying a Cas9 / guide RNA (gRNA) aligned to intron 2 of the LAMB3 gene and an integrase-deficient lentiviral vector carrying a promoterless, nearly complete LAMB3 cDNA flanked by homology arms.

[0005] In this field, we have recently achieved high indel efficiency (nearly 95%) in primary RDEB keratinocytes using the CRISPR / Cas9 system as a ribonucleoprotein complex delivered by electroporation. Meanwhile, adeno-associated virus (AAV) has emerged as a leading vector for donor template delivery, offering higher efficiency than IDLV and increasing the HDR ratio without compromising biosafety. Therefore, the combination of AAV and CRISPR / Cas9 may be an interesting option as a genome editing tool for exploiting HDR in primary keratinocytes and, ultimately, as a gene correction strategy for RDEB primary cells.

[0006] Although an efficient NHEJ-based approach was recently tested by our group for exon 80-containing mutant RDEB patients, HR correction can cover a large number of exons within the designed donor length, providing one therapeutic system for correcting different mutant exons in COL7A1 and allowing a large cohort of RDEB patients to benefit.

[0007] Beyond the main cell types of the skin, allogeneic bone marrow transplantation (BMT) has been considered as an alternative treatment for EB in recent years (Non-Patent Document 5, Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8). Indeed, Non-Patent Document 8 demonstrated this year the benefit of subsequent infusion of systemic MSCs in RDEB patients after BMT, with acceptable safety for the recipients. Some of these patients showed increased primitive anchor fibrils (AF) and increased C7 immunostaining. Given the potential of BMT in the treatment of RDEB, the present inventors investigated the effects of CB-isolated CD34 cells from three healthy donors. + This study also included proof of principle of HDR-based gene editing with the same CRISPR / Cas9 system in cells and primary MSCs, confirming the potential of our approach as a platform for gene correction and cell therapy. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Osborn et al. [Non-patent document 2] Izmyrian (2016) and coworkers [Non-patent document 3] Hainzl et al. [Non-patent document 4] Izmiryan et al. [Non-patent document 5] Fujita 2010 [Non-patent document 6] Petrof et al., 2015 [Non-Patent Document 7] Kaneda et al. 2015 [Non-patent document 8] C. L. Ebens et al. 2019 Summary of the Invention

[0009] Thus, the present invention is evidence of an efficient marker-free HR-based strategy ex vivo for gene correction of different relevant cell types for RDEB treatment. [Brief explanation of the drawings]

[0010] [Figure 1] A) Schematic of the HDR-based strategy for precise COL7A1 correction. AAV6-delivered donor template combined with CRISPR / Cas9 as a gene editing strategy for RDEB. CRISPR / Cas9-induced double-strand breaks near the pathogenic mutation are used to trigger HDR repair. B) TIDE analysis of indel generation within intron 79 of COL7A1. This analysis revealed highly efficient indel generation (close to 90%) in primary RDEB keratinocytes. [Figure 2]A) AAV serotype testing in primary keratinocytes. We evaluated eight different serotypes of AAV, with AAV6 showing the highest transduction efficiency (39.7%). B) HDR-based correction genotyping in primary RDEB keratinocytes. This analysis revealed accurate gene correction efficiencies approaching 40% when two different donor templates (symmetric and asymmetric arms) were tested. [Figure 3] Figure 1 shows collagen VII expression in a gene-corrected RDEB polyclonal keratinocyte population. RDEB primary keratinocytes were treated with CRISPR / Cas9 and AAV6-containing donor template, and C7 expression restoration was assessed by immunofluorescence and Western blot from cell extracts. A) C7 immunofluorescence analysis. Top left panel: Positive control, healthy donor keratinocytes. Top right panel: Untreated RDEB P1 keratinocytes showing null C7 expression. Bottom left panel: AAV6 symmetric donor + RNP-treated RDEB keratinocytes. Bottom right panel: AAV6 asymmetric donor template + RNP-treated RDEB keratinocytes. Bar: 50 μm. B) Western blot analysis of C7 restoration in untreated, healthy, and treated patient RDEB cells shows good C7 expression consistent with the immunofluorescence images. [Figure 4] Figure 4 shows restoration of epidermal-dermal adhesion and C7 expression in HDR-corrected RDEB grafts. Skin equivalents containing bulk-edited RDEB keratinocyte populations, untreated RDEB keratinocytes, and healthy keratinocytes were grafted onto nude mice. Histological analysis (H&E staining) of the grafts (Figures 4A, 4D, and 4G) shows epidermal detachment in the P1 graft. C7 expression analysis shows continuous C7 deposition in the BMZ in HDR-corrected (P1 HDR) and healthy donor (HD) keratinocytes, and no C7 detection in grafts from untreated RDEB keratinocytes (P1) (Figures 4B, 4E, and 4H). Human involucrin (h-Inv) assessment demonstrates normal epidermal differentiation in all grafts shown (Figures 4C, 4F, and 4I). [Figure 5]A) PCR genotyping of P2 RDEB-treated cells using AAV6-containing RNP+ donor template. Similar rates of gene correction were observed between the two RDEB-treated patients. B) Immunofluorescence for C7 expression detection. P2 was null for C7 expression. After treatment, C7 expression is restored in a significant amount of cells. [Figure 6] A) HDR-based gene editing in CD34+ cells from three healthy donors. B) HDR-based gene editing in MSCs in three healthy donors. DETAILED DESCRIPTION OF THE INVENTION

[0011] definition As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0012] As used herein, "a," "an," or "the" includes not only embodiments having one member, but also embodiments having two or more members. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, reference to "the agent" includes reference to one or more agents known to those of skill in the art, and so forth.

[0013] The term "gene" refers to a combination of polynucleotide elements that, when operably linked, either naturally or recombinantly, provide a product or function. The term "gene" should be interpreted broadly and can encompass mRNA, cDNA, cRNA, and genomic DNA forms of a gene.

[0014] The term "homology-directed repair" or "HDR" refers to the mechanism within cells that correctly and precisely repairs double-stranded DNA breaks using a homologous template to guide the repair. The mechanism underlying HDR is homologous recombination (HR).

[0015] 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 correctly repair harmful breaks that occur in both strands of DNA (known as double-strand breaks or other breaks that create overhanging sequences).

[0016] The term "single guide RNA" or "sgRNA" refers to a DNA-targeting RNA that includes 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.

[0017] The term "Cas polypeptide" or "Cas nuclease" refers to a clustered regularly interspaced short palindromic repeat-associated polypeptide or nuclease that cleaves DNA at a site specified by a 20-nucleotide guide sequence contained within the crRNA molecule, generating a double-stranded break and blunt ends. Cas nucleases require both the crRNA and tracrRNA for site-specific DNA recognition and cleavage. The crRNA associates with the tracrRNA through a region of partial complementarity and guides the Cas nuclease to a region of homology to the crRNA in the target DNA, called the "protospacer."

[0018] The term "ribonucleoprotein complex" or "RNP complex" refers to a complex comprising an sgRNA and a Cas polypeptide.

[0019] The terms "adeno-associated viral vector-delivered donor template" or "donor template-containing adeno-associated viral vector" refer to an adeno-associated viral particle capable of delivering a recombinant donor template for CRISPR-based gene editing via homology-directed repair in target cells, e.g., primary cells.

[0020] The term "recombinant donor template" refers to a nucleic acid strand, e.g., a DNA strand that is the donor strand, during homologous recombination strand invasion initiated by damaged DNA repair mechanisms, sometimes resulting from a double-strand break. The donor polynucleotide serves as template material to direct the repair of the damaged DNA region. In the present invention, we preferably design and construct a DNA donor fragment or recombinant donor template that lacks one or more introns, particularly introns containing guide RNA target sequences. Specifically, the donor template does not contain an intron region of the targeting nucleic acid that contains a Cas-recognition protospacer adjacent motif (PAM) sequence. By using this donor DNA design, we avoid the possibility of gene splicing or alteration of the coding sequence that may result from undesired nuclease activity. Indel generation near an exon of the COL7A1 gene can result in a change in the gene's open reading frame, resulting in a mutation in the amino acid sequence of type VII collagen, potentially resulting in a non-functional protein variant. Furthermore, the absence of intron sequences is easily detected by PCR, facilitating genotyping of recombinant alleles. Preferably, the recombinant donor template is a fusion of exon 79 and exon 80 lacking intron 79, where the guide RNA includes its target sequence (sg2 cleaves). Even more preferably, the recombinant donor template is a fusion of exon 79 and exon 80 lacking intron 79 such that it does not have a PAM sequence to avoid an NHEJ event after the HDR repair event.

[0021] The term "sequence identity" or "percent identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are the same ("identical") or have a specified percentage of amino acid residues or nucleotides that are identical when compared and aligned for maximum correspondence with a second molecule ("percent identity"), as determined by a sequence comparison algorithm (e.g., by BLAST alignment, or any other algorithm known to one of skill in the art), or alternatively, by visual inspection.

[0022] The term "homologous" refers to two or more amino acid sequences when they are derived, naturally or artificially, from a common ancestral protein or amino acid sequence. Similarly, nucleotide sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid.

[0023] The term "primary cells" refers to cells isolated directly from a multicellular organism. Primary cells typically have undergone few population doublings and therefore are more representative of the primary functional components of the tissue from which they are derived than continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells are cells that are isolated and used immediately thereafter. In other cases, primary cells cannot divide indefinitely and therefore cannot be cultured in vitro for long periods of time.

[0024] The terms "genetically modified primary cell" or "genome-edited primary cell" refer to a primary cell into which heterologous nucleic acid has been introduced, as the case may be, into its endogenous genomic DNA.

[0025] The term "pharmaceutical composition" refers to a physiologically and pharmacologically acceptable composition. In some instances, the composition may include agents for buffering and preservation during storage, and may include buffers and carriers for delivery appropriate for the route of administration.

[0026] The term "pharmaceutically acceptable carrier" refers to a substance that aids in the administration of an agent (e.g., a Cas nuclease, modified single guide RNA, genetically modified primary cells, etc.) to a cell, organism, or subject. A "pharmaceutically acceptable carrier" refers to a carrier or excipient that can be included in a composition or formulation and that does not cause significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, saline, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavors, and colorants, etc. One of skill in the art will recognize that other pharmaceutical carriers are useful in the present invention.

[0027] The term "administer" or "administration" refers to the process by which an agent, composition, dosage form, and / or combination disclosed herein is delivered to a subject for therapeutic or prophylactic purposes. The compositions, dosage forms, and / or combinations disclosed herein are administered in accordance with good medical practice, taking into consideration the subject's clinical condition, the site and method of administration, the dosage, the subject's age, sex, weight, and other factors known to a physician. For example, the term "administer," or "administration" includes providing, giving, dispensing, and / or prescribing an agent, composition, dosage form, and / or combination disclosed herein by a clinician or other clinical professional.

[0028] The term "treating" refers to an approach to obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms being treated. In the case of prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject who experiences one or more physiological symptoms of the disease, even if the disease, condition, or symptom has not yet manifested.

[0029] The terms "subject," "patient," and "individual" are used interchangeably herein to include a human or an animal. For example, an animal subject may be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a horse, cow, sheep, pig, or goat), a companion animal (e.g., a dog, cat), a laboratory animal (e.g., a mouse, rat, guinea pig, bird), an animal of veterinary importance, or an animal of economic importance.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. Although exemplary methods, devices, and materials are described herein, any methods and materials similar or equivalent to those explicitly described herein can be used in the practice or testing of the technology. For example, the reagents described herein are merely exemplary; such equivalents are known in the art. The practice of the technology may employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of those of ordinary skill 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 See Mammalian Cells (Cold Spring Harbor Laboratory).

[0031] Detailed Description of the Invention Genetically modified stem cells offer a new field of therapeutic solutions for untreatable diseases. + Gene-corrected cells have shown great benefit in treating severe blood disorders. Furthermore, the latest gene modification techniques have reached the clinical trial stage (CRISPR), revolutionizing modern medicine. MSC therapy has also shown clinical benefits in wound healing and the treatment of immune disorders, providing a safe approach for regenerative medicine.

[0032] In skin disorders, keratinocytes and fibroblasts are the cellular source of these therapies, and numerous approaches have been developed with the aim of paving the way for clinical application. Epidermolysis bullosa is one of the most devastating rare skin diseases, and the RDEB- subtype, in which C7 expression is completely absent, is considered the most severe subtype. Numerous mutations in these patients have been described within the COL7A1 gene, making this gene a prime target for gene therapy strategies to correct RDEB. Recently, a phase I ex vivo clinical trial demonstrated benefit in patients receiving skin equivalents containing autologous epidermal stems treated with a gammaretrovirus expressing the cDNA C7 sequence, a classic gene therapy approach. Patients demonstrated improved wound healing and C7 deposition and anchor fibril formation.

[0033] However, new gene editing tools are paving the way for more precise gene correction therapy. Indeed, we previously demonstrated a highly efficient gene editing-based approach for E80 correction in RDEB patient cells. This study revealed that CRISPR / Cas9, as an RNP, is the most efficient tool for genome editing in primary keratinocytes, a cell type that is considered difficult to transfect. In this study, we tested a large collection of AAV serotypes for the first time to find the highest possible transduction efficiency and found that the AAV6 serotype provided the best performance after electroporation. Thus, we demonstrated that donor template + RNP containing AAV6 delivered by electroporation is an efficient tool for genome editing in keratinocytes.

[0034] When different homologous donor designs were tested, we found similar HR-based correction ratios in primary keratinocytes, regardless of the degree of symmetry in the homologous arms relative to the cleavage site. In our study, the symmetric donor template covered E74 to E84, while the asymmetric donor template covered E77 to E88. Therefore, each design could cover patients with mutations within 10 different exons of COL7A1. Furthermore, we developed a large collection of AAVs containing different gene regions of COL7A1, allowing us to correct any mutation along the gene. This is an important advantage over previously proposed NHEJ, as there are several exons that are not amenable to exon removal, which can be corrected by precise HR-mediated gene correction, such as exons 1, 2, 3, 24, 27, or 113. Beyond EB treatment, this approach can be transferred to any genome editing application in primary keratinocytes to treat mutations from other skin diseases, or even knock-in genes (reporters, therapeutic molecules) to create skin with novel functional properties.

[0035] Previous studies have demonstrated HR-based gene correction in various cell types relevant to RDEB treatment. Indeed, previous studies have demonstrated the feasibility of achieving HR correction in patient-derived keratinocyte cell lines using AAVs containing TALENs and selection cassettes. After selection treatment of treated keratinocytes, 32 of 34 clones isolated were genetically corrected. However, although we demonstrated the feasibility of C7 recovery by HR in RDEB keratinocyte cell lines, drug selection and clonal isolation make the translation of this type of therapy into the clinic extremely difficult. However, others have demonstrated an indel generation frequency approaching 30% in RDEB keratinocytes using integrase-deficient lentivirus-guided delivery of exon 2-containing mutations. In combination with donor template delivery, we achieved COL7A1 corrected transcripts in RDEB cells at 11% and 15% in keratinocytes and fibroblasts, respectively, and up to 19% in skin grafts, sufficient to enable AF formation without dermal-epidermal separation.

[0036] [Table 1]

[0037] In this study, we provide a marker-free, highly efficient approach in primary keratinocytes, achieving nearly 40% corrected transcripts in primary keratinocytes, surpassing previous HR-based gene correction ratios. Furthermore, we generated a population of edited bulk keratinocytes that can generate normal human skin regeneration with restoration of dermal-epidermal adhesion when transplanted into nude mice. The use of a polyclonal population facilitates translation to the clinic and avoids the time-consuming and labor-intensive isolation of epidermal clones.

[0038] Based on wild-type (WT) fibroblast injection experiments in a DEB hypomorphic mouse model, 35% normal C7 levels are believed to be required for mechanical stability of the skin (see Georgiadis, C., Syed, F., Petrova, A., Abdul-Wahab, A., Lwin, S.M., Farzaneh, F., Chan, L., Ghani, S., Fleck, R.A., Glover, L., et al. (2016). Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB. J. Invest. Dermatol. 136, 284-292).

[0039] Therefore, in contrast to other methods, the methodology presented herein exceeds the percentage of corrected transcripts in primary keratinocytes required to adequately treat or prevent EB. Overall, our invention provides evidence of an effective ex vivo genome editing tool that can achieve gene correction in many different cell types, providing a foundation for developing various cell therapies aimed at curing EB. This technology will enable coverage of a wider range of EB mutations, paving the way for clinical benefit in large cohorts of EB patients.

[0040] Thus, in a first aspect of the present invention, the inventors herein provide a method for inducing stable genetic modification of a target nucleic acid comprising one or more epidermolysis bullosa, preferably recessive dystrophic epidermolysis bullosa (RDEB), pathogenic mutations in the COL7A1 gene via homologous recombination in primary cells, preferably primary keratinocytes, fibroblasts or skin stem cells. The method includes introducing into a primary cell a homologous donor, preferably wild-type, adeno-associated virus serotype 6 (AAV-6) or serotype 1 (AAV-1), comprising: (a) a modified single guide RNA (sgRNA) comprising a nucleotide sequence complementary to a target nucleic acid and a nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein the RNA components can be two individual RNA molecules (crRNA and tracrRNA) or a single RNA molecule (sgRNA); (b) a recombinant expression vector comprising a Cas polypeptide, an mRNA encoding the Cas polypeptide, and / or a nucleotide sequence encoding the Cas polypeptide, which separately provides the modified sgRNA component or the crRNA component and the tracrRNA component to guide the Cas polypeptide to the target genomic sequence to be corrected; and (c) a recombinant donor template comprising two nucleotide sequences comprising two non-overlapping homologous portions of the target nucleic acid, the nucleotide sequences being located at the 5' and 3' ends of nucleotide sequences corresponding to the target nucleic acid to undergo homologous recombination. The stable genetic modification of the target nucleic acid comprises replacing a pathogenic mutation, preferably a pathogenic mutation with a high heterozygous frequency in the human population, in the COL7A1 gene (target nucleic acid), preferably in any of exon 73, exon 74, exon 75, exon 80, or exon 105 of the COL7A1 gene, by introducing a homologous donor AAV-6 or AAV-1 vector containing a corrected donor template.

[0041] The above-described genetic modification strategy is performed in primary cells, preferably primary keratinocytes, for the purpose of treating subjects with or suffering from epidermolysis bullosa, preferably recessive dystrophic epidermolysis bullosa (RDEB). Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary, genetic, bullous skin disorder caused by mutations in the COL7A1 gene (collagen VII, C7), leading to a lack of C7 function. Type VII collagen (C7) is a large homotrimeric, triple-helical collagen molecule that undergoes antiparallel dimerization at the NC2 end, followed by supramolecular assembly into attachment structures called anchor fibrils that connect the lamina densa of the basement membrane (BMZ) to the papillary dermis. C7 contains a large NC1 domain that binds to laminin-332 in the lamina densa of the basement membrane and a collagenous domain that encases interstitial collagen fibers in the papillary dermis. Therefore, the absence of C7 in RDEB results in blistering between the papillary dermis and the lamina densa of the basement membrane. The human type VII collagen gene, COL7A1, has a complex structure consisting of a total of 118 separate exons. However, the gene is relatively compact, and most introns are relatively small. As a result, the entire human COL7A1 gene is only 32 kb in size, encoding an 8.9 kb messenger RNA. COL7A1 has been mapped to the short arm of human chromosome 3, region 3p21.1. The structure of the type VII collagen gene and the primary sequence encoding the protein are well conserved; for example, the mouse gene shows 84.7% homology at the nucleotide level and 90.4% identity at the protein level.

[0042] Type VII collagen is synthesized by both epidermal keratinocytes and dermal fibroblasts in culture. During synthesis of the complete pro-al(VII) polypeptide, three polypeptides associate via their carboxy termini into a trimeric molecule, which then folds in its collagenous portion to form a triple helix. The triple helical molecule is then secreted into the extracellular environment, where two type VII collagen molecules align into an antiparallel dimer with the amino-terminal domains present at opposite ends of the molecule. This dimeric assembly is followed by proteolytic removal of the carboxy-terminal portions of both type VII collagen molecules and stabilization by intermolecular disulfide bond formation. Subsequently, many of these antiparallel dimers laterally aggregate to form anchored fibrils.

[0043] Glycine substitution mutations in the triple-helical domain of COL7A (particularly exons 73, 74, and 75) predominate in dominant dystrophic epidermolysis bullosa (DDEB). Mutations p.Gly2034Arg and p.Gly2043Arg are the most common DDEB-causing mutations, accounting for 50% of dominant mutations reported in the largest US cohort. Glycine substitutions, as well as other amino acid substitutions and splice junction mutations outside this region, can also be found in dominant DEB. Over 400 recessive DEB-causing mutations spanning the entire gene have been described in all forms of DEB. However, each mutation accounts for less than 1% to 2% of the total number of mutations described. Null mutations predominate in RDEB, but glycine substitutions and other amino acid substitutions have been described. Milder forms of RDEB are often caused by splice junction mutations or other missense mutations.

[0044] Therefore, in a further embodiment, stable genetic modification of a target nucleic acid involves replacing any of the above-mentioned pathogenic mutations in the COL7A1 gene by introducing a homologous donor AAV-6 or AAV-1 vector containing a corrected donor template. As previously mentioned, the term "recombinant donor template" or "donor template" refers to the nucleic acid strand, e.g., the DNA strand that is the donor strand, upon homologous recombination strand invasion initiated by the damaged DNA repair mechanism resulting from a double-strand break. The donor polynucleotide serves as template material to direct the repair of the damaged DNA region. In the present invention, the inventors preferably design and construct a DNA donor fragment or recombinant donor template that lacks one or more introns, particularly introns containing guide RNA target sequences. More specifically, the donor template does not contain an intron region of the target nucleic acid that contains a Cas-recognition protospacer adjacent motif (PAM) sequence. By using this donor DNA design, the inventors avoid the possibility of gene splicing or alterations in the coding sequence that may result from undesired nuclease activity. Indels near exons in the COL7A1 gene can result in changes in the gene's open reading frame, resulting in mutations in the amino acid sequence of type VII collagen and potentially resulting in non-functional protein variants. Furthermore, the absence of intron sequences can be easily detected by PCR, facilitating genotyping of recombinant alleles. Preferably, the recombinant donor template is a fusion of exon 79 and exon 80 that lacks intron 79, where the guide RNA contains its target sequence (cleaved by sg2). Even more preferably, the recombinant donor template is a fusion of exon 79 and exon 80 that lacks intron 79 and does not have a PAM sequence to avoid NHEJ events after HDR repair.

[0045] In some embodiments, the primary cells are selected from the group consisting of primary keratinocytes or fibroblasts, and combinations thereof. In some embodiments, the primary cells are isolated from a mammal before introducing the modified sgRNA, Cas polypeptide, and homologous donor AAV vector into the primary cells. For example, the primary cells can be harvested from a human subject. In some instances, the primary cells or their progeny are returned to the mammal after introducing the modified sgRNA, Cas polypeptide, and homologous donor AAV vector into the primary cells. That is, the genetically modified primary cells undergo autologous transplantation. In other instances, the genetically modified primary cells undergo allogeneic transplantation. For example, primary cells that have not undergone stable genetic modification are isolated from a donor subject, and then the genetically modified primary cells are transplanted into a recipient subject that is different from the donor subject.

[0046] The primary cells can comprise a population of primary cells. In some cases, the population of primary cells comprises a heterogeneous population of primary cells. In other cases, the population of primary cells comprises a homogeneous population of primary cells.

[0047] In some examples, the homologous donor AAV-6 vector has at least about 90% sequence identity to AAV6. In other examples, the homologous donor is wild-type AAV6 or an AAV6 variant having at least 95% sequence identity to wild-type AAV6, for example, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to wild-type AAV6. In some embodiments, polynucleotides encoding one or more of the various components of the AAV-6 vector are operably linked to an inducible promoter, a repressible promoter, or a constitutive promoter. Additionally, regulatory sequences operably linked to the components can include activator-binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor-binding sequences, stem-loop structures, translation initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, etc. Commonly used promoters include constitutive mammalian promoters CMV, EF1a, SV40, PGK1 (mouse or human), Ubc, CAG, CaMKI1a, 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). Additionally, mammalian RNA polymerase III promoters, including HI and U6, can also be used.

[0048] In some embodiments, a recombinant mammalian expression vector can direct expression of a nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express a polynucleotide). 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), pancreatic-specific promoters, mammary gland-specific promoters (e.g., whey promoters), and T-cell receptor and immunoglobulin promoters, among others. Developmentally regulated promoters, such as mouse hox promoters and alpha-fetoprotein promoters, are also encompassed.

[0049] Methods for introducing 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.

[0050] In some embodiments, stable genetic modifications of the target nucleic acid are induced in greater than about 30% of the primary cell population, e.g., about 35%, about 40%, about 50%, about 60%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, 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 about 100% of the primary cell population. In other embodiments, stable genetic modifications of the target nucleic acid are induced in greater than about 80% of the primary cell population, e.g., 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 about 100% of the primary cell population. In yet other embodiments, stable genetic modifications of the target nucleic acid are induced in greater than about 90% of the primary cell population, e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the primary cell population.

[0051] In some embodiments, the sequences of the first aspect of the present invention may include modified nucleotides, such as modifications in the ribose group, the phosphate group, the nucleobase, or a combination thereof. In some instances, the modification in the ribose group includes a modification at the 2' position of the ribose group. In some instances, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, 2'-O-(2-methoxyethyl), and combinations thereof. In other instances, the modification in the phosphate group includes a phosphorothioate modification. In other embodiments, the modified nucleotide is 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.

[0052] In some embodiments, the Cas polypeptide is a Cas9 polypeptide, a variant thereof, or a fragment thereof. In certain instances, the Cas polypeptide variant comprises a high-fidelity or enhanced-specificity Cas9 polypeptide variant. In certain embodiments, the modified sgRNA and Cas polypeptide are introduced into the primary cells simultaneously. In other embodiments, the modified sgRNA and Cas polypeptide are introduced into the 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.

[0053] In some embodiments, the modified sgRNA and Cas polypeptide can be incubated together to form a ribonucleoprotein (RNP) complex before being introduced into primary cells. For example, the modified sgRNA and Cas polypeptide can be mixed together in a container to form an RNP complex, and then the RNP complex can be introduced into primary cells. In other embodiments, the Cas polypeptide described herein can be an mRNA encoding the Cas polypeptide, where the Cas mRNA is introduced into primary cells together with the modified sgRNA as an "All RNA" CRISPR system. In certain instances, the modified sgRNA and Cas mRNA are introduced into primary cells simultaneously. In other instances, the modified sgRNA and Cas mRNA are introduced into primary cells sequentially. In some cases, the modified sgRNA is introduced first, followed by the Cas mRNA. In other cases, the Cas mRNA is introduced first, followed by the modified sgRNA.

[0054] In some embodiments, the RNP complex and the homologous donor AAV-6 or AAV-1 vector are introduced into the primary cells simultaneously. In other embodiments, the RNP complex and the homologous donor AAV-6 vector are introduced into the primary cells sequentially. In some examples, the RNP complex is introduced into the primary cells before the homologous donor AAV vector. In other examples, the homologous donor AAV vector is introduced into the primary cells before the RNP complex. For example, the RNP complex can be introduced into the primary cells about 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, or vice versa. In certain embodiments, the RNP complex is introduced into the primary cells about 15 minutes (e.g., about 10 to about 20 minutes) before the homologous donor AAV-6 vector.

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

[0056] In some embodiments, any of the methods described herein can also include purifying primary cells having stable genetic modifications of the target nucleic acid using a marker. In some cases, the composition isolated by the purification step comprises at least about 80% of the primary cells having stable genetic modifications of the target nucleic acid, e.g., 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 of the primary cells having stable genetic modifications of the target nucleic acid.

[0057] In some embodiments, introducing the modified sgRNA and Cas polypeptide into the primary cells comprises electroporating the modified sgRNA and Cas polypeptide into the primary cells. In some embodiments, introducing the homologous donor AAV-6 or AAV-1 vector into the primary cells comprises transducing the primary cells.

[0058] In another aspect, 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 primary keratinocytes or fibroblasts, and combinations thereof.

[0059] In yet another aspect, provided herein is a pharmaceutical composition comprising any of the genetically modified primary cells described herein and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition comprises one type of genetically modified primary cell. In other embodiments, the pharmaceutical composition comprises two or more different types of genetically modified primary cells, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different types of genetically modified primary cells.

[0060] In a further aspect, provided herein is a method for inducing stable genetic modification of a target nucleic acid comprising one or more epidermolysis bullosa, preferably recessive dystrophic epidermolysis bullosa (RDEB), pathogenic mutations in the COL7A1 gene via homologous recombination in primary cells, preferably primary keratinocytes or fibroblasts obtained from a subject, the method comprising: (a) a single guide RNA (sgRNA) comprising a first nucleotide sequence complementary to the target nucleic acid and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide; (b) the sgRNA (c) a recombinant expression vector comprising a Cas polypeptide, an mRNA encoding the Cas polypeptide, and / or a nucleotide sequence encoding the Cas polypeptide, which directs the Cas polypeptide to a target nucleic acid; (d) a homologous donor, adeno-associated virus (AAV6) or AAV-1 vector, which comprises a recombinant donor template comprising two nucleotide sequences comprising two non-overlapping homologous portions of a target nucleic acid, the nucleotide sequences being located at the 5' and 3' ends of a nucleotide sequence corresponding to the target nucleic acid to undergo homologous recombination; and, optionally, instructions for use in vitro.

[0061] In some examples, the kit also includes reagents for harvesting or isolating primary cells from a subject. The subject can be a mammalian subject, for example, a human subject.

[0062] In still a further aspect, provided herein is a method of preventing or treating epidermolysis bullosa, preferably recessive dystrophic epidermolysis bullosa (RDEB), in a subject in need thereof, the method comprising administering to the subject any of the genetically modified primary cells described herein or any of the pharmaceutical compositions described herein to prevent or ameliorate one or more symptoms of the disease.

[0063] In some embodiments, the administering step comprises a delivery route selected from the group consisting of intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, intrathecal, intraosseous, or a combination thereof.

[0064] In certain embodiments, the genetically modified primary cells or pharmaceutical compositions of the invention are administered to a subject in an amount sufficient to correct a mutation in a target nucleic acid associated with a disease, hi some instances, the mutation is corrected by replacing the mutant allele in the target nucleic acid with a wild-type allele.

[0065] In further embodiments of the invention, the genetically modified primary cells or pharmaceutical compositions of the invention are used in an in vitro, preferably in a method for producing a skin equivalent or artificial skin. A still further embodiment of the invention thus relates to a skin equivalent obtainable or obtained according to the aforementioned in vitro use. A still further embodiment relates to the use of such a skin equivalent obtainable or obtained according to the aforementioned in vitro use or method in a method for treating epidermolysis bullosa, in particular the recessive dystrophic subtype (RDEB), in a subject in need thereof.

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

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

[0068] The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. [Example]

[0069] Materials and Methods Keratinocyte cell culture and clonal isolation Patient keratinocytes were originally obtained from skin biopsies of three RDEB patients with mutations in the COL7A1 gene (RDEB-sev gen). Skin biopsies were obtained from the patients with informed consent and with approval from the ethics committee of the affiliated hospital.

[0070] Primary human RDEB and healthy donor keratinocytes were cultured. Human primary RDEB keratinocytes from three patients were seeded onto lethally irradiated 3T3-J2 cells and supplemented with fetal bovine serum (Hyclone, GE Healthcare, Logan, UT) (10%), penicillin-streptomycin (1%), glutamine (2%), insulin (5 μg / ml; Sigma-Aldrich), adenine (0.18 mmol / l; Sigma-Aldrich), hydrocortisone (0.4 μg / ml; Sigma-Aldrich), cholera toxin (0.1 nmol / l; Sigma-Aldrich), triiodothyronine (2 nmol / l; Sigma-Aldrich), EGF (10 ng / ml; Sigma-Aldrich), and 10 μM Y-27632 ROCK inhibitor (Sigma-Aldrich). The cells were cultured in keratinocyte growth cFAD medium (KCa), a 3:1 mix of Dulbecco's modified Eagle's medium containing 10% FAAD (Aldrich) and Ham's F12 medium (GIBCO-BRL, Barcelona, ​​Spain). To obtain isolated clones, the cells were trypsinized and plated at 2 × 10 cells / well. 6 100 mm plates containing lethally irradiated 3T3 feeder cells at low density (10 3 Cell clones were then harvested using polystyrene cloning cylinders (Sigma, St. Louis, MO) and expanded for further analysis.

[0071] Production of AAV6-containing donor templates Homology arms were amplified by PCR from wild-type genomic DNA. The symmetric donor is a fusion of exon 79 and exon 80, lacking intron 79 (sg2 truncation). Thus, the left homology arm (LHA) is 1008 bp 5' from the end of exon 79 of the COL7A1 gene, and the right homology arm (RHA) is 798 bp 3' from the start of exon 80 of the same gene. The asymmetric donor followed the same strategy, but the LHA is 556 bp and the RHA is 1461 bp. Both arms were then assembled using the AAV backbone plasmid by Gibson assembly technology.

[0072] For production of AAV6-containing donor templates, the backbone vector plasmid was propagated in E. coli and isolated using an Endotoxin-Free Maxi Plasmid Purification Kit (Invitrogen, Cat. No. A33073). Five 15 cm plates were then purified using 120 μL of 1 mg / mL PEI (molecular weight 25K) (Polysciences) per plate, mixed with 6 mg of ITR-containing plasmid and 22 mg of pDGM6 (harboring AAV6 cap, AAV2 rep, and adenovirus helper genes). 2 293 cells were transfected in a dish (gift from D. Russell). 72 hours after transfection, the vector was purified using the Takara Bio AAVpro Purification Kit (catalog no. 6666) according to the manufacturer's protocol. Vector titer was assessed by ddPCR using a probe on the ITR region.

[0073] CRISPR / Cas9 delivery and AAV6 transduction The sg2 gRNA was previously described (Bonafont et al.). In this approach, instead of the crRNA:tracrRNA system, sg2 is a single guide RNA that has been chemically modified (Synthego, CA, USA). 1.6 μg of sgRNA mixed with 6 μg of Cas9 protein was used for 1 × 10 5Delivery was by electroporation (Integrated DNA Technologies, Iowa, USA) into primary keratinocytes in each reaction. The electroporation platform used for RNP delivery was the 4D-Nucleofector™ System (Lonza Bioscience, Switzerland), electroporation code CM137.

[0074] After electroporation, cells were transduced in suspension with donor AAV6 (MOI 30K) in a final volume of 50 μl using Opti-MEM (ThermoFisher Scientific) for 1 h, and then plated onto feeder layer-containing plates.

[0075] MSCs and CD34 + For cells, 3.2 μg of sgRNA and 6 μg of Cas9 were used. The electroporation code used for MSC electroporation was CM119, and CD34 + The code used for cell transfection was DZ100. For transduction, MSCs were incubated with AAV6 in suspension for 15 minutes and then plated onto CD34 medium. + For cells, AAV6 was added directly to the wells.

[0076] Gene-targeted keratinocyte genotyping Six days after treatment, genomic DNA was isolated by isopropanol precipitation of keratinocyte lysates (lysis buffer was 100 mM Tris pH 8, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 1 mg / ml proteinase K (Roche Diagnostics, Mannheim, Germany)) and resuspended in TE buffer. Approximately 50 ng of genomic DNA was used for PCR amplification. A PCR fragment spanning the target region was generated using primers S1 F / R outside the homology arms: F: 5'-CACCAGCATTCTCTCTTCC-3'; R: 5'-GTTCTT GGG TAC TCACCA C-3'. The PCR program was as follows: 98°C for 1 minute, five cycles of 98°C for 30 seconds, 68°C for 30 seconds, and 72°C for 45 seconds, with the annealing temperature decreasing by 1°C per cycle, followed by 30 cycles of 94°C for 30 seconds, 63°C for 30 seconds, and 72°C for 45 seconds, followed by 72°C for 10 minutes. PCR products were analyzed in a 1.5% agarose gel. The molecular weight marker was IX (Sigma-Aldrich). For sequencing, PCR products were treated with illustra™ ExoProStar™ (GE Healthcare, UK), sequenced using the Big Dye Terminator V.1.1 Cycle Sequencing Kit (Thermo Fisher, Waltham, MA), and examined on a 3730 DNA Analyzer (Life Technologies, Carlsbad, CA). Chromatograms were analyzed using Sequencher (Gene Codes, Ann Arbor, MI). Bio-Rad Image Lab Software 6.0 was used for PCR band concentration measurements.

[0077] Western blot analysis Keratinocytes were lysed in protein extraction buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% Nonidet P-40, 4 mM EDTA) containing a proteinase inhibitor cocktail (Complete Mini, EDTA-free; Roche Diagnostics, Mannheim, Germany). The lysates were incubated on ice for 30 minutes and centrifuged at 15,000 × g for 30 minutes at 4°C. The supernatants were collected, and protein concentrations were measured using a Bradford assay (BioRad, Hercules, CA). For each sample, 40 μg of total protein was separated by NuPAGE™ Novex 3-8% Tris-acetate gel electrophoresis (Invitrogen, Carlsbad, CA) and electrophoretically transferred to a nitrocellulose membrane (Invitrogen, Carlsbad, CA). For type VII collagen analysis, blots were probed with a monospecific polyclonal anti-C7 antibody (a gift from Dr. A. Nystrom; University of Freiburg). Antibody against GAPDHA antibody to vinculin was used as a loading control. Visualization was performed by incubation with HRP-conjugated anti-rabbit antibody (Amersham, Burlington, MA) and West Pico chemiluminescent substrate (Pierce, Rockford, IL).

[0078] Immunofluorescence and immunohistochemical staining For immunofluorescence detection of C7 in keratinocytes, cells grown on glass coverslips were fixed in methanol / acetone (1:1) at -20°C for 10 minutes. After three washes in phosphate-buffered saline (PBS) and one wash for 30 minutes in PBS containing 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO), cells were incubated with a monospecific polyclonal anti-C7 antibody at a 1:5000 dilution. The secondary antibody (AlexaFluor 488, Invitrogen, Carlsbad, CA) was used at a 1:1000 dilution. After a final wash step in PBS, preparations were mounted with Mowiol (Hoechst, Somerville, NJ) mounting medium and DAPI 20 μg / ml (Sigma-Aldrich, St. Louis, MO) for nuclear visualization. Immunoperoxidase detection of C7 in paraffin-embedded, formalin-fixed sections was performed as described in 33. 33 Immunoperoxidase staining for human involucrin and p63 was performed on paraffin sections without antigen retrieval using rabbit SY5 monoclonal antibody (Sigma) and 4A4 monoclonal antibody, respectively. The ABC peroxidase kit (Vector) with diaminobenzidine as the substrate was used as the developing reagent.

[0079] Electron microscopy Specimens of approximately 0.4 cm × 0.3 cm were fixed in 3% glutaraldehyde solution in 0.1 M cacodylate buffer, pH 7.4, at room temperature for at least 2 hours and then cut into approximately 1 mm pieces. 3 The tissue was cut into 100 mm pieces, washed in buffer, post-fixed in 1% osmium tetroxide at 4°C for 1 hour, rinsed in water, dehydrated through graded ethanol solutions, transferred to propylene oxide, and embedded in epoxy resin (glycidether 100). Semi-thin and ultrathin sections were cut with an ultramicrotome (Reichert Ultracut E). Ultrathin sections were treated with uranyl acetate and lead citrate and examined with an electron microscope (JEM 1400) equipped with a 2k CCD camera (TVIPS).

[0080] Generation of skin equivalents, transplantation into immunodeficient mice, and graft analysis Animal experiments were approved by our Institutional Animal Care and Use Committee in accordance with national and European legal regulations.

[0081] Gene-edited keratinocytes were then cultured as previously described. 34 The fibroblasts were plated onto fibrin dermal equivalents containing RDEB fibroblasts null for C7 expression, prepared as previously described. 30 Bioengineered skin equivalents were grafted onto the backs of 7-week-old female immunodeficient mice (nu / nu, NMRI background) purchased from Elevage-Janvier (France) as described in [1]. The grafting was performed under sterile conditions, and the mice were kept under pathogen-free conditions for the duration of the experiment at the CIEMAT Laboratory Animal Facility (Spanish registration number 28079-21A). The animals were individually housed in ventilated type II cages with 25 air changes per hour, and 10 kGy gamma-irradiated soft wood pellets were used as bedding. All manipulations were performed under sterile conditions, and all experimental procedures were performed in accordance with European and Spanish laws and regulations. The mice were sacrificed 10 weeks after grafting, and the grafts were harvested for skin histology, immunohistochemistry, and electron microscopy studies.

[0082] HDR-based correction of polyclonal keratinocytes was performed as previously described. 34 The fibroblasts were plated onto fibrin dermal equivalents containing RDEB fibroblasts null for C7 expression, prepared as previously described. 30Bioengineered skin equivalents were grafted onto the backs of 7-week-old female immunodeficient mice (nu / nu, NMRI background) purchased from Elevage-Janvier (France) as described in [1]. The grafting was performed under sterile conditions, and the mice were kept under pathogen-free conditions throughout the experiment at the CIEMAT Laboratory Animal Facility (Spanish registration number 28079-21A). The animals were individually housed in ventilated type II cages with 25 air changes per hour, and 10 kGy gamma-irradiated soft wood pellets were used as bedding. All manipulations were performed under sterile conditions, and all experimental procedures were performed in accordance with European and Spanish laws and regulations. The mice were sacrificed at various time points after grafting, and the grafts were harvested for skin histology, immunohistochemical analysis, and electron microscopy studies.

[0083] In vivo skin fragility test A suction device developed in our laboratory was installed, and negative pressure of 10 ± 2 kPa was applied to a 3 mm diameter area for 5 minutes to induce blister formation in human skin grafts regenerated in immunodeficient mice 12 weeks after transplantation. Two mice with unedited grafts and two mice with sg2 + sg3 RNP-treated keratinocytes were used. Suction was applied to two different sites for each graft. Before applying suction, an incandescent light bulb was placed over the graft area approximately 2 cm apart for 2 minutes to promote blister formation. 35 The bulb was then left on for the entire duration of the experiment. The aspirated area was photographed 10 minutes after aspiration and excised for histological analysis.

[0084] A suction device developed in our laboratory was installed to apply a negative pressure of 10 ± 2 kPa to a 3 mm diameter area to induce blister formation in human skin grafts regenerated in immunodeficient mice 10 weeks after transplantation. Before applying suction, an incandescent light bulb was placed over the graft area approximately 2 cm away for 2 minutes to promote blister formation. 35 The bulb was then left on for the entire duration of the experiment. The aspirated area was photographed 10 minutes after aspiration and excised for histological analysis.

[0085] result CRISPR / Cas9 RNP complexes delivered to primary RDEB cells achieved highly efficient indel generation To demonstrate the indel-generating ability of sgRNA-modified guides, we evaluated the cleavage efficiency of the "sg2" guide used in our previous NHEJ-based double-guide strategy (Bonafont et al. 2019), since it demonstrated good efficiency and biosafety. In this case, instead of the crRNA:tracrRNA molecule in the RNP complex, we tested a chemically modified sgRNA (Synthego, CA).

[0086] This guide targets intron 79, which is very close to pathogenic exon 80 (Figure 1A). We excluded the sequence of intron 79 from having a PAM sequence in our donor template construct to avoid NHEJ events after the HDR repair event.

[0087] Sg2 was electroporated as an RNP complex in primary RDEB keratinocytes under the conditions of the Amaxa 4D Nucleofector platform with the CRISPR / Cas9 system under the code CM137 condition. The indel generation capacity was assessed by TIDE analysis of NHEJ events in the target region, achieving 82.6% and 90.8% in each technical replicate in primary keratinocytes (Figure 1B).

[0088] AAV serotype testing for primary keratinocyte transduction and HDR-based RDEB correction in primary cells AAV, CD34 + It has been shown to be a highly efficient and safe vector for donor template delivery in HDR-based gene editing for different cell types, such as cells or iPSCs, and has promising therapeutic benefits for untreatable diseases.

[0089] To evaluate AAV transduction efficiency in primary keratinocytes, we evaluated a wide collection of AAV serotypes (AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, and DJ; Figure 2A) packaging GFP-based constructs and selected the best-performing subtype. Flow cytometry analysis of GFP expression in different AAVs revealed that AAV6 was the most potent serotype achieving keratinocyte transduction. Therefore, we packaged our constructs into AAV vectors with serotype 6.

[0090] After optimizing the transduction protocol, we electroporated RDEB primary keratinocytes using the sg2 sgRNA as an RNP and then transduced them with AAV6-loaded donor templates. We tested two different donor designs: one with symmetric and one with asymmetric homology arms. The symmetric donor covered E74 to E84 of the COL7A1 gene, while the asymmetric donor covered E77 to E88. We analyzed the repair events that occurred by PCR, TOPO cloning, and Sanger sequencing. We found a nearly 40% HDR frequency at E80, correcting the I79 deletion and the c6527insC mutation (Figure 2B), with no difference in HDR efficiency between the two donors.

[0091] De novo C7 expression after HDR-based correction in RDEB primary keratinocytes High-efficiency gene correction in COL7A1 should restore C7 expression in a high percentage of cells within the edited keratinocyte bulk population. Therefore, we analyzed C7 expression by immunofluorescence and Western blot in RDEB keratinocytes electroporated with sg2 RNP and transduced with two different donor templates carrying AAV6. The number of C7-expressing cells detected by immunofluorescence analysis (Figure 3) was consistent with the observed HDR frequency demonstrated by PCR and Sanger sequencing (Figure 2). Thus, Western blot analysis from cell extracts further demonstrated high expression of restored C7 with two types of AAV6 (symmetric and asymmetric), similar to healthy donor samples. This suggests that we could use both donors to treat RDEB patient cells and increase the population of RDEB patients who could benefit from this gene therapy treatment.

[0092] Long-term engraftment test of gene-corrected RDEB keratinocytes A high percentage of cells expressing C7 after AAV6 and RNP treatment should be sufficient to achieve skin adhesion recovery. To assess the grade of healthy skin regeneration potential, healthy, untreated, and gene-edited bulk keratinocyte populations were combined with C7-null fibroblasts to generate skin equivalents that were transplanted into nude mice. H&E histological analysis showed normal skin architecture in grafts from healthy and gene-edited keratinocytes, while some blisters were observed in the graft from untreated patient 1. Immunohistochemical C7 detection showed no C7 expression in regenerated tissue from untreated keratinocytes from RDEB patient 1 (Figure 4A). On the other hand, grafts from RDEB keratinocytes (P1-edited keratinocytes) after AAV6 + RNP treatment revealed C7 expression in the basement membrane of the regenerated skin (Figure 4B), similar to grafts regenerated from healthy donor keratinocytes (Figure 4C). All tissue samples showed correct suprabasal human involucrin expression, demonstrating normal epidermal differentiation (FIGS. 4D, 4E, and 4F).

[0093] HDR-based correction in RDEB patients with mutations at E79 The donor template covers a larger number of exons within COL7A1, allowing for feasible gene correction at different points in the gene. Therefore, after demonstrating relevant correction efficiency in patient cells harboring a mutation in exon 80 (Patient 1), we tested the exon 79-exon 80 fusion strategy for homozygous correction in an RDEB patient harboring a mutation in E79 (Patient 2; P2). We tested only the symmetric arm containing AAV6 for this transduction. Genotyping demonstrated similar HDR-based correction ratios by PCR compared to previously treated P1 (Figure 5). We also assessed C7 expression restoration by immunofluorescence in RDEB P2-treated cells, demonstrating a significant proportion of positive C7 cells in the bulk edited population.

[0094] CD34 as a cell source for bone marrow transplantation in EB + and MSC gene-edited cells Recently, HSCT has been explored as a therapeutic option for the treatment of EB. Although HSCT offers the benefit of improving symptoms, it has several complications. Allogeneic HSCT containing gene-corrected cells may overcome this barrier and provide a safer therapeutic solution. CD34 + MSCs are the major stem cell type in bone marrow, and therefore, we used the RNP+AAV6 strategy to isolate umbilical cord blood CD34 cells from three healthy donors. + and MSC cells to test the potential of our approach to target another relevant cell type for RDEB treatment.

[0095] Five days later, we detected CD34 + When gene correction was analyzed, we found similar gene correction ratios to those observed in HK P1-treated cells with the same donor template containing AAV6 (Figure 6). +For cells, we tested two different MOIs, 5K and 10K, and found no difference in HDR-based correction efficiency. Furthermore, comparison of different cell donors showed no differences in HDR events. Similarly, MSCs from three healthy donors demonstrated accurate correction rates approaching 50% across all cell donors tested. No significant differences in editing efficiency were observed between cell donors, supporting the robustness of this genome editing approach. This study provides proof-of-concept that bone marrow stem cells are suitable for gene correction therapy using the proposed strategy, which may offer various potential benefits for EB treatment.

Claims

1. Inducing stable genetic modification of a target nucleic acid containing one or more mutant alleles containing a pathogenic mutation in the COL7A1 gene via homologous recombination in primary cells selected from the group consisting of keratinocytes or skin fibroblasts. The in vitro method includes introducing into the primary cell: (a) a modified single guide RNA (sgRNA) comprising a nucleotide sequence complementary to a target nucleic acid and a nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein the RNA components can be two individual RNA molecules (crRNA and tracrRNA) or a single RNA molecule (sgRNA); (b) a recombinant expression vector comprising a Cas polypeptide, an mRNA encoding the Cas polypeptide, and / or a nucleotide sequence encoding the Cas polypeptide, wherein the modified sgRNA component, or the crRNA component and the tracrRNA component, provided separately, guide the Cas polypeptide to the target genomic sequence to be corrected; and (c) a donor template DNA carried by a serotype 6 adeno-associated virus vector (AAV-6), which is homologous to the genomic sequence comprising the mutation site to be repaired; the stable genetic modification of the target nucleic acid occurs based on replacement of one or more mutant alleles (target nucleic acids) comprising a pathogenic mutation in the COL7A1 gene by providing an AAV-6 vector carrying the corrected donor template comprising a wild-type allele corresponding to the mutant allele; The method, wherein the donor template does not contain an intron region of the targeting nucleic acid that includes a Cas recognition protospacer adjacent motif (PAM) sequence.

2. 2. The method of claim 1, wherein the epidermolysis bullosa pathogenic mutation is a recessive dystrophic epidermolysis bullosa (RDEB) pathogenic mutation.

3. The method of claim 1 or 2, wherein one or more mutant alleles (target nucleic acids) containing a pathogenic mutation in the COL7A1 gene are located in any of exon 73, exon 74, exon 75, exon 80, or exon 105 of the COL7A1 gene, and these mutations are repaired using a corrected donor template containing wild-type exon 73, wild-type exon 74, wild-type exon 75, wild-type exon 80, or wild-type exon 105 of the COL7A1 gene.

4. 4. The method of any one of claims 1 to 3, wherein the primary cells are isolated from a mammal prior to introducing an AAV-6 vector carrying the modified sgRNA, the Cas polypeptide, and the homologous donor template into the primary cells.

5. The method of any one of claims 1 to 4, wherein the Cas polypeptide is a Cas9 polypeptide.

6. 6. The method of any one of claims 1 to 5, wherein the RNA components and / or the Cas polypeptide are introduced into the primary cells by electroporation, and optionally an AAV-6 vector carrying the homologous donor template is introduced into the primary cells by transduction.

7. 7. The method of any one of claims 1 to 6, wherein the RNA component and the Cas polypeptide are incubated together to form a ribonucleoprotein (RNP) complex prior to introduction into the primary cells, and optionally the RNP complex and the homologous donor AAV-6 vector are sequentially introduced into the primary cells.

8. A method for inducing stable genetic modification of a target nucleic acid comprising one or more mutant alleles comprising a pathogenic mutation in the COL7A1 gene through homologous recombination in primary cells selected from keratinocytes or fibroblasts obtained from a subject, the method comprising: (a) a modified single guide RNA (sgRNA) comprising a nucleotide sequence complementary to the target nucleic acid and a nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein the RNA components can be two individual RNA molecules (crRNA and tracrRNA) or a single RNA molecule (sgRNA); (b) a recombinant expression vector comprising a Cas polypeptide, an mRNA encoding a Cas polypeptide, and / or a nucleotide sequence encoding a Cas polypeptide, wherein the modified sgRNA components, or the crRNA component and the tracrRNA component, provided separately, guide the Cas polypeptide to the target genomic sequence to be corrected; and (c) a recombinant donor template comprising two nucleotide sequences comprising two non-overlapping homologous portions of the target nucleic acid to undergo homologous recombination, In vitro use of a kit comprising an adeno-associated virus (AAV6) or AAV-1 vector, wherein the donor template does not contain an intron region of a targeting nucleic acid that includes a Cas recognition protospacer adjacent motif (PAM) sequence.

9. 8. A pharmaceutical composition comprising primary cells comprising a stable genetic modification of a target nucleic acid obtained by or obtainable by the method of any one of claims 1 to 7, or a cell population comprising said primary cells, for use in a method for preventing or treating epidermolysis bullosa in a subject in need thereof, wherein said population comprises at least about 30% primary keratinocytes with a stable genetic modification of said target nucleic acid.

10. 8. A method for producing a skin equivalent by using the primary cells comprising a stable genetic modification of the target nucleic acid obtained by or obtainable by the method of any one of claims 1 to 7, or a cell population comprising the primary cells, wherein the population comprises at least about 30% primary keratinocytes having a stable genetic modification of the target nucleic acid.

11. 11. A skin equivalent obtainable or obtainable according to claim 10 for use in the treatment of epidermolysis bullosa, in particular the recessive dystrophic subtype (RDEB).

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