Gene therapy for recessive dystrophic epidermolysis bullosa using genetically modified autologous keratinocytes

Abstract

To provide pharmaceutical compositions to treat Epidermolysis Bullosa (EB) and corneal erosion, and methods to produce pharmaceutical compositions.SOLUTION: Methods are provided for the cell-based delivery of collagen VII for the treatment of Epidermolysis Bullosa and corneal erosion. The disclosure also provides a composition and a pharmaceutical composition comprising, or alternatively consisting essentially of, or yet further consisting of a keratinocyte sheet or a corneal cell sheet.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This disclosure generally relates to methods and compositions for treating epidermolysis bullosa (EB) and corneal erosion. [Background technology]

[0002] Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary vesicular cutaneous disorder caused by mutations in the COL7A1 gene (collagen VII, C7), resulting in a deficiency of C7 function. Patients with this disorder are characterized by widespread vesicular formation and erosion of skin and mucous tissue, including the oropharynx, conjunctiva, esophagus, and the distal surfaces of the genitourinary tract and gastrointestinal tract. Painful vesicular formation and erosion are the primary impairments, but scarring from healed wounds also leads to significant morbidities, including mitten finger deformities (pseudomygdactyly), eyelid fusion, esophageal stenosis, microstomia, tongue-tie, and limb stenosis. Chronic wounds and scarring predispose individuals to invasive squamous cell carcinoma invasion, which is a major problem in RDEB and a leading cause of death in this population, beginning in adolescence. Therefore, the optimal treatment for this disease would be one that can be implemented early to prevent the development of dysfunctional scarring and blister formation. Furthermore, the ability to systemically repair both skin and mucous membrane tissue would be highly desirable in an RDEB treatment approach.

[0003] Type VII collagen (C7) is a large homotrimeric triple-helical collagen molecule that undergoes antiparallel dimerization at its NC2 terminus, followed by supramolecular assembly into a binding structure called an anchoring fibril, which connects the lamina compacta of the basement membrane zone (BMZ) to the dermal papillary layer. C7 contains a large NC1 domain, which binds laminin-332 in the lamina compacta and collagen domain, and which encapsulates interstitial collagen fibers within the dermal papillary layer. Therefore, the absence of C7 in RDEB results in blister formation between the dermal papillary layer and the lamina compacta.

[0004] Despite advances in molecular diagnostics for this disease, current treatments are limited to palliative care. Several alternative approaches to C7 have been proposed, but all have limitations. When applied topically, rC7 cannot penetrate intact skin and is restricted to the damaged area. Intradermal rC7 protein injection in RDEB patients is another alternative. However, limitations in diffusion from conventional needle injection necessitate rC7 microneedle array delivery, which is not yet available for clinical use.

[0005] For therapeutic purposes, topical delivery of C7 to the skin is desirable. This invention addresses this problem. Systemic therapy with C7 can cause systemic toxicity. (See Hou et al. (2015), Journal of Investigative Dermatology 135, 3060-3067.) [Overview of the Initiative]

[0006] Compositions and methods for the treatment of epidermolysis bullosa (EB) in human subjects are provided. In the treatment method of the present invention, a population of human keratinocytes is engineered to express C7 by integrating a gene construct encoding wild-type human C7. In some embodiments, the expression level is higher than the expression level of normal human keratinocytes. In some embodiments, the expression level is lower than, similar to, or the same as the expression level of normal human keratinocytes. The present invention includes an isolated population of keratinocytes engineered by the method of the present invention to express wild-type C7, which is provided in pharmaceutical unit dose compositions. This is possible. In some embodiments, the subjects are humans with a C7 genetic defect that causes epidermolysis bullosa (EB). In embodiments, the genetic defect is RDEB.

[0007] In some embodiments, the keratinocytes used for treatment are autologous keratinocytes. Ex vivo manipulation methods may be selected from, but are not limited to, virus-induced methods, including retroviruses (e.g., gamma retroviruses), AAV viruses, and lentiviruses, or virus-free integration methods, including non-viral vectors, transposons, minicircle integration, and CRISPR / Cas9 genome editing systems. In some embodiments, gamma retroviruses include, essentially, or further include, mouse leukemia virus (MLV or MuLV), feline leukemia virus (FeLV), gibbon leukemia virus (GALV), and heterologous mouse leukemia virus-associated virus (XMRV). In some embodiments, non-viral vectors include, essentially, or further include, episomal or embedded vectors having genome editing capabilities. In some embodiments, GMP-grade GalV-pseudotyped LZRSE-COL7A1 virus containing functional COL7A1 cDNA under the control of MLV LTR is used to integrate the C7 gene into keratinocytes. In some embodiments, the functional COL7A1 cDNA is full-length wild-type human COL7A1 cDNA. In one embodiment, the functional COL7A1 cDNA includes genetic modification from full-length wild-type human COL7A1 cDNA. In some embodiments, viral transduction is carried out by overlaying the viral supernatant onto cell cultures. In some embodiments, keratinocytes thus treated meet pre-release criteria of a viral transduction efficiency (VTE) greater than 50% and a proviral genome copy number (PGCN) of 3 or less. In some embodiments, the PGCN is greater than 3, 10, 20, 40, or 60. In some embodiments, the PGCN is less than 2, 1.5, 1, or 0.5.

[0008] In some embodiments, an endogenous mutation, dysfunction, or truncated C7 gene is replaced using a CRISPR / Cas system (or a vector encoding the CRISPR / Cas system) as described herein and a “donor” sequence (e.g., a functional COL7A1 cDNA or C7 gene) that is inserted into the gene after targeted cleavage.

[0009] In some embodiments of the present invention, a method for treating EB is provided, which comprises, essentially, or further comprises, obtaining a population of keratinocytes derived from an EB-affected subject, modifying the keratinocytes by retroviral transduction to express wild-type human C7, and reintroducing the keratinocytes into an individual. In some embodiments, the keratinocytes are obtained from a skin punch biopsy cultured in vitro in a serum-containing or serum-free keratinocyte medium. In some embodiments, the keratinocytes are cultured in a medium with or without a cell feeder layer. The epidermis is separated from the dermis, and the keratinocytes are obtained from the epidermal layer. At least about 10 6 Each cell, at least approximately 2 × 10⁻¹⁶ 6 pieces, and / or at least 4 × 10 6 Individual cells are used for transduction to provide a population of genetically modified cells. The cells are approximately 25 cm long for transplantation. 2 ~about 100 2 The cells are cultured to produce a sheet approximately 50 cm in size. The genetically modified keratinocyte sheet is placed on an uninfected, eroded, and / or scarred wound site that does not show clinical signs of squamous cell carcinoma (SCC). The wound site is approximately 50 cm in size. 2 , about 100cm 2 , and / or approximately 200cm 2 This may be the case. In some embodiments, a wound is created for the graft. In some such embodiments, the wound is electrocautered to excise any remaining uncorrected wound bed keratinocytes. The graft is fixed to the wound bed with dissolvable sutures after the wound bed has been prepared.

[0010] In another embodiment, the present disclosure provides a composition comprising, consisting essentially of, or further comprising a population of genetically modified keratinocytes that express wild-type human C7 at an effective dose for reducing symptoms of EB, and a pharmaceutically acceptable carrier. In one aspect of the present disclosure, the composition is frozen. In some embodiments, the keratinocytes are autologous to the individual selected for treatment.

[0011] In another embodiment, the present disclosure provides a pharmaceutical composition comprising, consisting essentially of, or further comprising a keratinocyte sheet, which comprises, consists essentially of, or further comprises skin cells in which a gene construct encoding a functional COL7A1 protein has been integrated ex vivo. In some embodiments, the keratinocyte sheet is placed on a skin equivalent produced by biotechnology. In some embodiments, the keratinocyte sheet is placed on an acellular matrix, a collagen matrix, an ECM protein, or a chemical layer, or a biocompatible mesh. In one embodiment, the acellular matrix is made from human and / or animal dermis. In some embodiments, the biocompatible mesh is made of thermoplastic resin, polyethylene, ultrahigh molecular weight polyethylene, high molecular weight polyolefin, uncoated monofilament polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, silicon, or any combination thereof.

[0012] As described herein, autologous RDEB keratinocytes were isolated from a skin biopsy and transduced with a retrovirus having functional (e.g., full-length) human COL7A1. For each subject, an autologous epidermal sheet (about 35 cm 2Grafts were manufactured and transplanted into prepared wound beds. Endpoints included safety as a percentage of wound healing compared to baseline, efficacy, and evidence of C7 expression at 3 and 6 months post-transplantation. All grafts were well tolerated by all subjects, and no serious adverse events (systemic viral infection, autoimmunity, or development of skin cancer within the grafts) were reported. Between 3 and 6 months, the majority of grafts showed 75% healing. Biopsies of the transplant sites showed strong C7 expression at the dermal-epidermal junction at 3 and 6 months in the presence of normal anchoring fibrils. COL7A1 ex vivo gene transfection had a favorable safety profile and showed promising efficacy in subjects with inherited RDEB. [Brief explanation of the drawing]

[0013] [Figure 1]A shows the RDEB gene modification flowchart and represents autologous epidermal sheet grafts modified with KC-epidermal keratinocytes and LEAES-LZRSE-COL7A1. B shows indirect immunofluorescence (IIF) of RDEB KCs transduced with LZRSE-COL7A1 virus, representing anti-VII collagen polyclonal antibody (orange) and Hoechst33342 nuclei (blue), with a scale bar of 100 μm. C shows the quantification of viral transduction efficiency (VTE) in modified KCs from four RDEB subjects. D shows the quantification of mean proviral copy number (PGCN) in modified KCs from four RDEB subjects. E shows the clinical representation of the RDEB phenotype of the grafts before and after transplantation, noting blistering in pre-modified skin graft and untreated wounds compared to 3 months and 6 months before LEAES transplantation. F shows IIF analysis of type VII collagen expression in skin grafts, noting the linear green staining of type VII collagen at the dermal-epidermal junction of the modified tissue graft, with anti-type VII collagen NC2 Mab LH24 and NC1 Pab FNC1 (green), Hoechst33342 nuclei (blue), and the scale bar being 100 μm. G shows immunoEM analysis of modified RDEB skin grafts, with tissue sections collectively labeled with anti-type VII collagen NC2 Mab LH24, followed by anti-mouse IgM conjugate immunogold particles (black dots, indicated by arrows), with the scale bar being 200 nm. [Figure 2] This is a CONSORT diagram of participant registration in the Phase I trial. [Figure 3] This report presents Western blot analysis of cultured KC supernatant using an anti-type VII collagen polyclonal antibody specific to the NC1 domain, highlighting the expression of the truncated C7 protein containing the NC1 domain in all subjects registered for the study. [Figure 4] Mature leaves before harvest, and the assembled final leaves grafts are shown. [Figure 5]A shows the clinical representation of the RDEB phenotype of the graft before and after transplantation. B shows the IIF analysis of type VII collagen expression in the skin graft, representing anti-type VII collagen NC2 Mab LH24 (green), Hoechst33342 nuclei (blue), keratin 14 (anti-K14 Pab, orange), keratin 1 (anti-K1 Pab, orange), and loricrin (anti-loricrin Pab, orange). Note the linear green staining of type VII collagen at the dermal-epidermal junction of the corrected tissue graft at all time points. The scale bar is 100 μm. [Figure 6] A shows the clinical representation of the RDEB phenotype of the wound and graft of Subject 2 before and after transplantation. B shows the IIF analysis of type VII collagen expression in the wound and skin graft of Subject 2 before and after transplantation, representing anti-type VII collagen NC2 Mab LH24 (green), Hoechst33342 nuclei (blue), keratin 14 (anti-K14 Pab, orange), keratin 1 (anti-K1 Pab, orange), and loricrin (anti-loricrin Pab, orange). Note the linear green staining of type VII collagen at the dermal-epidermal junction of the corrected tissue graft at 3 months. The scale bar is 100 μm. [Figure 7] A shows the clinical representation of the RDEB phenotype of the wound and graft of Subject 3 before and after transplantation. B shows the IIF analysis of type VII collagen expression in the wound and skin graft of Subject 3 before and after transplantation, representing anti-type VII collagen NC2 Mab LH24 (green), Hoechst33342 nuclei (blue), keratin 14 (anti-K14 Pab, orange), keratin 1 (anti-K1 Pab, orange), and loricrin (anti-loricrin Pab, orange). Note the linear green staining of type VII collagen at the dermal-epidermal junction of the corrected tissue graft at all time points, and the scale bar is 100 μm. [Figure 8]A shows the wounds of Subject 4 before and after transplantation, and the clinical manifestations of the RDEB phenotype of the graft before and after transplantation. B shows the IIF analysis of type VII collagen expression in the wounds of Subject 4 before and after transplantation and in the skin graft, representing anti-type VII collagen NC2 Mab LH24 (green), Hoechst33342 nuclei (blue), keratin 14 (anti-K14 Pab, orange), keratin 1 (anti-K1 Pab, orange), and loricrin (anti-loricrin Pab, orange). Note the linear green staining of type VII collagen at the dermal-epidermal junction of the corrected tissue graft at 3 months using LH24 Mab and the corrected tissue graft at 6 months using NC1 Pab. The scale bar is 100 μm. [Figure 9] Shows the characterization of the anti-C7 LH24 monoclonal antibody, a Western blot analysis of enzymatically digested C7 showing the cross-reactivity of the LH24 Mab with a carboxyl-terminal peptide containing the NC2 domain, representing the presence of the NC2 domain in the pepsin-digested C7 fraction confirmed by NC2-specific pAb (NC2-10)5, and representing the NC1 domain in the collagenase-digested C7 fraction identified using FNC1 pAb 6. [Figure 10] Shows the clinical manifestation of the uncorrected wound of Subject 4, the characteristic spontaneous blistering in the untreated wound. [Figure 11] A shows a Western blot analysis showing the serum reactivity of Subject 4 against type VII collagen and the cross-reactivity of the serum of Subject 4 before and after transplantation (3 months) against full-length C7. B shows the serum reactivity of Subject 4 against type VII collagen. The serum of Subject 4 obtained before and 3 months after transplantation is specific for the enzymatically (pepsin) digested C7 protein containing the NC2 domain. The control (right lane) confirms the presence of the NC2 domain in the C7 fraction digested using NC2-specific Pab (NC2-10)5. [Figure 12]Wound of Subject 1 before transplantation and 12 months after transplantation, clinical manifestations of the wound at baseline and 12 months after transplantation, IIF analysis of type VII collagen expression in skin grafts, showing anti-type VII collagen NC1 Pab (green), Hoechst33342 nuclei (blue), keratin 14 (anti-K14 Pab, orange), keratin 1 (anti-K1 Pab, orange), and loricrin (anti-loricrin Pab, orange), note the linear green staining of type VII collagen at the dermal-epidermal junction of the modified tissue graft, scale bar is 100 μm. [Figure 13] Shows the map of the pLZRSE-COL7A1 retroviral plasmid.

Mode for Carrying Out the Invention

[0014] It should be understood that the present invention is not limited to the specific methodologies, protocols, cell lines, animal species or genera, and reagents described, and such things can vary. It should also be understood that the terms used herein are for the purpose of describing only specific embodiments and do not limit the scope of the present invention, which is limited only by the appended claims.

[0015] As used herein, 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, and reference to "a culture" includes reference to one or more cultures known to those skilled in the art and their equivalents. 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 invention belongs, unless otherwise expressly defined.

[0016] Any embodiment of the Method, Apparatus, and System may consist of or essentially consist of the described steps and / or functions rather than including / containing / containing / having. Accordingly, in any of the claims, the terms "consist of" or "essentially consist of" may be replaced with any of the unrestricted linking verbs cited above, from those using the unrestricted linking verbs separately, in order to modify the scope of a given claim.

[0017] The use of the term “or” in the claims is used to mean “and / or” unless expressly intended to refer only to substitutes, or substitutes are mutually exclusive. However, this disclosure supports the definition of substitutes and “and / or” only.

[0018] Throughout this application, the term “about” is used to indicate that the value includes the standard deviation of the error of the apparatus or method used to determine the value.

[0019] The target conditions for treatment with the manipulated keratinocytes of the present invention include, but are not limited to, various forms of epidermolysis bullosa, including acquired and congenital forms, the latter of which may be recessive or dominant.

[0020] Based on recent classification systems, dysphagia epidermolysis bullosa (DEB) includes three subtypes: recessive DEB, severe systemic (RDEB-sev gen) (formerly called Hallopeau-Siemens type (RDEB-HS)), recessive DEB, other systemic (RDEB-O) (formerly called non-Hallopeau-Siemens type (non-HS type RDEB)), and dominant DEB (DDEB). In RDEB-sev gen, systemic blisters may be present in the neonatal period. Oral lesions can result in blister formation in the mouth, fusion of the tongue to the floor of the mouth, and a progressive reduction in oral size. Esophageal erosion can lead to webs and stenosis, which can cause severe dysphagia. Thus, severe malnutrition and secondary problems are common. Corneal erosion can lead to scarring and vision loss. The formation of blisters on the hands and feet, followed by scarring and fusion of fingers and toes, is characteristic of this disease. The hands and feet develop a "mitten" appearance. The lifetime risk of active squamous cell carcinoma is over 90%. In DDEB, blister formation is often mild and limited to the hands, feet, knees, and elbows, but nevertheless heals with scarring. Dystrophy of the nails, especially the toenails, is common and may be the only symptom of DDEB.

[0021] Standard treatment of symptoms, including wound dressings and nutritional support, is generally effective. Occupational therapy may help prevent hand contractures. Surgical release of the fingers often needs to be repeated.

[0022] Keratinocytes engineered to express wild-type C7 may find use in the treatment of dystrophic epidermolysis bullosa.

[0023] In addition to hereditary forms of EB, acquired forms of epidermolysis bullosa (EBA) include pathological conditions in type VII collagen, which can be treated with engineered keratinocytes in this disclosure. In EBA patients, autoantibodies in circulating blood recognize epitopes in the type VII collagen molecule, and molecular cloning of type VII collagen cDNA provides a tool for identifying dominant immunoepitopes within the amino-terminal NC-1 domain of type VII collagen. The antigenic properties of the NC-1(VII) domain are further highlighted by the fact that monoclonal antibodies such as H3A and L3D, which are clinically used to map type VII collagen in the skin of patients with acquired forms of EB, also identify epitopes in this portion of the protein. In addition to autoantibodies in circulating blood that recognize type VII collagen epitopes in EBA, bullous lesions in some patients with systemic lupus erythematosus are also associated with anti-type VII collagen antibodies.

[0024] collagen As used herein, the term “collagen” refers to a composition in which at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and at least about 95% or more of the proteins present are collagen in a triple helix conformation. The folding of individual α-chains into the triple helix conformation is based on a characteristic primary sequence containing repeating Gly-XY triplet sequences. Collagen is widely found in vertebrate species, and sequences have been determined for many different species. Due to the high degree of sequence similarity between species, for example, between mammalian species, collagen from different species can be used for biological purposes; however, human proteins may be preferred.

[0025] FACIT collagen (fiber-associated collagen with a suspended triple helix) includes types IX, XII, XIV, XIX, XX, and XXI. Some of the latter types of collagen associate with and connect to larger collagen fibers, functioning as molecular bridges and stabilizing the structure of the extracellular matrix. Collagen VII (COL7A1, chromosome 3, NC_000003.10 (48576510..48607689, complement)) is of particular interest. Type VII collagen is a major component of anchoring fibrils.

[0026] Type VII collagen is a long 424 nm triple helix domain with adjacent non-collagenous sequences. The type VII collagen molecule contains a major collagen, triple helix segment flanked by non-collagenous NC-1 and NC-2 domains. Unlike interstitial collagen, the repeating Gly-XY sequence is interrupted by 19 defects resulting from amino acid insertions or deletions within the Gly-XY repeat sequence. Most notably, in the center of the triple helix domain is a 39-amino acid non-collagenous "hinge" region, which is sensitive to proteolytic digestion using pepsin. The amino-terminal NC-1 domain of type VII, approximately 145 kDa in size, is a segment homologous to cartilage matrix protein (CMP), with nine consecutive fibronectin type III-like (FN-III) domains, von It contains a segment homologous to the A domain of the Willebrand factor, as well as a submodule homologous to known adhesion proteins, including a short cysteine ​​and proline-rich region. The carboxyl-terminal non-collagenous domain NC-2 is relatively small at approximately 30 kDa and contains a segment homologous to the Kunitz protease inhibitor molecule.

[0027] The human type VII collagen gene, COL7A1, has a complex structure with a total of 118 separate exons. However, the gene is relatively compact, and most of the introns are relatively small. Therefore, the overall size of the human COL7A1 gene is only about 32kb, encoding approximately 8.9kb of messenger RNA. COL7A1 maps to region 3p21.1, which is on the short arm of human chromosome 3. The gene structure and the primary sequence encoding the protein of type VII collagen are well conserved; for example, the mouse gene shows 84.7% homology at the nucleotide level and 90.4% identity at the protein level.

[0028] Type VII collagen is synthesized by both epidermal keratinocytes and dermal fibroblasts in culture. During the synthesis of the complete pro-α1(VII) polypeptide, the three polypeptides associate via their carboxyl terminals to form a trimer molecule in which the collagen moieties fold to form a triple helix. The triple helix molecule is then secreted into the extracellular environment, where the two types of type VII collagen molecules align to form an antiparallel dimer, with the amino-terminal domains located at both ends of the molecule. This dimer assembly involves proteolytic removal of portions of the carboxyl terminals on both ends of the type VII collagen molecule and stabilization by the formation of intermolecular disulfide bonds. Subsequently, a number of these antiparallel dimers aggregate laterally to form anchoring fibrils.

[0029] In dystrophic epidermolysis bullosa dominant (DDEB), glycine substitution mutations in the triple helix domain of COL7A1 (particularly exons 73, 74, and 75) are dominant. Mutations p.Gly2034Arg and p.Gly2043Arg are the most common DDEB-causing mutations, accounting for 50% of the dominant mutations reported in the largest US cohort. Glycine substitutions, as well as other amino acid substitutions and splice mutations outside this region, can also be found in dominant DEB.

[0030] For all forms of recessive DEB, more than 400 gene-wide mutations causing recessive DEB have been described. However, each mutation accounts for less than 1% to 2% of the total number of mutations. Glycine substitutions and other amino acid substitutions have been described, but null mutations are dominant in RDEB. Milder forms of RDEB are often caused by splice junction mutations or other missense mutations.

[0031] A “natural sequence” polypeptide is one that has the same amino acid sequence as a naturally occurring polypeptide. Such natural sequence polypeptides can be produced by recombinant means according to the methods described herein. Thus, natural sequence polypeptides may have amino acid sequences such as naturally occurring human polypeptides, mouse polypeptides, or polypeptides derived from any other mammalian species. The term “natural sequence collagen VII protein” includes natural proteins that have or do not have an initiating N-terminal methionine (Met).

[0032] A “variant” polypeptide means a biologically active polypeptide that has less than 100% sequence identity with the native sequence polypeptide, as defined below. Such a variant comprises a polypeptide in which one or more amino acid residues are added at the N-terminus or C-terminus of the native sequence, or within the native sequence, and in which approximately 1 to 40 amino acid residues are deleted, and 1 The present invention includes polypeptides optionally substituted with one or more amino acid residues, and derivatives of the polypeptides in which amino acid residues are covalently modified such that the resulting product has amino acids not found in nature. Typically, biologically active collagen VII variants will have an amino acid sequence that has at least about 90%, preferably at least about 95%, and more preferably at least about 99% amino acid sequence identity with the natural collagen VII polypeptide.

[0033] Functional derivatives of the naturally occurring collagen VII polypeptide are compounds that share qualitative biological properties with the naturally occurring collagen VII polypeptide. Functional derivatives include, but are not limited to, fragments of the natural sequence, as well as derivatives and fragments of the naturally occurring collagen VII polypeptide. However, they share biological activity with the corresponding naturally occurring collagen VII polypeptide. The term "derivative" encompasses both amino acid sequence variants of collagen VII polypeptide and their covalent modifications.

[0034] The term "wound bed" refers to the viable outermost layer of a wound. In one embodiment, the wound bed is covered with slough or escar. In another embodiment, the wound bed can be assessed for the presence of granulation tissue fibers, slough, escar, bone, tendons, and / or other underlying structures.

[0035] The term "viral transduction efficiency (VTE) test" refers to a test that measures the ratio of the number of cells transduced by a virus to the total number of transduced cells. In one embodiment, VTE is measured by immunofluorescence staining using an antibody that targets a protein expressed on the transduced virus. In another embodiment, VTE is measured by real-time PCR or quantitative PCR.

[0036] The term "proviral genome copy number" or "PGCN" refers to the number of proviral DNA copies in transduced cells. Therefore, a PGCN test measures the number of proviral DNA copies in cells after transduction or infection. In one embodiment, the copy number is measured by real-time PCR or quantitative PCR. In another embodiment, PGCN is measured by Southern blotting or a high-throughput method. In some embodiments, PGCN is less than 3, 2, 1, or 0.5. In some embodiments, PGCN is greater than 3, 10, 100, or 1,000.

[0037] The term "sterility test" refers to a test attempted to determine the presence or absence of viable contaminating microorganisms in a sample, and is often used to eliminate false positive results. In one embodiment, a false positive result results from contamination from the environment or errors.

[0038] The term “endotoxin” refers, but is not limited to, toxins associated with the outer membrane of certain Gram-negative bacteria, including Brucella, Neisseria, and Vibrio species. In one embodiment, endotoxins are not secreted but are released only when cells are destroyed. Endotoxins can be measured or tested by gel-clot, colorimetric, turbidimetric, or a combination thereof.

[0039] The term "mycoplasma" refers to a group of bacteria that lack a cell wall around their cell membrane, making them less susceptible to or immune to certain types of antibiotics. Mycoplasma testing includes, but is not limited to, agar and blotting procedures, DNA detection, enzymes, ELISA methods, and PCR.

[0040] The term "Gram stain sterility test" is used to detect bacteria and / or fungi in a sample. This refers to the procedure. In one embodiment, a Gram staining sterilization test can indicate the presence or absence of bacteria or fungi in the sample, and / or their general type.

[0041] The term “viability test” refers to a test that determines the ability of an organ, cell, or tissue to maintain or restore viability, including, but not limited to, the mechanical activity, motility, contractility, and mitotic activity of the organ, cell, or tissue. In one embodiment, an autologous epidermal sheet (LEAES) viability test performed with LZRSE-COL7A1 is to test the ability of cells or tissues on a LEAES to maintain or restore viability.

[0042] In this disclosure, the term “reproducible retrovirus (RCR)” refers to a retrovirus that is capable of replicating even if the retroviral vector is designed to be replication-deficient. In one embodiment, the RCR is generated during production by homologous or non-homologous recombination between the packaging components of the introduction vector and endogenous retroviral elements in producer cells. RCR testing involves detecting the RCR in a sample.

[0043] The term "cytotoxic T cell assay" refers to an assay used to evaluate cell-mediated immune function.

[0044] In this disclosure, the term “post-release testing” refers to one or more tests performed after the epidermal sheet has been released from the plate, including, but not limited to, sterility testing, RCR testing, mycoplasma testing, viability testing, and Gram staining sterility testing.

[0045] The term “genetic modification” refers to the process of altering the genes of an organism or inserting a gene from one organism into another. In one embodiment, genetic modification includes, is essentially, or is more than, insertions, deletions, and / or mutations. The term “insertion” means adding one or more nucleotide base pairs to a nucleotide sequence. The term “deletion” refers to a portion of a chromosome or nucleotide sequence that is removed or missing. The term “mutation” is a change in a nucleotide sequence (e.g., a DNA sequence). Mutations can occur in various sizes, including, but are not limited to, a single base pair of a chromosome (i.e., a point mutation), several base pairs, or at most a large segment.

[0046] The term "conservative genetic modification" refers to a genetic modification that maintains the same or similar biochemical properties of the polypeptide encoded by the modified gene. For example, both aspartic acid and glutamic acid are small, negatively charged residues. In some embodiments, this is a conservative genetic modification that involves mutating aspartic acid to glutamic acid in the polypeptide.

[0047] Prolyl 4-hydroxylase (P4HA; EC 1.14.11.2) plays a central role in collagen synthesis. It catalyzes the formation of 4-hydroxyproline in collagen by hydroxylating proline residues in peptide linkages. The 4-hydroxyproline residue is essential for the folding of the newly synthesized procollagen polypeptide chain into a triple helix molecule. The active enzyme is a tetramer of 2α and 2β subunits with a molecular weight of approximately 240,000. The β subunit (P4HB) is identical to that of the enzyme disulfide isomerase (EC 5.3.4.1) and the major cell thyroid-binding protein. The α subunit is involved in most of the catalytic site of the enzyme. The polypeptide consists of a 517-amino acid residue and a 17-amino acid signal peptide.

[0048] The P4HA gene encompasses more than 69 kilobases and consists of 16 exons. Evidence for mutually exclusive alternative splicing of RNA transcripts has been presented previously. These data show that mutually exclusive sequences found in mRNA are encoded by two consecutive homologous 71bp exons 9 and 10. These exons are identical in their first five base pairs, and their overall identity is 61% at the nucleotide level and 58% at the encoded amino acid level. Both types of mRNA were found to be expressed in all tissues studied, but in some tissues, the type encoding either exon 9 or exon 10 sequences was more abundant than the other.

[0049] A "nucleic acid construct" refers to a nucleic acid sequence constructed to contain one or more functional units that are not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences derived from lambda phages), and viral genomes containing non-natural nucleic acid sequences.

[0050] In this method, collagen VII is typically produced by introducing a viral expression construct into a cell population during integration. The DNA encoding the collagen VII polypeptide can be obtained from any cDNA library prepared from tissues expressing collagen VII polypeptide mRNA, prepared from various sources. The collagen VII polypeptide encoding gene can also be obtained from a genomic library or by oligonucleotide synthesis. Another means of identifying the genetic code is to use PCR methodology.

[0051] A nucleic acid encoding collagen VII polypeptide (e.g., cDNA or genomic DNA) is inserted into an expression construct that is operably linked to the elements necessary for expression. Many such constructs are available. The components generally include, but are not limited to, one or more of the following: coding sequences, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[0052] A "vector" is capable of introducing a nucleic acid sequence into target cells. For example, a vector may contain a coding sequence that can be expressed in target cells. For the purposes of this invention, "vector construct," "expression vector," and "gene transfer vector" generally refer to any nucleic acid construct capable of inducing the expression of a target gene, which is useful for introducing the target gene into target cells. Therefore, this term includes cloning and expression vehicles, as well as embedded vectors.

[0053] An "expression cassette" comprises any nucleic acid construct capable of inducing the expression of a target gene / coding sequence and any RNA transcript, including non-coding RNA, such as shRNA, microRNA, siRNA, and antisense RNA. Such a cassette can be constructed into a "vector," "vector construct," "expression vector," or "gene transfer vector" for introducing the expression cassette into target cells. Thus, the term encompasses cloning and expression vehicles, as well as viral vectors.

[0054] Nucleic acids are "operably ligated" when they are placed in a functional relationship with another nucleic acid sequence. For example, signal sequence DNA is operably ligated to polypeptide DNA when expressed as a preprotein involved in polypeptide secretion; promoters or enhancers are operably ligated to coding sequences when they affect sequence transcription; or ribosome binding sites are operably ligated to coding sequences when they are positioned to facilitate translation. Generally, "operably ligated" means that the ligated DNA sequences are contiguous, and in the case of secretion readers, contiguous and in the reading stage. However, enhancers do not need to be contiguous. Ligation is achieved by ligation at a convenient restriction site. If such a site does not exist, synthetic oligonucleotides... The adapter or linker is used according to normal practice.

[0055] The expression vector will contain a promoter that is recognized by the autologous leukocyte or host cell for mRNA expression and is operably ligated to the collagen VII coding sequence. The promoter is an untranslated sequence located upstream (5′) of the start codon of a structural gene (generally within about 100 bp to 1000 bp) that controls the transcription and translation of a specific nucleic acid sequence to which it is operably ligated. Such promoters are usually classified into two classes: inducible and constitutive. Inducible promoters are those that initiate an increased level of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients or changes in temperature. A large number of promoters recognized by various potential host cells are well known. Heterogeneous promoters are generally preferred because they allow for greater transcription and higher yields.

[0056] Transcription from vectors in mammalian host cells can be controlled by heterogeneous mammalian promoters, such as actin promoters, PGK (phosphoglyceride kinase), or immunoglobulin promoters, or heat shock promoters, from the genomes of viruses, e.g., polyomaviruses, fowlpox virus, adenoviruses (e.g., adenovirus 2), bovine papillomavirus, arowanasarcoma virus, cytomegalovirus, retroviruses, hepatitis B, and Simianvirus 40 (SV40). However, such promoters must be compatible with the host cell system. Early and late promoters of the SV40 virus can be conveniently obtained as SV40 restriction fragments that also contain the SV40 virus origin of replication. The immediate-type early promoter of human cytomegalovirus can be conveniently obtained as a HindIII E restriction fragment.

[0057] Transcription in higher eukaryotes is often increased by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically around 10 bp to 300 bp in length, that act on promoters to increase transcription. Enhancers are relative orientation and position-independent, found at the 5′ and 3′ positions of transcription units within introns and within the coding sequence itself. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, enhancers derived from eukaryotic cell viruses are typically used. Examples include the SV40 enhancer posterior to the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer posterior to the origin of replication, and the adenovirus enhancer. Enhancers can be spliced ​​into expression vectors at the 5′ or 3′ position of the coding sequence, but are preferably located 5′ from the promoter.

[0058] Expression vectors used in eukaryotic host cells will also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are generally available from the 5′ and sometimes 3′ untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of mRNA.

[0059] Vectors and systems for the integration of expression cassettes into cells are known in the art, but are not limited to retroviral vectors. Many vectors useful for introducing foreign genes into target mammalian cells are available. Retroviral vectors have been shown to be particularly useful. A combination of retrovirus and a suitable packaging strain may be used, and the capsid protein will function to infect the target cells. Typically, the cells and virus are incubated in culture medium for at least about 24 hours. The cells are then expressed at short intervals, for example, in some applications. The vectors are grown in culture medium for 24-73 hours, or at least two weeks, and can be grown for five weeks or more before analysis. Commonly used retroviral vectors are "defective," meaning they cannot produce the viral proteins necessary for productive infection. Vector replication requires growth in a packaging cell line.

[0060] The host cell specificity of retroviruses is determined by the envelope protein, env(p120). Envelope proteins are provided by packaging cell lines. There are at least three types of envelope proteins: ecotropic, amphotropic, and xenotropic. Retroviruses possessing amphotropic envelope proteins, such as 4070A (Danos et al., cited above), can infect most mammalian cell types, including human, dog, and mouse. Amphotropic packaging cell lines include PA12 (Miller et al. (1985) Mol.Cell.Biol.5:431B437); PA317 (Miller et al. (1986) Mol.Cell.Biol.6:2895B2902); and GRIP (Danos et al. (1988) PNAS 85:6460B6464). Retroviruses packaged with xenotropic envelope proteins, such as AKR env, can infect most mammalian cell types except mouse cells. The 5′ and 3′ sequences of retroviruses are long terminal repeats (LTRs). A number of LTR sequences, including MMLV-LTR, HIV-LTR, AKR-LTR, FIV-LTR, and ALV-LTR, are known in the art and can be used. The 5′ LTR acts as a strong promoter, triggering the transcription of the introduced gene after integration into the target cell genome.

[0061] Keratinocyte Newly collected primary keratinocytes can be obtained from skin punch biopsies, isolated, and transduced after a culture period to isolate keratinocytes from dermal cells. Epithelial keratinocytes grown in vitro can form sheets. In some embodiments, transduced cells are administered to the patient approximately 1–100 days, 1–50 days, 1–20 days, 1–10 days, 1–5 days, 1–3 days, 1–2 days, or within 1 day from the time the cells were transduced.

[0062] As is known to those skilled in the art, any of the various culture media may be used in this method (see, for example, Current Protocols in Cell Culture, 2000-2009 by John Wiley & Sons, Inc.). Examples of media include, but are not limited to, keratinocyte medium, which may be serum-free and may contain appropriate keratinocyte supplements.

[0063] This disclosure provides a method for treating epidermolysis bullosa (EB) in a subject, the method comprising, obtaining a population of skin cells of the subject, ex vivo modifying the skin cells by integration of a gene construct encoding a functional (e.g., full-length wild-type) human collagen VII (COL7A1) protein, culturing the gene-modified cells to form a keratinocyte sheet, and transplanting a graft of the keratinocyte sheet into a skin wound bed, or further therefrom. This disclosure also relates to the use of a population of skin cells for treating epidermolysis bullosa (EB) in a subject, the population of skin cells being modified by transduction with a virus containing a gene construct encoding a full-length wild-type human collagen VII (COL7A1) protein to obtain a population of transduced skin cells having a proviral genome copy number (PGCN), the PGCN of the transduced population of skin cells being 3 or less. In some embodiments, the gene-modified cells are cultured in DFF31 medium, which comprises, or is essentially therefrom, or further therefrom, Dulbecco's Modified Eagle Medium and F12 Medium. In one embodiment, skin cells contain an expression construct or The keratinocytes are modified by transduction with a virus that is essentially or further derived from them, and the virus includes, essentially or further derived from, a retrovirus, AAV (adeno-associated virus), or a lentivirus. In another embodiment, the retrovirus is an LZRSE virus. In one embodiment, the retrovirus is a GalV pseudotype. In further embodiments, the keratinocytes thus transduced meet pre-release criteria of a viral transduction efficiency (VTE) greater than 50% and a proviral genome copy number (PGCN) of 3 or less. In some embodiments, the PGCN is 2.5, 2, 1.5, or less than 1. In another embodiment, the PGCN is 3-20, 20-40, 40-60, 60-80, or 80-100. In another embodiment, the PGCN is greater than 100. In some embodiments, the keratinocyte sheets are of different sizes. Those skilled in the art can determine the size of the keratinocyte sheets.

[0064] In some embodiments, endogenous mutations, dysfunctions, or truncated C7 genes are replaced using a CRISPR / Cas system (or a vector encoding the CRISPR / Cas system) as described herein, and a “donor” sequence (e.g., functional COL7A1 cDNA or C7 gene or full-length wild-type COL7A1 cDNA or gene) that is inserted into the gene after targeted cleavage. CRISPR / Cas systems are found in 40% of bacteria and 90% of archaea, and the complexity of these systems varies. See, for example, U.S. Patent No. 8,697,359, incorporated by reference as a whole. CRISPR loci (regularly clustered, short-spacing palindromic repeats) are regions in the genome of an organism in which short segments of foreign DNA are integrated between short-repeat palindromic sequences. These loci are transcribed, and the RNA transcript ("pre-crRNA") is processed into short CRISPR RNA (crRNA). There are three types of CRISPR / Cas systems that incorporate all of these RNAs and proteins known as "Cas" proteins (CRISPR-related). Both types I and III have a Cas endonuclease that processes pre-crRNA and, when fully processed into crRNA, assembles a multi-Cas protein complex capable of cleaving nucleic acids complementary to crRNA. A CRISPR / Cas system that binds to a target site in a desired region of an endogenous gene in the genome (e.g., an endogenous or safe harbor gene, or a regulatory gene or its DNA target), the CRISPR / Cas system includes one or more engineered single guide RNAs that recognize the target gene and its functional domain (e.g., a transcriptional regulatory domain and / or nuclease domain).In some embodiments, the CRISPR / Cas system described herein may bind to and / or cleave a region of interest (e.g., an endogenous C7 gene derived from EB tissue) within or adjacent to a gene, such as a leader sequence, trailer sequence, or intron, or within a non-transcriptional region either upstream or downstream of a coding region. In certain embodiments, CRISPR / Cas binds to and / or cleaves a gene, such as a mutated, dysfunctional, or truncated C7 gene.

[0065] In one embodiment, the wound does not contain uncorrected wound bed keratinocytes. In one embodiment, the wound is treated to excise uncorrected wound bed keratinocytes. In another embodiment, the subject has recessive dystrophic epidermolysis bullosa (RDEB). In a different embodiment, the subject is human.

[0066] In another embodiment, the keratinocyte sheet undergoes one or more tests selected from the group consisting of VTE testing, PGCN testing, sterility testing, endotoxin testing, mycoplasma testing, Gram staining sterility testing, LEAES viability testing, post-release testing, RCR testing, cytotoxic T cell assay, anti-C7 LH24 mAb characterization, electron microscopy, immunoelectron microscopy, immunofluorescence staining, C7 expression, and AF analysis. In one embodiment, immunofluorescence staining is performed directly This includes, is essentially, or is further derived from, direct or indirect immunofluorescence staining.

[0067] In some embodiments, the keratinocyte sheet is placed on a cell-free matrix, a collagen matrix, or a biocompatible mesh. In one embodiment, the biocompatible mesh is made from thermoplastic resin, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofiber polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, foamed polytetrafluoroethylene, nylon, silicon, or any combination thereof.

[0068] In some embodiments, skin cells include, are essentially, or are further derived from keratinocytes. In one embodiment, skin cells include, are essentially, or are further derived from stem cells. In another embodiment, the method further includes, are essentially, or are further derived from differentiating stem cells into keratinocytes. In one embodiment, stem cells differentiate before, after, or during transduction. In some embodiments, stem cells differentiate before transduction (e.g., into keratinocytes or corneal epithelial cells). In some embodiments, stem cells differentiate after transduction. In further embodiments, stem cells differentiate during transduction.

[0069] Patients with RDEB may frequently develop debilitating and painful corneal erosions. Therefore, methods for treating corneal erosions in subjects are also provided in this disclosure, the methods comprising, or essentially, or further comprising, obtaining a population of corneal cells from a subject; modifying the corneal cells ex vivo by integration of a gene construct encoding a functional (e.g., full-length wild-type) human collagen VII (COL7A1) protein; culturing the gene-modified cells to form a corneal cell sheet; and transplanting a graft of the corneal cell sheet onto the corneal surface. In some embodiments, the corneal cells include, or essentially, or further comprise corneal epithelial cells. In other embodiments, the corneal cells include, or essentially, or further comprise stem cells. In some embodiments, the methods further comprise, or essentially, or further comprise differentiating stem cells into corneal epithelial cells.

[0070] In some embodiments, corneal cells are modified by transduction with a virus containing a genetic construct, the virus including, essentially, or further comprising a retrovirus, lentivirus, or AAV. In one embodiment, the retrovirus is the LZRSE virus. In some embodiments, the retrovirus is a GalV pseudotype. In some embodiments, the corneal cells thus transduced meet pre-release criteria of a viral transduction efficiency (VTE) greater than 50% and a proviral genome copy number (PGCN) of 3 or less. In some embodiments, the PGCN is 2.5, 2, 1.5, or less than 1. In another embodiment, the PGCN is 3-20, 20-40, 40-60, 60-80, or 80-100. In yet another embodiment, the PGCN is greater than 100. In some embodiments, the subjects have recessive dystrophic epidermolysis bullosa (RDEB). In different embodiments, the subjects are human.

[0071] In one embodiment, the corneal cell sheet undergoes one or more tests selected from the group consisting of VTE testing, PGCN testing, sterility testing, endotoxin testing, mycoplasma testing, Gram staining sterility testing, LEAES viability testing, post-release testing, RCR testing, cytotoxic T cell assay, anti-C7 LH24 mAb characterization, electron microscopy, immunoelectron microscopy, immunofluorescence staining, C7 expression, and AF analysis. In one embodiment, immunofluorescence staining includes, is essentially, or further than direct or indirect immunofluorescence staining. In some embodiments, the corneal cell sheet is a cell-free matrix, a collagen matrix or placed on a biocompatible mesh.

[0072] In one embodiment, the biocompatible mesh may be made from non-reabsorbable materials, including, but not limited to, biocompatible metals such as titanium alloys, stainless steel, cobalt-chromium alloys, and nickel-titanium alloys. In another embodiment, layers of the biocompatible mesh may be made from non-reabsorbable polymer materials, including, but not limited to, thermoplastic resins, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefins, uncoated monofilament polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, foamed polytetrafluoroethylene, nylon, any polymer or aliphatic hydrocarbon containing one or more double bonds, any other suitable porous material, or any other suitable porous material that can be bent or formed into other shapes.

[0073] In another embodiment, the biocompatible mesh may consist of synthetic or bioabsorbable polymer materials, including, but not limited to, polyglycolic acid, poly-L-lactic acid (PLLA), poly-D, L-lactic acid (PDLA), trimethylene carbonate (TMC), poly-ε-caprolactone, poly-P-dioxanone, lactide and glycoside copolymer (PLGA), polyhydroxy-3-butyrate, collagen, hyaluronic acid, silk, biocellulose, other protein-based polymers, polysaccharides, poly(DTE carbonate), polyalylate, PLLA, PLDA, or blends of PLGA and TMC, as well as other combinations of these polymers.

[0074] In one embodiment, the biocompatible mesh is made from thermoplastic resin, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofiber polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, foamed polytetrafluoroethylene, nylon, silicon, or any combination thereof.

[0075] Compositions comprising, essentially, or further comprising keratinocyte sheets are also provided in this disclosure, the keratinocyte sheets being prepared by a process comprising, essentially, or further comprising the steps of: obtaining a population of skin cells of interest; modifying the skin cells ex vivo by integrating a gene construct encoding a functional (e.g., full-length wild-type) human collagen VII (COL7A1) protein; and culturing the gene-modified cells to form a keratinocyte sheet. In some embodiments, the skin cells comprise, essentially, or further comprising keratinocytes. In one embodiment, the skin cells comprise, essentially, or further comprising stem cells. In another embodiment, the stem cells differentiate into keratinocytes. In some embodiments, the stem cells differentiate before, after, or during transduction.

[0076] Further provided are pharmaceutical compositions comprising, essentially, or further comprising keratinocyte sheets, wherein the keratinocyte sheets comprise, essentially, or further comprising skin cells into which a gene construct encoding a functional COL7A1 protein is ex vivo integrated. In one embodiment, the skin cells are obtained from a subject. In some embodiments, the subject is human. In some embodiments, the skin cells are differentiated from stem cells into which a gene construct encoding a functional COL7A1 protein is ex vivo integrated. In one embodiment, the subject is RDEB-affected. In some embodiments, the skin cells or stem cells are transduced with a virus containing the gene construct, the virus comprising a retrovirus, lentivirus, or AAV.

[0077] In one embodiment, the functional COL7A1 protein is the full-length wild-type human COL7A1 protein. In one embodiment, the functional COL7A1 protein comprises, essentially, or further comprises a genetic modification from the full-length wild-type human COL7A1 protein. In another embodiment, the functional COL7A1 protein comprises, essentially, or further comprises a genetic modification from the full-length wild-type human COL7A1 protein, and the genetic modification is conserved. In a further embodiment, the genetic modification comprises, essentially, or further comprises insertions, deletions, and / or mutations.

[0078] Pharmaceutical compositions comprising, essentially, or further comprising corneal cell sheets are also provided in this disclosure, wherein the corneal cells comprise corneal cells into which a gene construct encoding a functional COL7A1 protein has been ex vivo integrated. In some embodiments, the corneal cells are differentiated from stem cells into which a gene construct encoding a functional COL7A1 protein has been ex vivo integrated. In other embodiments, the corneal cells are obtained from a subject. In one embodiment, the subject is affected by RDEB. In some embodiments, the subject is human. In some embodiments, the corneal cells or stem cells are transduced with a virus comprising the gene construct, the virus comprising a retrovirus, lentivirus, or AAV. In one embodiment, the retrovirus is LZRSE virus. In some embodiments, the retrovirus is GalV-pseudotype. In further embodiments, the transduced cells meet pre-release criteria of a viral transduction efficiency (VTE) greater than 50% and a proviral genome copy number (PGCN) of 3 or less.

[0079] In one embodiment, the functional COL7A1 protein is the full-length wild-type human COL7A1 protein.

[0080] In certain embodiments, cells are cultured for 1 to 21 days. In further embodiments, cells are cultured for 7, 14, 21 days or longer. Thus, cells can be cultured under suitable conditions for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 days or longer. Cells may be replated, and the culture medium and supplements may be added or modified as necessary using techniques known in the art.

[0081] In certain embodiments, genetically modified keratinocytes can be cultured under conditions and for a sufficient period of time such that at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells express the C7 transgene.

[0082] In one embodiment, the cell composition of the present disclosure comprises, essentially, or further comprises a population of genetically modified autokeratinocytes expressing an effective amount of native human C7 protein for EB treatment. The target cell population is grown in a sheet for transplantation into a subject in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such a composition may comprise a buffer, e.g., neutral buffered saline, phosphate-buffered saline, etc.; carbohydrates, e.g., glucose, mannose, sucrose, or dextran, mannitol; proteins, polypeptides, or amino acids, e.g., glycine; antioxidants, chelating agents, e.g., EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0083] The cell composition of this disclosure is administered by a method appropriate for the treatment of EB. The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease. However, the appropriate dosage can be determined through clinical trials.

[0084] Cells can be administered to a subject, usually in the form of skin grafts, by methods well known to those skilled in the art. A practitioner may, in part, determine a suitable route of administration for a particular subject based on the type and location of the disease. Transfected cells can be administered topically to the wound site.

[0085] Pharmaceutical formulations of manipulated cells for administration to a target are considered in this invention. Those skilled in the art will be familiar with the techniques for administering cells to a target. Furthermore, those skilled in the art will be familiar with the techniques and pharmaceuticals required to prepare these cell sheets before administration to a target.

[0086] In certain embodiments of the present invention, the pharmaceutical formulation is an aqueous composition comprising, essentially, or further comprising engineered cells modified to overexpress C7 and optionally prolyl-4-hydroxylase. In certain embodiments, the transduced cells are prepared using cells obtained from a subject (i.e., autologous cells).

[0087] The pharmaceutical compositions of the present invention comprise an effective amount of solution of transfected cells in a pharmaceutically acceptable carrier or aqueous medium. As used herein, “pharmaceutical formulation” or “pharmaceutical composition” includes any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Their use in therapeutic compositions should be considered unless any common media or agent is incompatible with the cells. Auxiliary active ingredients may also be incorporated into the composition. For human administration, formulations should meet sterility, pyrogenicity, general safety, and purity requirements as required by the FDA Center for Biologics.

[0088] Those skilled in the art will be familiar with techniques for producing sterile solutions for application by any other route. The size of the cell graft and the number of cells on the graft will be determined by those skilled in the art. In certain embodiments, multiple doses may be administered over several days, weeks, months, or years. A subject may be administered, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fifteen, sixteen, seven, eighteen, nineteen, twenty grafts from the same or different regions. In one embodiment, the subject may be re-grafted in the same or different regions. In another embodiment, a biological sample of the subject (e.g., keratinocytes or corneal cells) is stored under appropriate conditions. If the biological sample is stored, punch biopsy is not required if the subject requires a new graft. The stored biological sample can provide sufficient or supplementary information to determine the graft required by the subject.

[0089] Where an "effective dose" or "therapeutic dose" is indicated, the exact amount of the composition of this disclosure to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, and condition. The cell compositions described herein include all integer values ​​within these ranges: 1 to 100, 1 to 10 3 , 1-10 4 , 1-10 5 , 1-10 6 , 1-10 7 , or 10 7 It can generally be stated that the cells may be administered in quantities exceeding a single cell. Cell compositions may also be administered multiple times at these dosages. Cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). Determining the optimal dosage and treatment regimen for a particular patient is easily done by those skilled in the medical field by monitoring the patient for signs of the disease and, accordingly, adjusting the treatment. It can be decided that way.

[0090] In certain embodiments of this disclosure, keratinocytes genetically engineered using the methods described herein or other methods known in the art are administered to a patient together with any number of relevant therapeutic modalities (e.g., before, simultaneously with, or after). [Examples]

[0091] The following examples are provided to those skilled in the art to provide a complete disclosure and explanation of the methods of preparation and use of the present invention and are not intended to limit the scope of what the inventors consider to be the invention, nor do they represent all or only the experiments in which the following experiments were performed. While efforts have been made to maintain accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental errors and deviations should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or approximately atmospheric pressure.

[0092] All publications and patent applications cited herein are incorporated herein by reference in such a way that each individual publication or patent application is specifically and individually incorporated by reference.

[0093] This invention describes specific embodiments found or proposed by the inventors to include preferred forms for carrying out the invention. Those skilled in the art will understand that, in light of this disclosure, many modifications and changes can be made in the specific embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes in the potential DNA sequence can be made without affecting the protein sequence. Furthermore, for consideration of biological functional equivalence, changes can be made to the protein structure without affecting the biological action in kind or quantity. All such modifications are intended to fall within the scope of the appended claims.

[0094] Example 1 Cellular reprogramming of autologous cells as a treatment for recessive dystrophic epidermolysis bullosa (RDEB)

[0095] Recessive dystrophic epidermolysis bullosa (RDEB) is a severe vesicular skin disease caused by a functional mutation deficiency in the COL7A1 gene, which encodes type VII collagen (C7). C7 is a major component of anchoring fibrils (AF) that fix the epidermal basement membrane zone (BMZ) to the dermis. C7 performs this function through the use of multiple domains, including an amino-terminal non-collagenous NC1 domain that binds to the BMZ ligand, a central collagenous domain that assembles into a triple helix, and an NC2 domain that catalyzes the assembly of C7 into AF. Deficiency of these functional C7 domains in RDEB results in severe BMZ separation. This leads to widespread and painful vesicular formation, erosion, and scarring, which can subsequently lead to a malignant, often fatal, form of SCC that appears in the 20s and 30s. Despite advances in molecular diagnostics of this disease, current treatments are limited to palliative care. Clinical trials of allogeneic fibroblasts (Venugopal et al, J.Am.Acad.Dermatol.2013;69(6):898-908), bone marrow transplantation (Wagner et al, N.Engl.J.Med.2010;363(7):629-639), intradermal and intravenous delivery of bone marrow-derived mesenchymal stromal cells (Conget et al, Cytotherapy 2010;12(3):429-431; Petrof et al, J Investig.Dermatol.2015;135(9):2319-2321; Gorell et al, Pediatr.Dermatol.2015;32(2):220-225), and skin substitutes (Fa Labella et al. (Arch Dermatol. 2000;136(10):1225-342) have conducted studies with variable rates of efficacy and safety. Preclinical studies have investigated potential therapeutic modalities, including intravenous and topical C7, inducible pluripotent stem cells, and aminoglycosides.

[0096] Gene therapy is a potentially powerful tool for treating unigenic diseases such as RDEB. However, significant safety concerns have been raised after gene transfection in patients with severe combined immunodeficiency and Wiskott-Aldrich syndrome. While insertional mutagenesis remains a potential concern, the advantage of cutaneous gene therapy is that neoplasms can be more easily clinically evaluated because the transplanted tissue is positioned on the surface. Furthermore, genetically modified skin grafts have been efficiently used to treat a single patient with junctional EB, requiring long-term modification and without adverse side effects. A platform for long-term C7 expression in regenerated human epidermis was established and transplanted into immunodeficient mice (Siprashvili et al, Hum. Gene Ther., 2010, 21(10):1299-1310). Currently, this disclosure provides the results of a Phase I clinical trial of ex vivo gene transfection of LZRSE-COL7A1-engineered autologous epidermal sheets (LEAES) transplanted into subjects with severe RDEB.

[0097] clinical results Target and Treatment Of the 38 subjects screened, 8 consented to participate in this study, and 4 subjects were enrolled and received grafts (Figure 2). The subjects had heterozygous COL7A1 mutations, which are various compounds that result in the expression of the shortened C7 (NC1 domain). These were detected in keratinocyte medium by Western blotting (Figure 3), but not in tissue by IIF (Figures 1F, 5B, 6B, 7B, 8B, and 12). All subjects were males with a mean age of 23 (range: 18-32) and affected 4%-30% of the body with systemic lesions. Each had a history of severe disease with skin symptoms, including anemia, esophageal stricture, and pseudosyndactyly (Table 1).

[0098] Primary RDEB keratinocytes were isolated from intact skin and transduced with the LZRSE-COL7A1 retroviral vector, which had an efficiency of 70% per cell and an average of 0.8 proviral genome copies (Figure 1B-D). These were transplanted into five scarred and / or eroded wounds, as well as one induced wound, in each patient. The majority of the transplanted chronic wounds had been present for more than 5 years (Table 3). All 24 grafts were continuously monitored for percentage wound healing, infection, pain, and itching.

[0099] Effectiveness All subjects reported improved wound healing and skin strength, as well as reduced pain and itching at the graft site. The grafts showed reduced blister formation compared to baseline, and representative photographs from each subject are shown (Figures 1E, 5A, 6A, 7A, 8A, and 12). In contrast, untreated wounds showed continued blister formation (Figure 10).

[0100] One month after transplantation, 20 out of 24 wounds (83%) showed over 75% healing, while only four wounds showed 50% to 74% healing (Table 3). At 3 months, 21 out of 24 wounds (87%) showed over 75% healing, while 3 out of 24 wounds (13%) showed 50% to 74% healing (Table 3). At 6 months, 16 out of 24 wounds (67%) showed over 75% healing, 5 out of 24 wounds (21%) showed 50% to 75% healing, and only 3 out of 24 (13%) of the transplant sites showed blistering and were considered to be transplant failure (0% to 49% healing).

[0101] Molecular analysis of LEAES grafts was performed on 9 / 10 (90%) of the samples at 3 months and 8 / at 6 months. Twelve (66%) of the samples showed potent C7 expression. IIF analysis of LEAES showed proper localization of C7 at the epidermal-cutaneous junction in contrast to unmodified skin controls (Figure 1F, Figure 5B, Figure 6B, Figure 7B, Figure 8B, Figure 12). LEAES grafts showed fully differentiated epidermis with stratum spinosum and stratum granulosum, which was positive for keratin 14, keratin 1, and loricrin, which are epidermal markers similar to those of normal skin (Figure 1F, Figure 5B, Figure 6B, Figure 7B, Figure 8B, Figure 12). Among the negative samples, C7 was not detected in analytical biopsies obtained from Subject 2 at 6 months. However, anchoring fibrils (AFs) were present in the corresponding biopsies. At 6 months, C7 was identified in Subject 4 using an antibody specific to the NC1 domain (Figure 1F).

[0102] To evaluate the molecular structure of BMZ in the modified samples, biopsies obtained from LEAES grafts were also analyzed by transmission electron microscopy. At 3 months, 5 out of 7 samples (71%) showed a morphologically normal appearance and the frequency of NC2-reactive AF was revealed (Figure 1G). At 6 months, AF was detected in 4 out of 12 (33%) biopsies, and no AF was detected in the biopsy obtained from Subject 4 (Figure 1G).

[0103] safety No serious adverse events were reported. The most common adverse events (grade 1 or 2) were itching at the transplant site (n=3), followed by increased drainage at the transplant site (n=2), and no clinical signs of malignancy were observed. RCR and cytotoxic T cell assays were negative at all time points (Table 2).

[0104] Increased wound drainage was observed in 2 out of 24 wounds, but there were no signs of infection, including the absence of erythema, edema, pain, or tenderness at the sites. At 6 months, site Z of subject 3 had wound colonization (grade 2) and was considered to be graft failure (Table 3). Itching was observed in 3 out of 24 graft sites and two surrounding areas (grade 1).

[0105] The C7 immune response was closely monitored throughout this study. Subjects did not exhibit systemic autoimmune symptoms or increased blister formation outside the transplant site. In Subject 1, no antibodies in circulating blood or tissue-bound antibodies were observed by IIF or DIF (Table 2). Subject 2 at 3 months showed 1+ (mild) linear IgG, IgM, and IgA without complement, as determined by DIF analysis of two wounds (A and E). These immunoreactives were not observed 6 months after transplantation. Subject 3 showed a transient elevation of linear serum IgA (1:320) by IIF at 3 months, along with faint traces of 1+ linear histological staining of IgM and IgA by DIF at 6 months. In contrast, Subject 4 revealed a 1:160 titer of linear IgG antibodies in circulating blood at 1 and 3 months by IIF, along with 1-2+ IgG, IgA, C3, and IgM staining detected in three grafts by DIF at 3 months (Table 2). However, no systemic autoimmune symptoms or increased blister formation outside the graft were observed, and the C7-specific cytotoxic T cell assay was negative. At 6 months, serum antibody IgG and C3 levels decreased (1:40), and tissue-bound immune complexes were not detected in the graft (Table 2). Following the discovery of subepidermal linear immunodeposition in Subject 4, we re-evaluated Subject 4's baseline plasma anti-C7 antibody levels using Western blot analysis of purified C7 protein. In contrast to the negative baseline IIF data, Western blot analysis showed that Subject 4 was receptive to purified C7 both at baseline (1:300) and 3 months after serum transplantation (1:1000), indicating that Subject 4 was sensitive to exogenous C7 before graft placement (Figure 11A). Serum antibody reactivity in Subject 4 was confirmed both before and after graft placement within previously characterized carboxyl-terminal C7 pepsin fragments (Figure 11B).

[0106] Consideration Genetic modification of RDEB presents substantial challenges, such as the efficient delivery of large transgenes containing COL7A1 cDNA exceeding 9kb. Here, this disclosure provides an in-human study of genetically modified autologous epidermal keratinocyte transplantation with promising efficacy and acceptable safety in four subjects with RDEB. Genetically modified epidermal cells regenerated functional autorenewing epidermis in over 67% of transplant sites tested 6 months after transplantation, and C7 was detectable up to 1 year per subject (Figure 12). This was a significant improvement over allogeneic keratinocyte grafts, where only 2 / 9 (22%) of chronic wounds healed at 18 weeks. LEAES grafts also resulted in better wound healing outcomes compared to allogeneic fibroblast injections. This showed some initial improvement in wound healing compared to placebo or intradermal or intravenous injection of BM-MSCs, but did not show long-term differences. Case reports of gene therapy for junctional epidermolysis bullosa using similar methodologies have shown modifications of up to six years, outweighing long-term therapeutic effects. The six-month sustained C7 expression observed by the inventors in LEAES grafts spans the duration of six epidermal turnover cycles, indicating that the inventors are successfully targeting stem cells with their gene transfer technology.

[0107] Our research has shown that improved wound healing and increased skin durability were directly associated with detectable full-length C7 production and AF formation in the BMZ. Both were present at each study time point, but were detected with variable efficiency, i.e., 66–90% for C7 expression and 33–71% for AF formation. Variability in biopsy sampling may be due to either heterogeneous populations of modified epidermal cells or partial graft uptake. In some embodiments, the graft region could be an area that the subject felt was beneficial to their quality of life. However, during the critical first few days after graft placement, parts of the region were difficult to fix or protect from mechanical friction (e.g., lower back E, left shoulder B, Table 3, posterior shoulder D, Figure 6A of subject 2). Furthermore, since Subject 2 had the shortest post-graft fixation time compared to other trial participants, shorter observed post-graft fixation times may negatively affect graft uptake, as indicated by the reduction in overall wound healing due to IF and the absence of detectable C7 in the 6-month biopsy sampled from Subject 2 (Table 1). In addition, the induced wound showed good healing ability but exhibited the lowest C7 expression. Without destructive methods such as electrocautery, residual uncorrected wound bed keratinocytes may have hindered the establishment of the covering LEAES graft. These findings suggest that wound bed preparation techniques that more efficiently excise wound bed keratinocytes may improve graft uptake.

[0108] Regarding safety, several adverse events were reported, all of which were mild. Subjects did not show any evidence of RCR in the blood or squamous cell carcinoma in the grafts at any given time, but long-term monitoring for potential adverse events is ongoing. All molecular substitution approaches, including gene transfer, carry the risk of undesirable immune responses to therapeutic products, particularly in subjects with null mutations. In this study, all subjects expressed a truncated C7 molecule containing the NC1 domain, which is considered to be the antigenic portion of the protein and therefore minimizes the risk of potential immune responses. No evidence of C7-related cytotoxic T cell activity was observed at any given time in any of the subjects in the study. However, the IIF and DIF studies of subject 4 revealed lower serum IgG titers at 1 and 3 months, complement C3 fixation at 3 months, and 6 months after transplantation (Table 2). While the NC1 domain has been reported as the most antigenic portion of C7, the carboxyl-terminal NC2 domain also contains a small antigenic epitope, and the presence of anti-C7 autoantibodies in RDEB patients has been reported numerous times in previous publications. The finding that Subject 4 had detectable anti-C7 antibodies before transplantation using Western blot analysis (which were not identified during the screening process with the CLIA-certified IIF assay) suggests that more sensitive and standardized methods should be developed to assess baseline immunocross-reactivity in future therapeutic studies.

[0109] In conclusion, genetically modified autologous epidermal skin grafts demonstrated increased C7 deposition and reduced blister formation in patients with severe RDEB, and the patient population had other specific treatment options available. Larger studies involving younger RDEB subjects are planned to evaluate the long-term efficacy and safety of this approach.

[0110] [Table 1]

[0111] [Table 2]

[0112] [Table 3]

[0113] [Table 4]

[0114] method Research design This is a Phase I open-label clinical trial (NCT01263379). The primary objective was to obtain safety and efficacy data in RDEB subjects transplanted with genetically modified autologous keratinocytes transduced with a retroviral vector containing the full-length COL7A1 coding sequence (Figure 1A). Between October 2013 and February 2015, LEAES was transplanted into four adult RDEB subjects. Data from all subjects will be collected with at least 6 months of follow-up. This study is being conducted by the Food and Drug Administration (IND#13708) and the Stanford IRB (Protocol#14563). Approved by ).

[0115] subject The target population is individuals aged 18 or older who have been clinically diagnosed with RDEB and are suitable for LEAES transplantation, with a total length of 100 cm². 2 ~200cm 2The subjects had erosions that were open to the extent of the area. The subjects were also able to undergo general anesthesia and were selected based on a screening protocol. In this, RDEB was confirmed by genetic testing (GeneDx, Gathersburg, Maryland). The presence of the NC1 domain of C7 was evaluated by Western blotting of cultured keratinocyte (KC) supernatant and indirect immunofluorescence microscopy (IIF) of skin biopsy specimens. The absence of full-length C7 and mature AF in the biopsy specimens was confirmed by IIF and immunoelectron microscopy (IEM) using an LH24 antibody specific to the carboxyl-terminal NC2 domain of C7 (Figure 9). IgG, IgA, IgM, and C3 bound to circulating blood and tissue were analyzed by IIF using direct immunofluorescence (DIF) of serum from primate esophagus and biopsy specimens, respectively. Subjects with significant non-RDEB complications, including HIV, hepatitis, systemic infection, or cardiac abnormalities, were excluded. Clinically significant anemia was treated pre-transplant.

[0116] Test treatment Two 8 mm punch biopsy materials were obtained from intact, non-scarred areas of skin for LEAES production. Baseline blood samples were obtained for CBC, complete biochemical testing (CMP), and replication-resistant tretrovirus (RCR) and C7-sensitive cytotoxic T cell assays. Autologous keratinocyte-derived skin biopsies were transduced with LZRSE-COL7A1 and used to generate eight LEAES grafts (Figure 4). Six grafts were applied to non-infected, eroded, and / or scarred wound sites lacking clinical findings of SCC. Of the six graft wound sites (A, B, C, D, E, Z, Table 3), one wound for each subject at the time of surgery was created by mechanical friction ("site Z"). Under general anesthesia, the wound beds were cauterized to minimize the possibility of retained epidermal stem cells. LEAES grafts were fixed to the wound beds via dissolvable sutures after wound bed preparation. Subjects 3 and 4 agreed to have small Indian ink tattoos placed on the corners of each graft to aid in graft identification during follow-up. The grafts were covered with standard wound dressings and topical mupirocin, which were removed 5–7 days after transplantation.

[0117] Endpoints and evaluation Subjects were followed up at 1, 3, 6, and 12 months after transplantation. At each study visit, serum samples were evaluated for autoantibodies in circulating blood by IIF, C7-sensitive cytotoxic T cell assay, CBC, and CMP. The presence of RCR in serum was assessed at 3 and 6 months. At each visit, representative grafts were biopsied and tissue-bound immunoglobulins and complement were evaluated by DIF, C7 expression by IIF, and the presence of anchoring fibrils by IEM. Wounds were clinically evaluated and assessed using digital photography and / or Canfield Vectra cameras as healed compared to baseline, with 100%–75% healed (defined as significant wound healing), 74%–50% healed, 49%–25% healed, and less than 25% healed.

[0118] Materials and Reagents All materials and reagents used in the production of LEAES were free of accidental viruses, based on the analytical certification provided by the manufacturer. Each lot of tissue medium containing bovine-derived reagents was tested for accidental viruses using a commercially available 9CFR absorbance test (American BioResearch, Pullman, Washington) and found to be negative.

[0119] Isolation and propagation of RDEB keratinocytes Skin samples were obtained as two 8mm punch biopsies, and 30 μg / mL amikacin (HIk The samples were introduced into 35 mL of biopsy collection medium 50 / 50A (50% keratinocyte medium 154 (Life Technologies, Carlsbad, California) containing human keratinocyte growth supplement, and 50% standardized keratinocyte serum-free medium (Life Technologies, Carlsbad, California) containing the supplement) along with 20 μg / mL vancomycin (Sigma Aldrich, St. Louis, Missouri) and 0.5 μg / mL amphotericin B (USBiological, Salem, Massachusetts) (ma Pharmaceuticals, London, UK). To separate the epidermis from the dermis, the skin samples were placed in a dispase solution (Life Technologies, Carlsbad, California) containing 25 casein-degrading units / mL of dispase for 16–20 hours at 5°C. The following day, the epidermis was carefully peeled from the dermis and placed in TrypLE Select 10X solution (Life Technologies, Carlsbad, California) for 20–30 minutes at 37°C. This solution was spun down to 1200 rpm to obtain a keratinocyte pellet. The cells were washed once with phosphate-buffered saline (PBS, Life Technologies, Carlsbad, California), and the keratinocytes were plated in 50 / 50A medium on PureCoat Collagen I Mimetic Cultureware (Corning Life Sciences, Tewkesbury, Massachusetts). After the keratinocytes reached 60-70% confluence, the cells were treated with TrypLE Select 10X and plated for viral transduction. At least 4 × 10⁶ cells were needed to initiate the transduction process. 6 It required individual cells.

[0120] Keratinocyte correction cGMP-grade GalV-pseudotyped LZRSE-COL7A1 virus containing full-length COL7A1 cDNA under MLVLTR control was prepared at the Indiana University Vector Production Facility using current Good Manufacturing Practices as described in Siprashvili et al 2010. A map of the pLZRSE-COL7A1 plasmid is shown in Figure 13, and the complete sequence is shown in Sequence ID No. 1. Viral transduction was performed by overlaying 12 mL of viral supernatant onto each plate and by centrifugation of cells at 1250 rpm and 32°C for 1 hour. After centrifugation, the viral supernatant was removed by washing with PBS and 50 / 50V medium, which was used for the growth of modified keratinocytes. Transduction was repeated as needed, as long as the modified KC continued to meet the pre-release criteria of a viral transduction efficiency (VTE) of more than 50% and a proviral genome copy number (PGCN) of 3 or less.

[0121] Pre-release testing VTE testing. VTE testing was performed using IF technology with anti-type VII collagen monoclonal antibodies NP32, NP185, or anti-type VII collagen polyclonal antibody FNC1. Cells were fixed in a methanol / acetone mixture solution, permeabilized with detergent, and incubated with anti-C7 primary antibody at room temperature for 1 hour. After multiple washes, secondary antibody conjugated with Alexa Fluor555 dye was added and incubated for another 1 hour. Cell nuclei were labeled with Hoechst 33342 for 10 minutes, washed, and mounted with gold anti-fade reagent (Life Technologies, Carlsbad, California). VTE was determined by counting the ratio of blue nuclei to C7-positive cells. At least 50% of cells were positive for C7 expression to meet the pre-release criteria.

[0122] PGCN test qP of genomic DNA isolated from modified RDEB KC after retroviral transduction PGCN testing was performed via CR analysis. Genomic DNA was collected using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Germany) in 3 × 10⁶ units. 6 The DNA was purified from individual modified cells. Standard spectrophotometric techniques were used to quantify the DNA, which was then used for qPCR analysis. Proviral dose was determined using standard curves for template threshold cycles (Ct) and Ct dependence from the amount of plasmid DNA control. The mean PGCN was calculated using the formula PGCN = (TPCN × 6.16 pg) / (Ctempl. × 10³ pg) (where TPCN = total proviral copy number). 6.16 pg = amount of genomic DNA in somatic cells. Ctempl. = amount of template used in PCR, in nanograms. To meet the pre-release criteria, an average of 3 or fewer proviral genome copies were present for each keratinocyte genome.

[0123] Sterility test Culture supernatant samples were tested for sterility by membrane filtration using a Millipore Steritest system (Pacific BioLabs, Hercules, California), designed to eliminate potential false negatives from antibiotics present in the culture medium. After filtration and washing, samples were placed in soybean casein digest medium and liquid thioglycolate medium and incubated for 14 days. Samples were observed daily for signs of microbial contamination.

[0124] Endotoxin test Culture supernatant samples were evaluated using the Limulus Amebocyte Lysate (LAL) QCL-1000 assay (Lonza, Switzerland, Basel) according to the manufacturer's recommendations. The results of this test were less than 1.0 EU / mL, which meets the product release requirements.

[0125] Mycoplasma test In accordance with the manufacturer's requirements, cell culture supernatants were tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza, Switzerland, Basel). The results of this test were less than 0.9, which is required to meet the product release requirements.

[0126] LEAES Start and Processing Once the modified keratinocytes reached 100% confluence, the LEAES initiation process was started, and the growth medium was changed from 50 / 50V to epidermal sheet-forming medium DFF31. This is Dulbecco's Modified Eagle Medium (Life) containing 10% fetal bovine serum (Lonza, Basel, Switzerland), 36 ng / mL hydrocortisone (Spectrum, New Brunswick, New Jersey), 25 μg / mL adenine (Sigma Aldrich, St. Louis, Missouri), 5 μg / mL recombinant human insulin (Sigma Aldrich, St. Louis, Missouri), 2 ng / mL liothyronine (Spectrum, New Brunswick, New Jersey), 5 μg / mL bovine transferrin (Millipore, Billrica, Massachusetts), 10 ng / mL recombinant epidermal growth factor (R&D Systems, Minneapolis, Minnesota), and 30 μg / mL amikacin (Hikma Pharmaceuticals, London, UK), as well as 20 μg / mL vancomycin (Sigma Aldrich, St. Louis, Missouri). It consists of Technologies (Carlsbad, California) and F12 medium (Lonza, Basel, Switzerland).

[0127] LEAES assembly and transport LEAES assembly was started on the day of transplantation. Enzymatic digestion with dispase (Life Technologies, Carlsbad, California) was performed on the epidermis. The sheets were released from the plate surface at 37°C for 20–30 minutes. To remove any residual culture medium and dispase, the epidermal sheets were washed at least five times with 50 / 50VC. They were then fixed to size-matched petroleum gauze with surgical hemoclips and marked on the basolateral side with sterile black sutures. The assembled LEAES were immersed in transport medium 50 / 50VC and sealed with a gas-permeable sterile membrane. The LEAES epidermal grafts were then transported to the operating room for transplantation.

[0128] Release test Gram staining sterilization test. On the day of LEAES release, culture medium samples were sent to the clinical laboratory at Stanford Hospital Clinical Laboratory for rapid Gram staining. Negative test results were used as the LEAES lot release criterion.

[0129] LEAES Survival Study LEAES samples were incubated for 20 minutes with a nuclear dye mixture containing Hoechst33342 and SYTOX Green stains to perform a viability test. Product release was induced when the ratio of SYTOX Green stain to Hoechst33342 stain was calculated to be 70% or higher.

[0130] Post-release testing Samples of culture media and LEAES grafts were submitted for release testing. Test results expected after graft implantation were anticipated due to the lengthy testing process. Safety plans were implemented if these "post-release" test results were substandard. Post-release criteria included additional sterility testing (see pre-release sterility testing), RCR testing (Indiana University Vector Production Facility), and mycoplasma testing (Bionique Testing Laboratories, Salanaclake, New York).

[0131] RCR (Randomized Controlled Research) LEAES and supernatant samples from LEAES cultures were subjected to the extended PG-4 S+L-cell plaque assay at the Indiana University Vector Production Facility, and the test result used as the release standard was "no evidence of RCR." At baseline, 3 months, and 6 months, blood samples were analyzed at the Indiana University Vector Production Facility, and the level of present GALV envelope (GALV-E) sequences was determined using quantitative polymerase chain reaction (Q-PCR). The validity of the blood sample volume was evaluated using a second probe and primer set for the human apolipoprotein B gene sequence. A standard curve using genomic 12 / 22 / 15 8DNA containing 10⁵, 10⁴, 10³, 10², and 10 copies of GALV-E sequences per 0.12 μg of genomic DNA was used as a positive control. The negative control contained untransduced human genomic DNA and water.

[0132] Cytotoxic T cell assay For the cytotoxic T cell assay, 15 mL of whole blood was collected at baseline, 1 month, 3 months, and 6 months. Peripheral blood mononuclear cells were isolated from the pia mater or whole blood using Ficoll-Paque (GE Healthcare) density gradient centrifugation. Adherent monocytes were then incubated in a petri dish for 2 hours and then harvested. CD4+ and CD8+ T lymphocytes were purified together by incubating non-adherent cells with anti-CD4 and anti-CD8 antibodies conjugated to paramagnetic microbeads using the MACS magnetic cell sorting kit (Miltenyi Biotech). 96-well PVDF filter plate Millipore cells were coated with monoclonal antibodies against IFN-γ (BD Pharmingen) or IL-4 (BD Pharmingen), blocked using RPMI medium containing 5% human AB serum, and washed with serum-free RPMI. CD4+ and CD8+ T lymphocytes (2 × 10⁶) were then processed. 5 Cells / well and gamma-irradiated monocytes (2.5 × 10⁻⁶) 4Cells (per well) were co-incubated on plates for 40 hours at 37°C in 5% CO2 humidified in an air incubator in the presence of 20 μg / ml IL-2. The culture medium contained either 10 μg / ml recombinant type VII collagen or 3 μg / ml concanavalin A (Sigma) to stimulate lymphocytes. After washing the plates, IFN-γ or anti-IL-4 secreted by individual cells was detected in situ by sequentially reacting each well with 1 μg / ml biotinylated anti-IFN-γ or anti-IL-4 monoclonal antibody (BD Pharmingen), followed by a 1:1000 dilution of streptavidin conjugate alkaline phosphatase (Roche). Detection was performed using BCIP / NBT chromogenic substrate (Promega). The reaction was stopped by washing with water, and spots were counted using a CTL ELISPOT reader. A negative control was performed in parallel using T cells without antigens, and the corresponding score was subtracted from the unknown.

[0133] Characterization of anti-C7 LH24 mAb LH24 mAbs were pre-identified and reacted with epidermal basement membrane 2. The specific absence of LH24 in the skin of C7 null RDEB patients in our study indicated that it recognized an epitope on C7. To further localize the LH24 reactivity on the C7 molecule, the LH24 reactivity to enzymatic digests of C7 containing NC1 and NC2 domains was tested by Western blotting. The NC1 domain containing the C7 fragment was generated from the digestion of purified C7 using highly purified bacterial collagenase (Worthington), as described above. Three NC2 12 / 22 / 15 9 containing the C7 fragment were generated after pepsin digestion of purified C7, as described above.

[0134] Electron microscopy 3 mm skin punch biopsies were prepared for electron microscopy by immersion for at least 1 hour in 1.5% glutaraldehyde / 1.5% paraformaldehyde in Dulbecco's serum-free medium (SFM) containing 0.05% tannic acid, followed by multiple rinses in SFM and post-fixation in 1% OsO4 for 60 minutes. The samples were washed in SFM, then dehydrated in a stepwise series of ethanol to 100%, rinsed in propylene oxide, and immersed in Spurr epoxy for a total of 2 hours, accelerated via microwave energy. The samples were polymerized at 70°C for 18 hours.

[0135] Immunoelectron microscopy 3 mm skin punch biopsy samples for immunoelectron microscopy were prepared by rinsing multiple times in SFM, then immersing overnight at 4°C in 1:5 diluted mouse IgM LH24 antibody specific to the NC2 region of collagen VII in SFM, rinsing multiple times in SFM, and then incubating overnight at 4°C in 1:3 diluted goat anti-mouse IgM miniature colloidal gold conjugate (Aurion) in SFM. After multiple rinsing in SFM, the samples were exposed on ice for 15 minutes in gold-sensitized solution (Nanoprobes), then rapidly warmed to 25°C and incubated for a further 5 minutes. The samples were then rinsed with ice-cold SFM and then fixed and embedded as described above. Indirect immunofluorescence (IIF): Human serum was placed on monkey esophagus and stained with antibodies against human IgA, IgM, IgG, and C3. Signals detected at antibody dilutions of 1:40 or higher are considered to be beyond the background.

[0136] Direct immunofluorescence (DIF) On December 22, 2015, 10 tissue samples were cut into 5-micrometer sections and stained with fluorophore-conjugated antibodies for human IgA, IgM, IgG, C3, and fibrinogen. Normal controls were performed concurrently. C7 expression and AF analysis: 3 mm skin punch biopsy samples were cut into 8-micrometer sections and analyzed by IIF using anti-type VII collagen polyclonal antibody FNC1 (against the NC1 domain of C7) or monoclonal antibody LH24 (against the NC2 domain of C7). In summary, sections were fixed in a methanol / acetone mixture solution, permeabilized with detergent, and incubated with anti-C7 primary antibody at room temperature (25°C) for 1 hour. After multiple washes, secondary antibody conjugated with Alexa Fluor 555 or 488 dye was added and incubated for 1 hour. Cell nuclei were labeled with Hoechst33342 for 10 minutes, washed, and mounted with gold anti-fade reagent (Life Technologies, Carlsbad, California). For epidermal markers, keratin 1, keratin 14, and loricrin antibodies obtained from Covance (Emeryville, California) were used. Biopsy material was considered positive for C7 expression if serial linear staining of type VII collagen was detected at the dermal-epidermal junction. Biopsy was considered positive for anchoring fibrils (AF) if gold conjugate particles representing NC2 domain-specific LH24 antibodies with ultrastructures exhibiting AF characteristics including density, thickness, curvature, arcuate, and loops were detected.

[0137] photograph For subjects 2–4, approximately five images of each graft site were taken from multiple angles using a Canfield Vectra 3D camera. These images were then stitched together to create a comprehensive 3D image. Using Mirror Software (Canfield, Fairfield, New Jersey), landmarks were selected, numbered to identify their corresponding locations in each image, and then fused into a single image. To accurately track the graft edges, fused images from follow-up visits were placed over the contour of the graft on day 0 and compared to the baseline image. Anatomical landmarks (e.g., tattoo dots) were reconfirmed to ensure correct contour placement. Additional images for subjects 2–4 and all images of subject 1 were obtained as needed using a digital camera (Canon Powershot).

[0138] Equal portions Although this disclosure is described in relation to the embodiments described above, it should be understood that the above description and examples are illustrative and not limiting the scope of this disclosure. Other aspects, advantages, and modifications within the scope of this disclosure will be apparent to those skilled in the art.

[0139] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in the 5′ to 3′ direction.

[0140] The embodiments described herein as exemplary may be adequately implemented in the absence of any elements(s) or limitations(s) not specifically disclosed herein. Therefore, terms such as “having,” “including,” and “containing” should be read broadly and without limitation. Furthermore, the terms and expressions used herein are used as descriptive and non-limiting terms, and there is no intention to use such terms and expressions except as any equivalents or parts thereof of the features shown and described, although it should be acknowledged that various modifications are possible within the scope of this disclosure.

[0141] Therefore, although this disclosure is specifically disclosed by specific embodiments, any features, modifications, improvements, and variations of the embodiments disclosed herein may be used by those skilled in the art, and such modifications, improvements, and variations should be understood to be within the scope of this disclosure. The materials, methods, and examples provided herein are specific These are representative and illustrative examples of embodiments and do not limit the scope of this disclosure.

[0142] The scope of this disclosure is broadly and generally described herein. Each of the narrower species and subgenera groups that fall under the general disclosure also forms part of this disclosure. This includes general descriptions with conditions or negative limitations that exclude any subject from a genus, whether or not the deleted material is specifically enumerated herein.

[0143] In addition, if any feature or aspect of the present disclosure is described in relation to the Markush group, those skilled in the art will recognize that embodiments of the present disclosure may also be described in relation to any individual member or subgroup of members of the Markush group.

[0144] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference as a whole, to the same extent that each is incorporated by reference individually. In case of any conflict, this specification shall prevail, including definitions.

[0145] This invention was made with government support under Contract AR055914, awarded by the National Institutes of Health. The government has certain rights to this invention.

[0146] Cross-reference of related applications This application claims priority under 119(e) of the United States Patent Act to U.S. Patent Application No. 62 / 274,700, filed on 4 January 2016, and U.S. Patent Application No. 62 / 414,533, filed on 28 October 2016, the contents of which are incorporated herein by reference.

Claims

1. A pharmaceutical composition comprising an engineered autologous epidermal sheet for use in a method of treating recessive dystrophic epidermolysis bullosa (RDEB) in human subjects who have one or more mutations in both copies of the human collagen VII (COL7A1) gene and are suffering from RDEB, The method described above is (a) The human subjects are subjected to one or more tests selected from a retroviral (RCR) test, a COL7A1-sensitive cytotoxic T cell assay, or a combination thereof. (b) Obtaining a population of skin cells including keratinocytes from the human subject, (c) Isolating a population of keratinocytes containing one or more mutations in the COL7A1 gene from the population of skin cells, and culturing the isolated population of keratinocytes on collagen 1 peptide in a first keratinocyte medium, (d) Transduction of isolated keratinocyte populations ex vivo with a retroviral vector containing a promoter operably linked to a gene construct encoding full-length wild-type human collagen VII (COL7A1) protein to generate genetically modified keratinocytes that meet the pre-release criteria of a viral transduction efficiency (VTE) of more than 50% and a proviral genome copy number (PGCN) of 3 or less, (e) Culturing the gene-modified keratinocytes in a first keratinocyte medium to form an autologous COL7A1 modified keratinocyte sheet, (f) The self-COL7A1 modified keratinocyte sheet is subjected to one or more pre-release tests selected from the group consisting of a VTE test, a PGCN test, and a retrovirus (RCR) test with replication ability. (g) Culturing and maturing the autologous COL7A1 modified keratinocyte sheet in a second medium containing DFF31 medium to form an engineered autologous skin sheet, (h) Assembling the manipulated autologous skin sheet, (i) Transplanting the assembled and manipulated autologous skin sheet graft into the RDEB-affected wound of the human subject. and Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the gene construct comprises nucleotides 409 to 11849 of SEQ ID NO:

1.

3. The pharmaceutical composition according to claim 1, wherein the pre-release standard for viral transduction efficiency (VTE) is approximately 70%, and the proviral genome copy number (PGCN) is approximately 0.

8.

4. The pharmaceutical composition according to claim 1, wherein the RDEB-affected wound does not contain uncorrected keratinocytes.

5. The pharmaceutical composition according to claim 1, further comprising cauterizing the RDEB-affected wound of the human subject to excise uncorrected keratinocytes before transplanting the assembled manipulated autologous skin sheet graft.

6. The pharmaceutical composition according to claim 1, wherein the keratinocyte sheet undergoes one or more tests selected from the group consisting of a sterility test, an endotoxin test, a mycoplasma test, a Gram stain sterility test, a manipulated autologous skin sheet viability test, a cytotoxic T cell assay, anti-C7 LH24 mAb characterization, electron microscopy, immunoelectron microscopy, immunofluorescence staining, C7 expression, and AF analysis.

7. The pharmaceutical composition according to claim 1, wherein the one or more pre-release tests are RCR tests.

8. The pharmaceutical composition according to claim 1, wherein the keratinocyte sheet is arranged on a cell-free matrix, a collagen matrix, or a biocompatible mesh.

9. The pharmaceutical composition according to claim 8, wherein the biocompatible mesh is made from a thermoplastic resin, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofiber polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, foamed polytetrafluoroethylene, nylon, silicon, or any combination thereof.

10. The pharmaceutical composition according to claim 1, wherein the population of skin cells includes stem cells.

11. The pharmaceutical composition according to claim 10, further comprising differentiating the stem cells into keratinocytes.

12. The pharmaceutical composition according to claim 1, further comprising subjecting a transplanted human subject to one or more tests selected from an RCR test, a COL7A1-sensitive cytotoxic T cell assay, or a combination thereof.

13. The pharmaceutical composition according to claim 1, wherein the retroviral vector is selected from Moloney mouse leukocyte virus (MoMLV), human immunodeficiency virus (HIV), AKR mouse leukocyte virus (AKR), feline immunodeficiency virus (FIV), or gibbon leukemia virus (ALV).

14. The pharmaceutical composition according to claim 1, wherein the retroviral vector is MoMLV.

15. The pharmaceutical composition according to claim 1, wherein the promoter is the MoMLVLTR promoter.

16. The pharmaceutical composition according to claim 1, wherein the retroviral vector is a MoMLV virus vector and the promoter is a MoMLVLTR promoter.

17. The pharmaceutical composition according to claim 1, wherein the average PGCN is 1 or less than 0.

5.

18. The pharmaceutical composition according to claim 1, wherein the average PGCN is 1.

5.

19. The pharmaceutical composition according to claim 1, wherein the average PGCN is 0.5 or more and 1.5 or less.

20. The pharmaceutical composition according to claim 1, wherein the first keratinocyte medium is a serum-free keratinocyte medium and / or comprises a human keratinocyte growth supplement.

21. The pharmaceutical composition according to claim 1, wherein the transduction of an isolated population of keratinocytes ex vivo is repeated at least twice.

22. A pharmaceutical composition comprising an engineered autologous epidermal sheet for use in a method of treating recessive dystrophic epidermolysis bullosa (RDEB) in human subjects who have one or more mutations in both copies of the human collagen VII (COL7A1) gene and are suffering from RDEB, The method described above is (a) The human subjects are subjected to one or more tests selected from a retroviral (RCR) test, a COL7A1-sensitive cytotoxic T cell assay, or a combination thereof. (b) Obtaining a population of skin cells including keratinocytes from the human subject, (c) Isolating a population of keratinocytes containing one or more mutations in the COL7A1 gene from the population of skin cells, and culturing the isolated population of keratinocytes in a first serum-free human keratinocyte medium, (d) Transduction of isolated keratinocyte populations ex vivo with a retroviral vector containing a promoter operably linked to a gene construct encoding functional human collagen VII (COL7A1) protein to generate genetically modified keratinocytes that meet the pre-release criteria of a viral transduction efficiency (VTE) of more than 50% and a proviral genome copy number (PGCN) of 1.5 or less, (e) Culturing the gene-modified keratinocytes in a first serum-free human keratinocyte medium to form an autologous COL7A1 modified keratinocyte sheet, (f) The self-COL7A1 modified keratinocyte sheet is subjected to one or more pre-release tests selected from the group consisting of a VTE test, a PGCN test, and a retrovirus (RCR) test with replication ability. (g) Culturing and maturing the autologous COL7A1 modified keratinocyte sheet in a second medium containing DFF31 medium to form an engineered autologous skin sheet, (h) Assembling the manipulated autologous skin sheet, (i) Transplanting the assembled and manipulated autologous skin sheet graft into the RDEB-affected wound of the human subject. and Pharmaceutical composition.

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