Gene therapy for recessive dystrophic epidermolysis bullosa using genetically corrected 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] The present disclosure relates generally to methods and compositions for treating epidermolysis bullosa (EB) and corneal erosions. [Background technology]

[0002] Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited, genetic, blistering skin disease caused by mutations in the COL7A1 gene (collagen VII, C7), resulting in a deficiency in C7 function. Patients with this disorder are characterized by widespread blistering and erosion of skin and mucosal tissues, including the oropharynx, conjunctiva, esophagus, and distal aspects of the genitourinary and gastrointestinal tracts. Painful blistering and erosion are the primary disability, but scarring from healed wounds also causes significant morbidity, including mitten finger deformity (pseudosyndactyly), ocular synechiae, esophageal strictures, microstomia, tongue synechiae, and limb stenosis. Chronic wounds and scarring predispose to invasive squamous cell carcinoma invasion, which is a major problem in RDEB and is the leading cause of death in this population starting in the teenage years. Therefore, an optimal treatment for this disease would be one that could be implemented early to prevent the development of dysfunctional scarring and prevent blister formation. Also, the ability to systemically modify both cutaneous and mucosal tissues 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 the NC2 end followed by supramolecular assembly into linkage structures called anchoring fibrils, which connect the lamina densa of the basement membrane zone (BMZ) to the papillary dermis. C7 contains a large NC1 domain that binds laminin-332 in the lamina densa and collagen domains that wrap around interstitial collagen fibers in the papillary dermis. Thus, the absence of C7 in RDEB results in blister formation between the papillary and lamina densa dermis.

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

[0005] For therapeutic purposes, local delivery of C7 to the skin is desirable. The present invention addresses this issue. Systemic therapy with C7 can cause systemic toxicity. (See Hou et al. (2015), Journal of Investigative Dermatology 135, 3060-3067.) Summary of the Invention

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

[0007] In some embodiments, the keratinocytes utilized for treatment are autologous keratinocytes. The ex vivo manipulation method may be selected from virus-driven, including but not limited to retrovirus (e.g., gamma retrovirus), AAV virus, and lentivirus, or virus-free integration methods, including but not limited to non-viral vectors, transposons, minicircle integration, CRISPR / Cas9 genome editing systems, and the like. In some embodiments, the gamma retrovirus comprises, consists essentially of, or further comprises murine leukemia virus (MLV or MuLV), feline leukemia virus (FeLV), gibbon ape leukemia virus (GALV), and xenogeneic murine leukemia virus-related virus (XMRV). In some embodiments, the non-viral vector comprises, consists essentially of, or further comprises episomal or integrative vectors capable of genome editing function. 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 comprises genetic modifications from full-length wild-type human COL7A1 cDNA. In some embodiments, viral transduction is performed by overlaying viral supernatant onto cell culture. In some embodiments, keratinocytes so treated meet the pre-release criteria of viral transduction efficiency (VTE) of more than 50% and proviral genome copy number (PGCN) of 3 or less. In some embodiments, the PGCN is more 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 mutated, dysfunctional, 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 the treatment of EB is provided, the method comprising, consisting essentially of, or consisting of obtaining a population of keratinocytes from a subject suffering from EB, modifying the keratinocytes by retroviral transduction to express wild-type human C7, and reintroducing the keratinocytes into the individual. In some embodiments, the keratinocytes are obtained from a skin punch biopsy cultured in vitro in a keratinocyte medium with or without serum. In some embodiments, the keratinocytes are cultured in a medium with or without a feeder layer of cells. The epidermis is separated from the dermis layer, and the keratinocytes are obtained from the epidermis layer. The method comprises the steps of: (1) obtaining a population of keratinocytes from a subject suffering from EB, (2) modifying the keratinocytes by retroviral transduction to express wild-type human C7, and (3) reintroducing the keratinocytes into the individual. In some embodiments, the keratinocytes are obtained from a skin punch biopsy cultured in vitro in a keratinocyte medium with or without serum. In some embodiments, the keratinocytes are cultured in a medium with or without a feeder layer of cells. The epidermis is separated from the dermis layer, and the keratinocytes are obtained from the epidermis layer. 6 At least about 2 x 10 cells 6 pcs, and / or at least 4 x 10 6 100 cells are used for transduction to provide a population of gene-corrected cells. The cells are grown to approximately 25 cm for transplantation. 2 ~about 100 2 The genetically modified keratinocyte sheets are then cultured to produce sheets measuring approximately 50 cm. The genetically modified keratinocyte sheets are placed onto non-infected, eroded, and / or scarred wound sites that are free of clinical evidence of squamous cell carcinoma (SCC). The wound sites are approximately 50 cm. 2 , about 100cm 2 , and / or approximately 200 cm 2 In some embodiments, a wound for the graft is created. In some such embodiments, the wound is electrocauterized to ablate the remaining unmodified wound bed keratinocytes. The graft is secured to the wound bed with dissolvable sutures after wound bed preparation.

[0010] In another embodiment, the present disclosure provides a composition comprising, consisting essentially of, or further consisting of a population of genetically modified keratinocytes to express wild-type human C7 in a dose effective to reduce symptoms of EB, and a pharma- ceutically 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 consisting of a keratinocyte sheet, which comprises, consists essentially of, or consists of skin cells ex vivo integrated with a genetic construct encoding a functional COL7 A1 protein. In some embodiments, the keratinocyte sheet is placed on a bioengineered skin equivalent. 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 from thermoplastics, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofilament polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, silicone, or any combination thereof.

[0012] Herein, autologous RDEB keratinocytes were isolated from skin biopsies and transduced with a retrovirus carrying functional (e.g., full-length) human COL7A1. Each subject received an autologous epidermal sheet (approximately 35 cm 2) were manufactured and grafted into the prepared wound bed. Endpoints included safety, efficacy as percent wound healing compared to baseline, and evidence of C7 expression at 3 and 6 months after grafting. All grafts were well tolerated by all subjects, and no significant adverse events (systemic viral infection, autoimmunity, development of skin cancer within the graft) were reported. At 3-6 months, the majority of grafts showed 75% healing. Biopsies of the graft 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 transfer had a favorable safety profile and showed promising efficacy in subjects with inherited RDEB. [Brief description of the drawings]

[0013] [Figure 1]A shows the RDEB gene correction flow chart, KC-epidermal keratinocytes, autologous epidermal sheet grafts engineered with LEAES-LZRSE-COL7A1. B shows indirect immunofluorescence (IIF) of RDEB KCs transduced with LZRSE-COL7A1 virus, anti-collagen type VII polyclonal antibody (orange), Hoechst33342 nuclei (blue), scale bar 100 μm. C shows quantification of viral transduction efficiency (VTE) in corrected KCs of four RDEB subjects. D shows quantification of mean proviral copy number (PGCN) in corrected KCs of four RDEB subjects. E shows clinical display of RDEB phenotype before and after graft transplantation, note the blisters in the corrected skin graft and untreated wounds compared to 3 and 6 months before LEAES transplantation. F shows IIF analysis of collagen VII expression in skin grafts, note anti-collagen VII NC2 Mab LH24 and NC1 Pab FNC1 (green), Hoechst33342 nuclei (blue), linear green staining of collagen VII at the dermal-epidermal junction of the corrected tissue grafts, scale bar 100 μm. G shows immunoEM analysis of the corrected RDEB skin grafts, tissue sections were labeled en bloc with anti-collagen VII NC2 Mab LH24 followed by anti-mouse IgM-conjugated immunogold particles (black dots, indicated by arrows), scale bar 200 nm. [Diagram 2] FIG. 1 is a CONSORT diagram of subject enrollment in the Phase I study. [Diagram 3] Western blot analysis of cultured KC supernatants using anti-collagen VII polyclonal antibody specific for the NC1 domain is shown, highlighting the expression of truncated C7 protein containing the NC1 domain in all subjects enrolled in the study. [Figure 4] Mature LEAES before harvesting and the final assembled LEAES graft are shown. [Diagram 5]A shows the clinical display of the RDEB phenotype of the grafts before and after transplantation. B shows IIF analysis of collagen VII expression in skin grafts, showing anti-collagen VII 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 collagen VII at the dermal-epidermal junction of the corrected tissue grafts at all time points, scale bar is 100 μm. [Figure 6] A shows the clinical display of the RDEB phenotype in the wound and graft of subject 2 before and after grafting. B shows IIF analysis of collagen VII expression in the wound and skin graft of subject 2 before and after grafting, anti-collagen VII 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 collagen VII at the dermal-epidermal junction of the corrected tissue graft after 3 months. Scale bar is 100 μm. [Figure 7] A shows the clinical display of the RDEB phenotype in the wounds and grafts of subject 3 before and after grafting. B shows IIF analysis of collagen VII expression in the wounds and skin grafts of subject 3 before and after grafting, anti-collagen VII 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 collagen VII at the dermal-epidermal junction of the corrected tissue grafts at all time points, scale bar is 100 μm. [Figure 8]A shows the clinical display of the RDEB phenotype in the wounds and grafts of subject 4 before and after grafting. B shows IIF analysis of collagen VII expression in the wounds and skin grafts of subject 4 before and after grafting, showing anti-collagen VII 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 collagen VII at the dermal-epidermal junction of the corrected tissue grafts at 3 months with LH24 Mab and at 6 months with NC1 Pab. Scale bars are 100 μm. [Figure 9] Figure 1 shows characterization of anti-C7 LH24 monoclonal antibody, Western blot analysis of enzymatically digested C7 showing LH24 Mab cross-reactivity to carboxyl-terminal peptides containing the NC2 domain, presence of the NC2 domain in pepsin-digested C7 fraction confirmed with NC2-specific pAb (NC2-10)5, and NC1 domain in collagenase-digested C7 fraction identified using FNC1 pAb6. [Figure 10] Clinical presentation of the unmodified wound of subject 4, showing the characteristic spontaneous blistering in untreated wounds. [Figure 11] A shows Western blot analysis of subject 4 showing serum type VII collagen seroreactivity and cross-reactivity to full-length C7 of subject 4 serum before and after (3 months) graft implantation. B shows serum reactivity to type VII collagen of subject 4, where serum of subject 4 obtained before and 3 months after implantation is specific for enzymatically (pepsin) digested C7 protein containing the NC2 domain, and the control (right lane) confirms the presence of the NC2 domain in the C7 fraction digested using the NC2-specific Pab (NC2-10)5. [Figure 12]Wound from subject 1 pre-transplant and 12 months after grafting, clinical presentation of wound at baseline and 12 months after grafting, IIF analysis of collagen VII expression in skin grafts showing anti-collagen VII NC1 Pab (green), Hoechst33342 nuclear (blue), keratin 14 (anti-K14 Pab, orange), keratin 1 (anti-K1 Pab, orange), and loricrin (anti-loricrin Pab, orange). Note linear green staining of collagen VII at the dermal-epidermal junction of the corrected tissue graft. Scale bar is 100 μm. [Figure 13] 1 shows a map of the pLZRSE-COL7A1 retroviral plasmid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to 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 indicates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells, and a reference to a "culture" includes a reference to one or more cultures and equivalents thereof known to those of skill in the art. 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 indicated.

[0016] Any embodiment of any of the present methods, apparatus, and systems may consist of, or consist essentially of, the recited steps and / or functions, rather than comprising / including / containing / having. Thus, in any of the claims, the terms "consisting of" or "consisting essentially of" may be substituted with any of the above-cited open-ended linking verbs in order to change the scope of a given claim from one that would otherwise use the open-ended linking verb.

[0017] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer only to alternatives or alternatives are mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives and "and / or."

[0018] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0019] Conditions of interest for treatment with the engineered 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 the recent classification system, dystrophic epidermolysis bullosa (DEB) includes three subtypes: recessive DEB, severe generalized (RDEB-sev gen) (previously called Hallopeau-Siemens type (RDEB-HS)), recessive DEB, other generalized (RDEB-O) (previously called non-Hallopeau-Siemens type (non-HS type RDEB)), and dominant DEB (DDEB). In RDEB-sev gen, blisters affecting the entire body may be present in the neonatal period. Oral involvement may result in blistering of the mouth, fusion of the tongue to the floor of the mouth, and a gradual reduction in the size of the oral cavity. Esophageal erosions may lead to webs and stenosis that may cause severe dysphagia. Thus, severe nutritional deficiencies and secondary problems are common. Corneal erosions may lead to scarring and vision loss. Blistering of the hands and feet, followed by scarring and fusion of the toes, is a hallmark of this disease. The patient develops "mitten" hands and feet. The lifetime risk of aggressive squamous cell carcinoma is greater than 90%. In DDEB, blistering is often mild and limited to the hands, feet, knees, and elbows, but nevertheless heals with scarring. Dystrophic nails, especially toenails, are common and may be the only symptom of DDEB.

[0021] Conventional treatment of the condition, including wound dressings and nutritional support, is largely effective. Occupational therapy can help prevent contractures of the hand. 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 the inherited form of EB, the acquired form of epidermolysis bullosa (EBA) involves pathology in collagen VII and can be treated with the engineered keratinocytes of the present disclosure. Circulating autoantibodies in EBA patients recognize epitopes in the collagen VII molecule, and molecular cloning of collagen VII cDNA has provided tools to identify dominant immune epitopes within the amino-terminal NC-1 domain of collagen VII. 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 used clinically to map collagen VII in the skin of patients with acquired forms of EB, also identify epitopes in this part of the protein. In addition to circulating autoantibodies that recognize collagen VII epitopes in EBA, bullous lesions in some patients with systemic lupus erythematosus are also associated with anti-collagen VII 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%, at least about 95% or more of the protein present is collagen in triple helical configuration. The folding of individual α-chains into triple helical conformation is based on a characteristic primary sequence that includes a repeating Gly-XY triplet sequence. Collagen is found widely in vertebrate species, and sequences have been determined for many different species. Due to the high degree of sequence similarity between species, e.g., between mammalian species, collagen from different species can be used for biological purposes. However, human protein may be preferred.

[0025] FACIT collagens (fibril-associated collagens with interrupted triple helices) include types IX, XII, XIV, XIX, XX, and XXI. Some of the latter types of collagens associate with and associate with larger collagen fibrils, functioning as molecular bridges and stabilizing the organization of the extracellular matrix. Collagen VII (COL7A1, chromosome 3, NC_000003.10 (48576510..48607689, complement)) is of particular interest. Collagen VII is the major constituent of anchoring fibrils.

[0026] Type VII collagen is a long 424 nm triple-helical domain with adjacent non-collagenous sequences. The type VII collagen molecule contains a major collagenous, triple-helical segment flanked by non-collagenous NC-1 and NC-2 domains. Unlike interstitial collagens, the repeating Gly-XY sequence is interrupted by 19 defects resulting from insertion or deletion of amino acids in the Gly-XY repeat sequence. Most notably, in the center of the triple-helical domain is a non-collagenous "hinge" region of 39 amino acids, which is sensitive to proteolytic digestion with pepsin. The amino-terminal NC-1 domain of type VII, approximately 145 kDa in size, contains a segment with homology to cartilage matrix protein (CMP), nine consecutive fibronectin type III-like (FN-III) domains, von It contains a segment with homology to the A domain of Willebrand factor, as well as a submodule with homology to known adhesion proteins that contains a short cysteine- and proline-rich region. The carboxy-terminal non-collagenous domain, NC-2, is relatively small at approximately 30 kDa, and it contains a segment with homology 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. Thus, the size of the entire human COL7A1 gene is only about 32 kb, encoding about 8.9 kb of messenger RNA. COL7A1 has been mapped to the region 3p21.1, the short arm of human chromosome 3. The type VII collagen gene structure and the primary encoded sequence of the protein are well conserved, e.g., 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. Upon synthesis of the complete pro-α1 (VII) polypeptide, the three polypeptides associate through their carboxy-terminal ends into a trimer molecule in which the collagen moieties fold to form a triple helix. The triple helical molecule is then secreted into the extracellular environment, where the two types of type VII collagen molecules align into antiparallel dimers, with the amino-terminal domains present at both ends of the molecule. This dimeric assembly is accompanied by proteolytic removal of portions of both carboxy termini of the type VII collagen molecule and stabilization by intermolecular disulfide bond formation. A number of these antiparallel dimers then aggregate laterally to form anchoring fibrils.

[0029] In dominant dystrophic epidermolysis bullosa (DDEB), glycine substitution mutations in the triple helical domain of COL7A1 (specifically exons 73, 74, and 75) predominate. The 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 junction mutations outside this region can also be found in dominant DEB.

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

[0031] A "native sequence" polypeptide is one that has the same amino acid sequence as a polypeptide derived from nature. Such native sequence polypeptides can be produced by recombinant means according to the methods described herein. Thus, a native sequence polypeptide can have the amino acid sequence of, for example, a naturally occurring human polypeptide, a mouse polypeptide, or a polypeptide derived from any other mammalian species. The term "native sequence collagen VII protein" includes naturally occurring proteins with or without an initial N-terminal methionine (Met).

[0032] A "variant" polypeptide refers to a biologically active polypeptide, as defined below, having less than 100% sequence identity with a native sequence polypeptide. Such variants include polypeptides 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, in which about 1-40 amino acid residues are deleted, and in which 1 The present invention also includes derivatives of the above polypeptides, in which an amino acid residue is optionally substituted with one or more amino acid residues, and in which the amino acid residue is covalently modified such that the resulting product contains a non-naturally occurring amino acid. Typically, a biologically active collagen VII variant will have an amino acid sequence having at least about 90%, preferably at least about 95%, and more preferably at least about 99% amino acid sequence identity with a native sequence collagen VII polypeptide.

[0033] A "functional derivative" of a native sequence collagen VII polypeptide is a compound that has qualitative biological properties in common with a native sequence collagen VII polypeptide. "Functional derivatives" include, but are not limited to, native sequence fragments and derivatives of native sequence collagen VII polypeptides and fragments thereof, provided that they have biological activity in common with the corresponding native sequence collagen VII polypeptide. The term "derivative" encompasses both amino acid sequence variants of collagen VII polypeptides and covalent modifications thereof.

[0034] The term "wound bed" refers to the viable top layer of a wound. In one embodiment, the wound bed is covered with a slough or eschar. In another embodiment, the wound bed can be evaluated for the presence of granulation tissue fibrous slough, eschar, bone, tendon, and / or other underlying structures.

[0035] The term "viral transduction efficiency (VTE) test" is a test to measure the ratio of the number of cells transduced with a virus to the total number of cells transduced. In one embodiment, VTE is measured by fluorescent antibody 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 a virally transduced cell. Thus, the PGCN test measures the number of copies of proviral DNA in a cell after viral transduction or infection. In one embodiment, the copy number is measured by real-time PCR or quantitative PCR. In another embodiment, the PGCN is measured by Southern blot or high-throughput methods. In some embodiments, the PGCN is less than 3, 2, 1, 0.5. In some embodiments, the PGCN is greater than 3, 10, 100, or 1,000.

[0037] The term "sterility testing" refers to a test that attempts to determine the presence or absence of viable contaminating microorganisms in a sample, and is often used to eliminate false positive results, which in one embodiment arise due to contamination or error from the environment.

[0038] The term "endotoxin" refers to a toxin associated with the outer membrane of certain Gram-negative bacteria, including, but not limited to, Brucella, Neisseria, and Vibrio species. In one embodiment, endotoxin is not secreted, but is released only upon cell rupture. Endotoxin can be measured or tested by gel-clot, chromogenic, turbidimetric, or a combination thereof.

[0039] The term "mycoplasma" refers to a population of bacteria that lack a cell wall around the cell membrane, making them less susceptible or insusceptible to certain types of antibiotics. Tests for mycoplasma include, but are not limited to, agar and blotting procedures, DNA detection, enzyme, and ELISA methods, and PCR.

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

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

[0042] In this disclosure, the term "replication-competent 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 packaging components of the transfer vector and endogenous retroviral elements in the producer cell. The RCR test is to detect the RCR in a sample.

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

[0044] The term "post-release test" in this disclosure refers to one or more tests after the epidermal sheet is released from the plate, including, but not limited to, a sterility test, an RCR test, a mycoplasma test, a viability test, and a Gram stain sterility test.

[0045] The term "genetic modification" refers to the process of modifying the genes of an organism or inserting genes from one organism into another. In one embodiment, the genetic modification comprises, consists essentially of, or further consists of an insertion, deletion, and / or mutation. The term "insertion" refers to the addition of 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 not limited to a single base pair (i.e., a point mutation), several base pairs, or at most a large segment of a chromosome.

[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 genetically modified gene. For example, both aspartic acid and glutamic acid are small negatively charged residues. In some embodiments, it is a conservative genetic modification by 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 hydroxylation of proline residues in the peptide linkage. The 4-hydroxyproline residue is essential for folding of the newly synthesized procollagen polypeptide chain into a triple helical molecule. The active enzyme is a tetramer of 2α and 2β subunits with a molecular weight of about 240,000. The β subunit (P4HB) is identical to the enzyme disulfide isomerase (EC 5.3.4.1) and the major cellular thyroid binding protein. The α subunit is responsible for most of the catalytic site of the enzyme. The polypeptide is 517 amino acid residues 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 had been presented previously. The present data showed that the mutually exclusive sequences found in the mRNAs are encoded by two contiguous homologous 71 bp exons 9 and 10. These exons are identical in the first five base pairs, and the overall identity between them is 61% at the nucleotide level and 58% at the coded amino acid level. Both types of mRNA were found to be expressed in all tissues studied, although in some tissues the types encoding exon 9 or exon 10 sequences were more abundant than the other types.

[0049] "Nucleic acid construct" refers to a nucleic acid sequence that has been 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 from lambda phage), viral genomes that contain non-naturally occurring nucleic acid sequences, etc.

[0050] In this method, collagen VII is usually produced by introducing a viral expression construct into a cell population at integration time.The DNA encoding collagen VII polypeptide can be obtained from any cDNA library prepared from tissue expressing collagen VII polypeptide mRNA, prepared from various sources.Collagen VII polypeptide coding gene can also be obtained from a genomic library or by oligonucleotide synthesis.Another way to identify gene code is to use PCR methodology.

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

[0052] A "vector" is capable of introducing a nucleic acid sequence into a target cell. For example, a vector may contain a coding sequence capable of being expressed in a target cell. For the purposes of the present invention, "vector construct", "expression vector" and "gene transfer vector" generally refer to any nucleic acid construct capable of directing the expression of a gene of interest, which is useful for introducing a gene of interest into a target cell. Thus, the term includes cloning and expression vehicles, as well as integration vectors.

[0053] An "expression cassette" includes any nucleic acid construct capable of directing the expression of any RNA transcript, including a gene / coding sequence of interest as well as non-translated RNA, e.g., shRNA, microRNA, siRNA, antisense RNA, etc. Such cassettes can be constructed into "vectors," "vector constructs," "expression vectors," or "gene transfer vectors" to introduce the expression cassette into a target cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0054] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If no such sites exist, synthetic oligonucleotides can be used. The adapter or linker is used in accordance with conventional practice.

[0055] The expression vector will contain a promoter recognized by the host cell for expression of autologous leukocytes or mRNA and operably linked to the collagen VII coding sequence. A promoter is a non-translated 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 particular nucleic acid sequence to which it is operably linked. Such promoters are usually divided into two classes: inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by a variety of potential host cells are well known. Heterologous promoters are preferred, as they generally allow for greater transcription and higher yields.

[0056] Transcription from the vector in mammalian host cells can be controlled by promoters derived, for example, from the genomes of viruses, such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B, Simian Virus 40 (SV40), from heterologous mammalian promoters, such as the actin promoter, PGK (phosphoglycerate kinase), or immunoglobulin promoters, from heat shock promoters, provided that such promoters are compatible with the host cell system. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment.

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

[0058] Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and stabilization of the mRNA. Such sequences are commonly available from the 5' and, occasionally, 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA.

[0059] Vectors and systems for integration of expression cassettes into cells are known in the art and may include, but are not limited to, retroviral vectors. Many vectors useful for introducing foreign genes into target mammalian cells are available. Retroviral-based vectors have been shown to be particularly useful. A combination of a retrovirus and an appropriate packaging line may be used, where the capsid protein will function to infect the target cells. Typically, the cells and virus are incubated in the medium for at least about 24 hours. The cells are then, in some applications, incubated for short intervals, e.g., 24 hours. For example, they may be grown in medium for 24-73 hours, or for at least 2 weeks, and may be grown for 5 weeks or more before analysis. Commonly used retroviral vectors are "defective," i.e., unable to produce viral proteins necessary for productive infection. Replication of the vector requires growth in a packaging cell line.

[0060] The host cell specificity of retroviruses is determined by the envelope protein, env (p120). The envelope protein is provided by the packaging cell line. There are at least three types of envelope proteins: ecotropic, amphotropic, and xenotropic. Retroviruses with amphotropic envelope proteins, such as 4070A (Danos et al., supra), are capable of infecting most mammalian cell types, including human, canine, and murine. 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); GRIP (Danos et al. (1988) PNAS 85:6460B6464). Retroviruses packaged with xenotropic envelope proteins, such as AKR env, are capable of infecting most mammalian cell types, except mouse cells. The sequences at the 5' and 3' ends of retroviruses are long terminal repeats (LTRs). A number of LTR sequences are known in the art and can be used, including MMLV-LTR, HIV-LTR, AKR-LTR, FIV-LTR, ALV-LTR, etc. The 5'LTR acts as a strong promoter, driving transcription of the introduced gene after integration into the target cell genome.

[0061] Keratinocytes Freshly harvested primary keratinocytes may be collected from a skin punch biopsy, isolated, and transduced after a culture period to isolate keratinocytes from dermal cells. Epidermal keratinocytes grown in vitro may form sheets. In some embodiments, the transduced cells are administered to the patient within about 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 known to those of skill in the art, any of a variety of media may be used in the present methods (see, e.g., Current Protocols in Cell Culture, 2000-2009 by John Wiley & Sons, Inc.). Illustrative media include, but are not limited to, keratinocyte medium, which may be serum-free and may contain appropriate keratinocyte supplements.

[0063] The present disclosure provides a method of treating epidermolysis bullosa (EB) in a subject, the method comprising, or consisting essentially of, or further consisting of, obtaining a population of skin cells of the subject, ex vivo correcting the skin cells by integration of a genetic construct encoding a functional (e.g., full-length wild-type) human collagen VII (COL7A1) protein, culturing the genetically corrected cells to form a keratinocyte sheet, and transplanting a graft of the keratinocyte sheet into a skin wound bed. The present disclosure also relates to the use of a population of skin cells to treat epidermolysis bullosa (EB) in a subject, the population of skin cells being corrected by transducing with a virus comprising a genetic 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 genetically corrected cells are cultured in DFF31 medium comprising, or consisting essentially of, or further consisting of Dulbecco's Modified Eagle's Medium and F12 medium. In one embodiment, the skin cells contain an expression construct or is modified by transduction with a virus consisting essentially of or further consisting of a retrovirus, AAV (adeno-associated virus), or lentivirus, the virus comprising, consisting essentially of, or further consisting of a retrovirus. In another embodiment, the retrovirus is an LZRSE virus. In one embodiment, the retrovirus is GalV-pseudotyped. In a further embodiment, the keratinocytes thus transduced meet the pre-release criteria of a viral transduction efficiency (VTE) of more than 50% and a proviral genome copy number (PGCN) of less than or equal to 3. In some embodiments, the PGCN is less than 2.5, 2, 1.5, or 1. In another embodiment, the PGCN is 3-20, 20-40, 40-60, 60-80, or 80-100. In another embodiment, the PGCN is more than 100. In some embodiments, the keratinocyte sheets vary in size. The size of the keratinocyte sheets can be determined by one skilled in the art.

[0064] In some embodiments, endogenous mutated, dysfunctional, 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., a functional COL7A1 cDNA or C7 gene or a 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, which is incorporated by reference in its entirety. CRISPR loci (clustered regularly interspaced short palindromic repeats) are regions in the genome of an organism where short segments of foreign DNA are integrated between short repeat palindromic sequences. These loci are transcribed and the RNA transcripts ("pre-crRNA") are processed into short CRISPR RNAs (crRNAs). There are three types of CRISPR / Cas systems that all incorporate these RNAs and proteins known as "Cas" proteins (CRISPR-associated). Both types I and III have a Cas endonuclease that processes the pre-crRNA and assembles a multi-Cas protein complex that is capable of cleaving nucleic acids that are complementary to the crRNA when fully processed into the crRNA. A CRISPR / Cas system that binds to a target site in a region of interest of an endogenous gene (e.g., an endogenous or safe harbor gene, or a regulatory gene or its DNA target) in the genome, the CRISPR / Cas system comprises one or more engineered single guide RNAs that recognize the target gene and a functional domain (e.g., a transcriptional regulatory domain and / or a nuclease domain).In some embodiments, a CRISPR / Cas system as described herein may bind to and / or cleave a region of interest (e.g., an endogenous C7 gene from EB tissue) in coding or non-coding regions within or adjacent to a gene, e.g., in leader sequences, trailer sequences, or introns, or in non-transcribed regions either upstream or downstream of the coding region. In certain embodiments, CRISPR / Cas binds to and / or cleaves a gene, e.g., a mutated, dysfunctional, or truncated C7 gene.

[0065] In one aspect, the wound does not contain non-modified wound bed keratinocytes. In one embodiment, the wound is treated to ablate non-modified wound bed keratinocytes. In another aspect, the subject is afflicted with recessive dystrophic epidermolysis bullosa (RDEB). In a different aspect, the subject is a human.

[0066] In another embodiment, the keratinocyte sheet is subjected to one or more tests selected from the group consisting of VTE test, PGCN test, sterility test, endotoxin test, mycoplasma test, Gram stain sterility test, LEAES viability test, post-release test, RCR test, cytotoxic T cell assay, anti-C7 LH24 mAb characterization, electron microscopy, immunoelectron microscopy, immunofluorescence staining, C7 expression, and AF analysis. In one aspect, immunofluorescence staining is performed directly. The method may comprise, consist essentially of, or further consist of direct or indirect immunofluorescence staining.

[0067] In some embodiments, the keratinocyte sheet is placed on an acellular matrix, a collagen matrix, or a biocompatible mesh. In one embodiment, the biocompatible mesh is made of thermoplastics, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefins, uncoated monofilament polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, silicone, or any combination thereof.

[0068] In some embodiments, the skin cells comprise, or consist essentially of, or consist of keratinocytes. In one embodiment, the skin cells comprise, or consist essentially of, or consist of stem cells. In another embodiment, the method further comprises, or consist essentially of, or consist of differentiating the stem cells into keratinocytes. In one aspect, the stem cells differentiate before, after, or during transduction. In some embodiments, the stem cells differentiate (e.g., into keratinocytes or corneal epithelial cells) before transduction. In some embodiments, the stem cells differentiate after transduction. In a further embodiment, the stem cells differentiate during transduction.

[0069] RDEB patients may frequently develop debilitating and painful corneal erosions. Thus, the present disclosure also provides a method for treating corneal erosions in a subject, comprising, or consisting essentially of, or further comprising: obtaining a population of corneal cells from a subject; modifying the corneal cells ex vivo by integrating 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 the corneal cell sheet graft onto the corneal surface. In some aspects, the corneal cells comprise, or consist essentially of, or further comprise corneal epithelial cells. In another aspect, the corneal cells comprise, or consist essentially of, or further comprise stem cells. In some embodiments, the method further comprises, or consist essentially of, or further comprise differentiating the stem cells into corneal epithelial cells.

[0070] In some aspects, the corneal cells are modified by transducing with a virus comprising the genetic construct, the virus comprising, or consisting essentially of, or consisting of a retrovirus, lentivirus, or AAV. In one embodiment, the retrovirus is an LZRSE virus. In some embodiments, the retrovirus is GalV-pseudotyped. In some embodiments, the corneal cells so transduced meet the pre-release criteria of greater than 50% viral transduction efficiency (VTE) and a proviral genome copy number (PGCN) of 3 or less. In some embodiments, the PGCN is less than 2.5, 2, 1.5, or 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 aspects, the subject is afflicted with recessive dystrophic epidermolysis bullosa (RDEB). In a different aspect, the subject is a human.

[0071] In one aspect, the corneal cell sheet is subjected to one or more tests selected from the group consisting of VTE testing, PGCN testing, sterility testing, endotoxin testing, mycoplasma testing, Gram stain 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, the immunofluorescence staining comprises, consists essentially of, or further consists of direct or indirect immunofluorescence staining. In some embodiments ... an acellular matrix, a collagen matrix, , or placed on a biocompatible mesh.

[0072] In one aspect, the biocompatible mesh can be made from a non-resorbable material, including, but not limited to, biocompatible metals, such as titanium alloys, stainless steel, cobalt-chromium alloys, and nickel-titanium alloys. In another aspect, the layers of the biocompatible mesh can be made from a non-resorbable polymeric material, including, but not limited to, thermoplastics, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefins, uncoated monofilament polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, expanded 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 otherwise formed into a shape.

[0073] In another aspect, the biocompatible mesh can be composed of synthetic or bioabsorbable polymeric 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, copolymers of lactide and glycolide (PLGA), polyhydroxy-3-butyrate, collagen, hyaluronic acid, silk, biocellulose, other protein-based polymers, polysaccharides, poly(DTE carbonate), polyarylates, PLLA, PLDA, or blends of PLGA with TMC, as well as other combinations of these polymers.

[0074] In one embodiment, the biocompatible mesh is made of thermoplastics, polyethylene, ultra high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofilament polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, silicone, or any combination thereof.

[0075] Also provided in the present disclosure are compositions comprising, or consisting essentially of, or consisting of keratinocyte sheets, which are prepared by a process comprising, or consisting essentially of, or consisting of obtaining a population of skin cells from a subject, modifying the skin cells ex vivo by integration of a genetic construct encoding a functional (e.g., full-length wild-type) human collagen VII (COL7A1) protein, and culturing the genetically modified cells to form keratinocyte sheets. In some embodiments, the skin cells comprise, or consist essentially of, or consist of keratinocytes. In one embodiment, the skin cells comprise, or consist essentially of, or consist of 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 is a pharmaceutical composition comprising, or consisting essentially of, or consisting of a keratinocyte sheet, the keratinocyte sheet comprising, or consisting essentially of, or consisting of skin cells ex vivo integrated with a genetic construct encoding a functional COL7A1 protein. In one aspect, the skin cells are obtained from a subject. In some embodiments, the subject is a human. In some embodiments, the skin cells are differentiated from stem cells ex vivo integrated with a genetic construct encoding a functional COL7A1 protein. In one embodiment, the subject is afflicted with RDEB. In some embodiments, the skin cells or stem cells are transduced with a virus comprising the genetic construct, the virus comprising a retrovirus, a lentivirus, or an AAV.

[0077] In one embodiment, the functional COL7A1 protein is a full-length wild-type human COL7A1 protein. In one aspect, the functional COL7A1 protein comprises, consists essentially of, or consists of genetic modifications from a full-length wild-type human COL7A1 protein. In another aspect, the functional COL7A1 protein comprises, consists essentially of, or consists of genetic modifications from a full-length wild-type human COL7A1 protein, wherein the genetic modifications are conservative. In a further aspect, the genetic modifications comprise, consists essentially of, or consist of insertions, deletions, and / or mutations.

[0078] Also provided in the present disclosure is a pharmaceutical composition comprising, or consisting essentially of, or further consisting of a corneal cell sheet, the corneal cells comprising corneal cells ex vivo integrated with a genetic construct encoding a functional COL7A1 protein. In some embodiments, the corneal cells are differentiated from stem cells ex vivo integrated with a genetic construct encoding a functional COL7A1 protein. In another embodiment, the corneal cells are obtained from a subject. In one embodiment, the subject is afflicted with RDEB. In some embodiments, the subject is human. In some embodiments, the corneal cells or stem cells are transduced with a virus comprising the genetic construct, the virus comprising a retrovirus, lentivirus, or AAV. In one embodiment, the retrovirus is an LZRSE virus. In some embodiments, the retrovirus is GalV-pseudotyped. In further embodiments, the transduced cells 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.

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

[0080] In certain embodiments, the cells are cultured for 1-21 days. In further embodiments, the cells are cultured for 7, 14, 21 days or more. Thus, the cells may be cultured under appropriate 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 more. The cells may be replated and media and supplements may be added or changed as needed using techniques known in the art.

[0081] In certain embodiments, the genetically altered keratinocytes may 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, or consists essentially of, or further consists of a genetically modified autologous keratinocyte population expressing an amount of native human C7 protein effective for treating EB. The target cell population is grown in a sheet in combination with one or more pharmacologic or physiologically acceptable carriers, diluents, or excipients for transplantation into a subject. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides, or amino acids, such as glycine, antioxidants, chelating agents, such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.

[0083] The cell compositions of the present disclosure are administered in a manner suitable for treating EB. The amount and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease. However, appropriate dosages can be determined through clinical trials.

[0084] The cells can be administered to a subject by methods well known to those skilled in the art, usually in the form of a skin graft. A practitioner will be able to determine the appropriate route of administration for a particular subject based, in part, on the type and location of the disease. The transfected cells can be administered locally to the wound site.

[0085] Pharmaceutical formulations of engineered cells for administration to a subject are contemplated by the present invention. Those skilled in the art will be familiar with techniques for administering cells to a subject. Furthermore, those skilled in the art will be familiar with the techniques and pharmaceuticals necessary to prepare these cell sheets prior to administration to a subject.

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

[0087] The pharmaceutical composition of the present invention comprises a solution of an effective amount of transfected cells in a pharma- ceutically acceptable carrier or aqueous medium. As used herein, a "pharmaceutical formulation" or "pharmaceutical composition" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the cells, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the composition. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards 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 aspects, multiple doses can be administered over a period of days, weeks, months, or years. A subject can be administered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 grafts of the same or different areas. In one embodiment, a subject can be re-grafted in the same or different areas. In another embodiment, the subject's biological sample (e.g., keratinocytes or corneal cells) is stored under appropriate conditions. Once the biological sample is stored, punch biopsy is not necessary if the subject requires a new graft. The stored biological sample can provide sufficient information or supplemental information to determine the graft required by the subject.

[0089] When an "effective amount" or a "therapeutic amount" is indicated, the exact amount of the composition of the present disclosure to be administered can be determined by a physician, taking into account individual differences in the age, weight, and condition of the patient (subject). 3 , 1 to 10 4 , 1 to 10 5 , 1 to 10 6 , 1 to 10 7 , or 10 7 It can generally be stated that the cell compositions may be administered in amounts of more than one cell. The cell compositions may also be administered multiple times at these dosages. The cells may be administered using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one of ordinary skill in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly. can be determined.

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

[0091] The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, or to represent all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0092] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0093] The present invention has been described with respect to specific embodiments found or proposed by the inventors to include preferred modes for carrying out the invention. In light of this disclosure, those skilled in the art will appreciate that 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, potential changes in the DNA sequence can be made without affecting the protein sequence. Furthermore, due to considerations of biological functional equivalence, changes can be made in the protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included 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 blistering skin disease caused by loss of function mutations in COL7A1, the gene encoding type VII collagen (C7). C7 is a major component of anchoring fibrils (AFs) that anchor the epidermal basement membrane zone (BMZ) to the dermis. C7 performs this function through the use of multiple domains, including an amino-terminal noncollagenous NC1 domain that binds BMZ ligands, a central collagenous domain that assembles into triple helices, and an NC2 domain that catalyzes C7 assembly into AFs. Loss of these functional C7 domains in RDEB results in severe BMZ dissociation. This results in extensive and painful blistering, erosion, and scarring that, in turn, can lead to malignant, often fatal forms of SCC that emerge in the second and third decades of life. Despite advances in the molecular diagnosis of the disease, current treatment is 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 been conducted with variable rates of efficacy and safety. Preclinical studies are investigating potential treatment modalities including intravenous and topical C7, induced pluripotent stem cells, and aminoglycosides.

[0096] Gene therapy is a potentially powerful tool for the treatment of monogenic diseases such as RDEB. However, significant safety concerns have been raised following gene transfer in patients with severe combined immunodeficiency and Wiskott-Aldrich syndrome. Although insertional mutagenesis remains a potential concern, an advantage of skin gene therapy is that the neoplasm can be more easily clinically evaluated because the transplanted tissue is located superficially. Furthermore, genetically modified skin grafts have been used efficiently to treat one patient with junctional EB, with long-term correction, and without adverse side effects. A platform for long-term C7 expression in reconstructed human epidermis has been established and grafted into immunodeficient mice (Siprashvili et al, Hum. Gene Ther., 2010, 21(10):1299-1310). The present disclosure now provides the results of a Phase I clinical trial of ex vivo gene transfer of LZRSE-COL7A1 engineered autologous epidermal sheets (LEAES) grafted into subjects with severe RDEB.

[0097] clinical results Subjects and Treatments Of the 38 subjects screened, 8 consented to the study and 4 subjects were enrolled and received grafts (Figure 2). The subjects had heterozygous COL7A1 mutations, a compound of various compounds that resulted in the expression of a truncated C7 (NC1 domain). This was detected in keratinocyte cultures by Western blot (Figure 3), but not in tissues by IIF (Figure 1F, Figure 5B, Figure 6B, Figure 7B, Figure 8B, Figure 12). All subjects were males with a mean age of 23 (range: 18-32) and affected with 4%-30% total body surface involvement. Each had severe disease with cutaneous manifestations including anemia, esophageal stricture, and a history of pseudosyndactyly (Table 1).

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

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

[0100] One month after grafting, 20 / 24 wounds (83%) showed 75% or greater healing, while only 4 wounds showed 50%-74% healing (Table 3). At 3 months, 21 / 24 (87%) wounds were 75% or greater healing, while 3 / 24 (13%) were 50%-74% healing (Table 3). At 6 months, 16 / 24 (67%) wounds were 75% or greater healing, 5 / 24 (21%) were 50%-75% healing, and only 3 / 24 (13%) graft sites showed blisters and were considered graft failures (0%-49% healing).

[0101] Molecular analysis of LEAES grafts was performed in 9 / 10 (90%) samples at 3 months and 8 / 10 (90%) at 6 months. Twelve (66%) samples showed strong C7 expression. IIF analysis of LEAES showed proper localization of C7 at the epidermal-dermal junction, in contrast to unmodified skin controls (Fig. 1F, 5B, 6B, 7B, 8B, 12). LEAES grafts showed fully differentiated epidermis with spinous and granular layers, which was positive for the epidermal markers keratin 14, keratin 1, and loricrin, resembling normal skin (Fig. 1F, 5B, 6B, 7B, 8B, 12). Among the negative samples, C7 was not detected in the analyzed biopsy obtained from subject 2 at 6 months. However, anchoring fibrils (AF) were present in the corresponding biopsy. At 6 months, C7 was identified in subject 4 using an antibody specific for the NC1 domain (Fig. 1F).

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

[0103] safety No serious adverse events were reported. Itching at the implant site (n=3), followed by increased implant site drainage (n=2) were the most common adverse events (grade 1 or 2); 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 noted in 2 / 24 wounds, but sites were noted to be free of signs of infection, including no erythema, edema, pain, or tenderness. At 6 months, subject 3, site Z, had wound colonization (grade 2) and was considered graft failure (Table 3). Pruritus was noted in 3 / 24 graft sites and 2 surrounding areas (grade 1).

[0105] C7 immune responses were closely monitored throughout the study. Subjects had no systemic autoimmune symptoms or increased blistering outside the transplant area. In subject 1, no circulating or tissue-bound antibodies were observed by IIF or DIF (Table 2). Subject 2 at 3 months showed 1+ (mild) linear IgG, IgM, IgA, without complement, by DIF analysis of two wounds (A and E). These immune reactants were not observed 6 months after transplantation. Subject 3 showed a transient elevation of linear serum IgA (1:320) by IIF at 3 months with slight evidence of 1+ linear tissue staining for IgM and IgA by DIF at 6 months. In contrast, subject 4 revealed circulating linear IgG antibodies at 1:160 titers by IIF at 1 and 3 months, with 1-2+ IgG, IgA, C3, and IgM staining detected in three grafts at 3 months by DIF (Table 2). However, there was no increase in systemic autoimmune symptoms or blister formation outside the graft, and the results of a C7-specific cytotoxic T cell assay were negative. At 6 months, serum antibody IgG and C3 levels were reduced (1:40), and tissue-bound immune complexes were not detected in the grafts (Table 2). Following the discovery of subepidermal linear immune deposits 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 demonstrated that both baseline (1:300) and 3 months post-transplant (1:1000) serum was reactive to purified C7, indicating that subject 4 was sensitive to exogenous C7 prior to graft placement (FIG. 11A). Subject 4's serum antibody reactivity was confirmed within the previously characterized carboxyl-terminal C7 pepsin fragment both prior to and after graft placement (FIG. 11B).

[0106] Consideration Genetic correction of RDEB poses substantial challenges, requiring efficient delivery of large transgenes encompassing over 9 kb of COL7A1 cDNA. Here, the present disclosure provides an in-human study of gene-corrected autologous epidermal keratinocyte transplantation with promising efficacy and acceptable safety in four subjects with RDEB. Gene-corrected epidermal cells regenerated functional self-renewing epidermis in over 67% of transplant sites tested 6 months after transplantation, and C7 was detectable for up to one year in one subject (Figure 12). This was a marked improvement over allogeneic keratinocyte transplants, where only 2 / 9 (22%) of chronic wounds were healed at 18 weeks. LEAES transplants also produced 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 no long-term differences. Case reports of gene therapy for junctional epidermolysis bullosa using similar methodology showed correction for up to 6 years, prior to long-term therapeutic efficacy. The 6 months of sustained C7 expression we observed in LEAES grafts spans the duration of six epidermal turnover cycles, implying that we have successfully targeted stem cells with our gene transfer technique.

[0107] In our study, improved wound healing and increased skin durability were directly related to detectable full-length C7 production and AF formation in the BMZ. Although absent before grafting, 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. The variability in biopsy sampling may be due to either a heterogeneous population of modified epidermal cells or partial graft incorporation. In some embodiments, the graft area can be an area that the subject perceives as beneficial to their quality of life. However, during the critical first few days of graft placement, some of the areas were difficult to immobilize or protect from mechanical friction (e.g., lower back E, left shoulder B, Table 3, posterior shoulder D, Figure 6A, of subject 2). Furthermore, as the subject had the shortest post-immobilization period compared to the other study participants, the short post-immobilization time as observed may have a negative impact on graft incorporation, as indicated by reduced overall wound healing by IF and the absence of detectable C7 in the 6-month sampled biopsy of subject 2 (Table 1). Furthermore, the induced wound showed good healing potential but the least C7 expression. In the absence of destructive methods such as electrocautery, the remaining unmodified wound bed keratinocytes may have prevented the establishment of the overlying LEAES graft. These findings suggest that wound bed preparation techniques that more efficiently excise wound bed keratinocytes could improve graft incorporation.

[0108] Regarding safety, several adverse events were reported and all observed were mild. No subjects showed evidence of RCR in the blood or squamous cell carcinoma at the graft at any time point, but long-term monitoring of potential adverse events is ongoing. All molecular replacement approaches, including gene transfer, carry the risk of undesired immune responses to the therapeutic product, especially in subjects with null mutations. In this study, all subjects expressed a truncated C7 molecule containing the NC1 domain, which is believed to be the antigenic portion of the protein, thus minimizing the risk of potential immune reactions. No evidence of C7-associated cytotoxic T-cell activity was observed at any time point in all subjects during the study. However, IIF and DIF studies of subject 4 revealed BMZ-reactive IgG at 1 and 3 months, complement C3 fixation at 3 months, and lower serum IgG titers 6 months after transplantation (Table 2). Although the NC1 domain has been reported as the most antigenic portion of C7, the carboxyl-terminal NC2 domain also contains small antigenic epitopes, and the presence of anti-C7 autoantibodies in RDEB patients has been reported many times in previous publications. The finding that subject 4 had detectable anti-C7 antibodies before transplant using Western blot analysis (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 immune cross-reactivity in future therapeutic studies.

[0109] In conclusion, gene-corrected autologous epidermal skin grafts showed increased C7 deposition and reduced blister formation in patients with severe RDEB, a patient population with other specific treatment options. Larger studies in 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 Study design This is a Phase I, open-label clinical trial (NCT01263379). The primary study objective was to obtain safety and efficacy data in RDEB subjects transplanted with genetically engineered autologous keratinocytes transduced with a retroviral vector containing the full-length COL7A1 coding sequence (Figure 1A). Four adult RDEB subjects were transplanted with LEAES between October 2013 and February 2015. Data for all subjects will be collected at a follow-up of at least 6 months. This study is being supported by Food and Drug Administration (IND#13708) and Stanford IRB (Protocol#14563 ) was approved by

[0115] subject The subjects were aged 18 years or older, had a clinical diagnosis of RDEB, and had a 100cm 2 ~200cm 2Subjects were also able to undergo general anesthesia and were selected based on a screening protocol in which RDEB was confirmed by genetic testing (GeneDx, Gaithersburg, MD). The presence of the NC1 domain of C7 was assessed by Western blot of cultured keratinocyte (KC) supernatants and indirect immunofluorescence microscopy (IIF) of skin biopsy samples. Absence of full-length C7 and mature AF in the biopsy samples was confirmed by IIF using the LH24 antibody specific for the carboxyl-terminal NC2 domain of C7 and immunoelectron microscopy (IEM) (Figure 9). Circulating and tissue-bound IgG, IgA, IgM, and C3 were analyzed by IIF using serum and direct immunofluorescence (DIF) of biopsy sections of the primate esophagus, respectively. Subjects with significant non-RDEB complications, including HIV, hepatitis, systemic infection, or cardiac abnormalities, were excluded. Clinically significant anemia was treated prior to transplantation.

[0116] Test procedure Two 8 mm punch biopsies were obtained for LEAES production from areas of intact, non-scarred skin. Baseline blood samples for CBC, complete biochemistry (CMP), and replication-competent retrovirus (RCR) and C7-sensitive cytotoxic T-cell assays were obtained. Skin biopsies from autologous keratinocytes were transduced with LZRSE-COL7A1 and used to generate eight LEAES grafts (Figure 4). Six grafts were applied to uninfected, eroded, and / or scarred wound sites lacking clinical evidence of SCC. Of the six graft wound sites (A, B, C, D, E, Z, Table 3), one wound was created for each subject at the time of surgery by mechanical friction ("site Z"). Under general anesthesia, the wound bed was cauterized to minimize the possibility of retained epidermal stem cells. The LEAES grafts were secured to the wound bed via dissolvable sutures after wound bed preparation. Subjects 3 and 4 consented to having a small India ink tattoo placed on the corner of each graft to aid in graft identification for follow-up. Grafts were covered with standard wound dressings and topical mupirocin, which were removed 5-7 days after grafting.

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

[0118] Materials and Reagents All materials and reagents used during LEAES manufacturing were free of adventitious viruses based on proof of analysis provided by the manufacturer. Each lot of tissue culture media containing bovine-derived reagents was tested by a commercially available 9CFR absorbance assay (American BioResearch, Pullman, WA) that tests for adventitious viruses and was found to be negative.

[0119] Isolation and expansion of RDEB keratinocytes Skin samples were obtained as two 8 mm punch biopsies and treated with 30 μg / mL amikacin (HIk The skin samples were then introduced into 35 mL of biopsy collection medium 50 / 50A (50% Keratinocyte Medium 154 with Human Keratinocyte Growth Supplement (Life Technologies, Carlsbad, CA) and 50% Defined Keratinocyte Serum-Free Medium with Supplement (Life Technologies, Carlsbad, CA)) with 20 μg / mL vancomycin (Sigma Aldrich, St. Louis, MO), and 0.5 μg / mL amphotericin B (USBiological, Salem, MA). To separate the epidermis from the dermis, the skin samples were placed in a dispase solution (Life Technologies, Carlsbad, CA) containing 25 casein digestion units / mL of dispase for 16-20 h at 5 °C. The following day, the epidermis was carefully peeled from the dermis and placed in TrypLE Select 10X (Life Technologies, Carlsbad, CA) solution for 20-30 min at 37 °C. The solution was spun down at 1200 rpm to obtain a keratinocyte pellet. The cells were washed once with phosphate-buffered saline (PBS, Life Technologies, Carlsbad, CA) and the keratinocytes were plated on PureCoat Collagen I Mimetic Cultureware (Corning Life Sciences, Tewksbury, MA) in 50 / 50A medium. After the keratinocytes reached 60-70% confluence, the cells were treated with TrypLE Select 10X and plated for viral transduction. At least 4 × 10 6 cells were required.

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

[0121] Pre-release testing VTE testing. VTE testing was performed using IF technique with anti-collagen VII monoclonal antibodies NP32, NP185, or anti-collagen VII polyclonal antibody FNC1. Cells were fixed in a solution of methanol / acetone mixture, permeabilized with detergent, and incubated with anti-C71 primary antibody for 1 h at room temperature. After extensive washing, secondary antibodies conjugated to Alexa Fluor555 dye were added and incubated for an additional 1 h. Cell nuclei were labeled with Hoechst33342 for 10 min, washed, and mounted with gold antifade reagent (Life Technologies, Carlsbad, CA). VTE was determined by counting the ratio of blue nuclei to C7-positive cells. To meet the pre-release criteria, at least 50% of cells were positive for C7 expression.

[0122] PGCN exam qP of genomic DNA isolated from corrected RDEB KCs after retroviral transduction PGCN test was performed via PCR analysis. Genomic DNA was extracted from 3 × 10 6 DNA was purified from somatic cells. DNA was quantified using standard spectrophotometric techniques and used for qPCR analysis. Proviral dose was determined using template threshold cycle (Ct) from the amount of plasmid DNA control and a standard curve of Ct dependence. Mean PGCN was calculated from PGCN=(TPCN×6.16pg) / (Ctempl.×103pg), where TPCN=total proviral copy number. 6.16pg=amount of genomic DNA in somatic cells. Ctempl.=amount of template used in PCR in nanograms. To meet the pre-release criteria, there were on average 3 or fewer proviral genome copies for each keratinocyte genome.

[0123] Sterility Testing Samples of culture supernatants were tested for sterility by membrane filtration using a Millipore Steritest system (Pacific BioLabs, Hercules, CA), designed to eliminate potential false negatives from antibiotics present in the medium. After filtration and washing, samples were placed into soyabean-casein digest medium and liquid thioglycollate medium and incubated for 14 days. Samples were observed daily for evidence of microbial contamination.

[0124] Endotoxin Testing Samples of culture supernatants were evaluated by Limulus Amebocyte Lysate (LAL) QCL-1000 assay (Lonza, Basel, Switzerland) according to the manufacturer's recommendations, and the results of this test were less than 1.0 EU / mL to meet the product release requirements.

[0125] Mycoplasma Test Cell culture supernatants were tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland) according to the manufacturer's requirements, and the results of this test were less than 0.9 to meet the product release requirements.

[0126] LEAES Initiation 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 generation medium DFF31. This was performed in Dulbecco's modified Eagle's medium (Life Technologies, London, UK) containing 10% fetal bovine serum (Lonza, Basel, Switzerland), 36 ng / mL hydrocortisone (Spectrum, New Brunswick, NJ), 25 μg / mL adenine (Sigma Aldrich, St. Louis, MO), 5 μg / mL recombinant human insulin (Sigma Aldrich, St. Louis, MO), 2 ng / mL liothyronine (Spectrum, New Brunswick, NJ), 5 μg / mL bovine transferrin (Millipore, Billerica, MA), 10 ng / mL recombinant epidermal growth factor (R&D Systems, Minneapolis, MN), and 30 μg / mL amikacin (Hikma Pharmaceuticals, London, UK), as well as 20 μg / mL vancomycin (Sigma Aldrich, St. Louis, MO). Technologies, Carlsbad, CA) and F12 medium (Lonza, Basel, Switzerland).

[0127] LEAES Assembly and Shipping LEAES assembly was initiated on the day of transplantation. The epidermis was digested with dispase (Life Technologies, Carlsbad, CA). The sheet was allowed to release from the surface of the plate for 20-30 min at 37 °C. The epidermal sheet was washed at least five times with 50 / 50 VC to remove residual amounts of medium and dispase. It was fixed to size-matched petroleum gauze with surgical hemoclips and marked with sterile black sutures on the basal surface. The assembled LEAES was immersed in 50 / 50 VC transport medium and sealed with a gas-permeable sterile membrane. The LEAES-epidermal graft was then transported to the operating room for transplantation.

[0128] Emission Testing Gram Stain Sterility Test. On the day of LEAES release, a sample of the medium was sent to the Stanford Hospital Clinical Laboratory for rapid Gram stain testing. A negative test result was used as the LEAES lot release criterion.

[0129] LEAES Viability Study A viability test was performed in which LEAES samples were incubated for 20 min with a nuclear dye mixture containing Hoechst 33342 and SYTOX Green staining. The ratio of SYTOX green staining to Hoechst 33342 staining was calculated to be 70% or higher to indicate the release product.

[0130] Post-release testing Samples of media and LEAES grafts were submitted for release testing. Test results were expected to be obtained after graft implantation due to the lengthy testing process. Safety plans were implemented in the event that these "post-release" test results were out of specification. Post-release criteria included additional sterility testing (see Sterility Testing in Pre-Release Testing), RCR testing (Indiana University Vector Production Facility), and mycoplasma testing (Bionique Testing Laboratories, Saranac Lake, NY).

[0131] RCR Test Samples of LEAES and LEAES culture supernatants were subjected to an extended PG-4 S+L-cell plaque assay at the Indiana University Vector Production Facility with a test result of "no evidence of RCR" used as a release criterion. At baseline, 3 months, and 6 months, blood samples were analyzed at the Indiana University Vector Production Facility to determine the level of GALV envelope (GALV-E) sequences present using quantitative polymerase chain reaction (Q-PCR). Adequacy of blood sample quantity was assessed with a second probe and primer set for human apolipoprotein B gene sequences. A standard curve using genomic 12 / 22 / 15 8 DNA 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. Negative controls included untransduced human genomic DNA and water.

[0132] Cytotoxic T cell assay For cytotoxic T cell assays, 15 mL of whole blood was collected at baseline, 1 month, 3 months, and 6 months. Peripheral blood mononuclear cells were isolated from buffy coats or whole blood using Ficoll-Paque (GE Healthcare) density gradient centrifugation. Adherent monocytes were then harvested after 2 hours of incubation in Petri dishes. CD4+ and CD8+ T lymphocytes were co-purified using a MACS magnetic cell sorting kit (Miltenyi Biotech) by incubating non-adherent cells with anti-CD4 and anti-CD8 antibodies coupled to paramagnetic microbeads. 96-well PVDF filter plates were used to sort the CD4+ and CD8+ T lymphocytes. Plates (Millipore) 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 × 10 5 cells / well) and γ-irradiated monocytes (2.5 × 10 4Cells / well) were co-incubated on plates for 40 h at 37°C in the presence of 20UI / ml IL-2 in a humidified 5% CO2 air incubator. The medium contained either 10 μg / ml recombinant collagen VII or 3 μg / ml concanavalin A (Sigma) to stimulate lymphocytes. Plates were washed and 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 antibodies (BD Pharmingen) followed by a 1:1000 dilution of streptavidin-conjugated 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. Negative controls were run in parallel using T cells without antigen and the corresponding scores were subtracted from those of the unknowns.

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

[0134] Electron microscopy Three-mm skin punch biopsies were prepared for electron microscopy by immersion in 1.5% glutaraldehyde / 1.5% paraformaldehyde in Dulbecco's serum-free medium (SFM) containing 0.05% tannic acid for a minimum of 1 hour, followed by multiple rinses in SFM and then post-fixation in 1% OsO4 for 60 minutes. Samples were washed in SFM and then dehydrated in a graded series of ethanol to 100%, rinsed in propylene oxide, and infiltrated in Spurr epoxy for a total of 2 hours and accelerated via microwave energy. 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 extensively in SFM, then immersing in mouse IgM LH24 antibody specific for the NC2 region of collagen VII diluted 1:5 in SFM overnight at 4°C, rinsing extensively in SFM, then incubating overnight at 4°C in goat anti-mouse IgM ultrasmall colloidal gold conjugate (Aurion) diluted 1:3 in SFM. After extensive rinsing in SFM, the samples were exposed to gold enhancing solution (Nanoprobes) for 15 min on ice, then rapidly warmed to 25°C and incubated for an additional 5 min. The samples were then rinsed in ice-cold SFM, then fixed and embedded as above. Indirect immunofluorescence (IIF): Human serum was placed on monkey esophagus and stained with antibodies against human IgA, IgM, IgG, and C3. Signal detected at antibody dilutions of 1:40 or higher was considered above background.

[0136] Direct immunofluorescence (DIF) 12 / 22 / 15 10 tissues were sectioned at 5 micrometers and stained with fluorophore-conjugated antibodies to human IgA, IgM, IgG, C3, and fibrinogen. Normal controls were run in parallel. C7 Expression and AF Analysis: 3 mm skin punch biopsy samples were sectioned at 8 micrometers and analyzed by IIF using anti-collagen VII polyclonal antibody FNC1 (against the NC1 domain of C7) or monoclonal antibody LH24 (NC2 domain of C7). Briefly, sections were fixed in a solution of methanol / acetone mixture, permeabilized with detergent, and incubated with anti-C71 primary antibody for 1 hour at room temperature (25°C). After extensive washing, secondary antibodies conjugated to Alexa Fluor 555 or 488 dyes were added and incubated for 1 hour. Nuclei were labeled with Hoechst33342 for 10 min, washed, and mounted with gold antifade reagent (Life Technologies, Carlsbad, CA). Epidermal markers were keratin 1, keratin 14, and loricrin antibodies obtained from Covance (Emeryville, CA). Biopsies were scored positive for C7 expression if continuous linear staining of collagen VII at the dermal-epidermal junction was detected. Biopsies were scored positive for anchoring fibrils (AF) if gold-conjugated particles representing the NC2 domain-specific LH24 antibody were detected with ultrastructural characteristics of AF including density, thickness, curvature, arcuate regions, and loops.

[0137] photograph For subjects 2-4, approximately five images of each implant 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, NJ), landmarks were selected and numbered to identify the corresponding location on each image, then fused into a single image. To accurately track the graft margins, the fused images from the follow-up visit were placed over the outline of the implant on day 0 and compared to the baseline image. Anatomical landmarks (e.g., tattoo dots) were rechecked to ensure the outline was correctly placed. Digital photography (Canon Powershot) was used to obtain additional photographs for subjects 2-4 as needed, and all images for subject 1.

[0138] Equivalent While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the above description and examples are illustrative and are not intended to limit the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

[0139] Unless otherwise defined, 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 disclosure belongs. All nucleotide sequences provided herein are presented in a 5' to 3' orientation.

[0140] The embodiments illustratively described herein may suitably be practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, terms such as "having", "including", "containing" and the like shall 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 excluding any equivalents or portions thereof of the features shown and described, but it is recognized that various modifications are possible within the scope of the present disclosure.

[0141] Thus, although the present disclosure has been specifically disclosed by specific embodiments, it should be understood that any features, modifications, improvements, and variations of the embodiments herein disclosed may be employed by those skilled in the art, and such modifications, improvements, and variations are deemed to be within the scope of the present disclosure. These are representative and illustrative embodiments and are not intended to limit the scope of the present disclosure.

[0142] The scope of the present disclosure is described broadly and generally herein. Each of the narrower species and subgeneric groupings that fall within the generic disclosure also form part of this disclosure. This includes any generic description with a condition or negative limitation that excludes any subject matter from the genus, regardless of whether the excised material is specifically recited herein.

[0143] In addition, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that embodiments of the disclosure can also be described thereby in terms of any individual members or subgroups of members of the Markush group.

[0144] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

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

[0146] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under U.S. Patent Act 119(e) to U.S. patent application Ser. No. 62 / 274,700, filed Jan. 4, 2016, and U.S. patent application Ser. No. 62 / 414,533, filed Oct. 28, 2016, the contents of each of which are incorporated herein by reference.

Claims

1. 1. A pharmaceutical composition comprising an engineered autologous epidermal sheet for use in a method of treating recessive dystrophic epidermolysis bullosa (RDEB) in a human subject suffering from RDEB, said subject having one or more mutations in both copies of the human collagen VII (COL7A1) gene, comprising: The method further comprising: (a) administering to the human subject one or more tests selected from replication-competent retrovirus (RCR), COL7A1-sensitive cytotoxic T cells, or a combination thereof; (b) obtaining a population of skin cells comprising keratinocytes from the human subject; and (c) isolating a population of keratinocytes from said population of skin cells, the population of keratinocytes comprising one or more mutations in the COL7A1 gene, and culturing the isolated population of keratinocytes on collagen 1 peptide in a first keratinocyte medium; (d) transducing the isolated population of keratinocytes ex vivo with a retroviral vector comprising a promoter operably linked to a genetic construct encoding full-length wild-type human collagen VII (COL7A1) protein to generate genetically corrected keratinocytes that meet pre-release criteria of greater than 50% viral transduction efficiency (VTE) 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) subjecting the autologous COL7A1-modified keratinocyte sheet to one or more pre-release tests selected from the group consisting of a VTE test, a PGCN test, and a replication-competent retrovirus (RCR) test; (g) culturing and maturing the autologous COL7A1-modified keratinocyte sheet in a second medium comprising DFF31 medium to form an engineered autologous skin sheet; (h) assembling the engineered autologous skin sheet; and (i) transplanting a graft of the assembled engineered autologous skin sheet into an RDEB-affected wound of the human subject. and Pharmaceutical compositions.

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

1.

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

8.

4. The pharmaceutical composition of claim 1 , wherein the RDEB-affected wound does not contain unmodified keratinocytes.

5. 2. The pharmaceutical composition of claim 1, wherein the method further comprises cauterizing the RDEB-affected wound of the human subject to excise non-modified keratinocytes prior to implanting a graft of the assembled engineered autologous skin sheet.

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

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

8. The method of claim 1 , wherein the keratinocyte sheet is disposed on an acellular matrix, a collagen matrix, or a biocompatible mesh.

9. 9. The pharmaceutical composition of claim 8, wherein the biocompatible mesh is made of thermoplastics, polyethylene, ultra high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofilament polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, silicone, or any combination thereof.

10. The method of claim 1 , wherein the population of skin cells comprises stem cells.

11. The pharmaceutical composition of claim 10 , wherein the method further comprises differentiating the stem cells into keratinocytes.

12. 2. The pharmaceutical composition of claim 1, wherein the method further comprises subjecting the 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. 13. The pharmaceutical composition of claim 12, wherein a blood sample from the transplanted human subject undergoes one or more tests selected from an RCR test, a COL7A1-sensitive cytotoxic T cell assay, or a combination thereof, at about 1, 3, 6, or 12 months post-transplant.

14. 2. The pharmaceutical composition of claim 1, wherein the retroviral vector is selected from Moloney murine leukemia virus (MoMLV), human immunodeficiency virus (HIV), AKR murine leukemia virus (AKR), feline immunodeficiency virus (FIV), or gibbon ape leukemia virus (ALV).

15. The pharmaceutical composition of claim 1 , wherein the retroviral vector is MoMLV.

16. The pharmaceutical composition of claim 1 , wherein the promoter is a MoMLV LTR promoter.

17. The pharmaceutical composition according to claim 1, wherein the retroviral vector is a MoMLV viral vector and the promoter is a MoMLV LTR promoter.

18. 2. The pharmaceutical composition of claim 1, wherein the average PGCN is less than 1 or 0.

5.

19. 2. The pharmaceutical composition of claim 1, wherein the average PGCN is 1.

5.

20. 2. The pharmaceutical composition of claim 1, wherein the average PGCN is greater than or equal to 0.5 and less than or equal to 1.

5.

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

22. The method of claim 1 , wherein the ex vivo transduction of the isolated population of keratinocytes is repeated at least twice.

23. 1. A pharmaceutical composition comprising an engineered autologous epidermal sheet for use in a method of treating recessive dystrophic epidermolysis bullosa (RDEB) in a human subject suffering from RDEB, said subject having one or more mutations in both copies of the human collagen VII (COL7A1) gene, comprising: The method further comprising: (a) administering to the human subject one or more tests selected from replication-competent retrovirus (RCR), COL7A1-sensitive cytotoxic T cells, or a combination thereof; (b) obtaining a population of skin cells comprising keratinocytes from the human subject; and (c) isolating from said population of skin cells a population of keratinocytes that comprises one or more mutations in the COL7A1 gene and culturing the isolated population of keratinocytes in a first serum-free human keratinocyte medium; (d) transducing the isolated population of keratinocytes ex vivo with a retroviral vector comprising a promoter operably linked to a genetic construct encoding a functional human collagen VII (COL7A1) protein to generate genetically corrected keratinocytes that meet pre-release criteria of greater than 50% viral transduction efficiency (VTE) 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) subjecting the autologous COL7A1-modified keratinocyte sheet to one or more pre-release tests selected from the group consisting of a VTE test, a PGCN test, and a replication-competent retrovirus (RCR) test; (g) culturing and maturing the autologous COL7A1-modified keratinocyte sheet in a second medium comprising DFF31 medium to form an engineered autologous skin sheet; (h) assembling the engineered autologous skin sheet; and (i) transplanting a graft of the assembled engineered autologous skin sheet into an RDEB-affected wound of the human subject. and Pharmaceutical compositions.

24. A method for producing an engineered autologous skin sheet, comprising: (a) isolating, from a population of skin cells comprising keratinocytes from a subject, a population of keratinocytes comprising one or more mutations in a gene encoding a protein of interest, and culturing the isolated population of keratinocytes on collagen 1 peptide in a first keratinocyte medium; (b) transducing the isolated population of keratinocytes ex vivo with a retroviral vector comprising a promoter operably linked to a genetic construct comprising a gene encoding a functional protein of interest to generate genetically corrected keratinocytes that meet pre-release criteria of greater than 50% viral transduction efficiency (VTE) and a proviral genome copy number (PGCN) of 3 or less; (c) culturing the gene-modified keratinocytes in a first keratinocyte medium to form a self-modifying keratinocyte sheet; (d) subjecting the self-correcting keratinocyte sheet to one or more pre-release tests selected from the group consisting of a VTE test, a PGCN test, and a replication-competent retrovirus (RCR) test; (e) culturing and maturing the autologous modified keratinocyte sheet in a second medium comprising DFF31 medium to form an engineered autologous skin sheet; (f) assembling the engineered autologous skin sheet; A method comprising:

25. 25. The method of claim 24, wherein the gene encoding the protein of interest is the collagen VIIA1 (COL7A1) gene.

26. 26. The method of claim 24 or 25, wherein the subject has one or more mutations in both copies of the human collagen VIIA1 (COL7A1) gene.

27. 27. The method of claim 26, wherein the subject is afflicted with recessive dystrophic epidermolysis bullosa (RDEB).

28. 28. The method of claim 27, wherein the subject is a human.

29. 28. The method of claim 27, wherein prior to step (a), the subject is subjected to one or more tests selected from replication-competent retrovirus (RCR), COL7A1-sensitive cytotoxic T cells, or a combination thereof.

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