Compositions and methods for the treatment of type vii collagen deficiency

Abstract

To provide a method for treating type VII collagen deficiency such as dominant dystrophic epidermolysis bullosa and recessive dystrophic epidermolysis bullosa.SOLUTION: A method of treating patients suffering from collagen VII (C7) deficiency, the method comprising: obtaining cells from the dermis or epidermis of C7 deficiency patients; contacting the cells with transducing lentiviral vector particles comprising a nucleotide sequence encoding COL7A1 or a functional variant thereof to form autologous genetically modified cells having a specific vector copy number, wherein the lentiviral vector has a transducing vector copy number ranging from 0.1 to 5.0 copies per cells, culturing the autologous genetically modified cells, and administering an effective amount of the genetically modified cells to C7 deficiency patients.SELECTED DRAWING: Figure 14

Description

[Technical Field]

[0001] Sequence Listing This application contains a sequence listing submitted electronically in ASCII format, The ASCII copy is dated March 2017, and is incorporated herein by reference in its entirety. It was created on the 9th of the month, called 0100-0016PR1_SL.txt, and is 92.9 It is 24 bytes.

[0002] The present invention relates to a nucleotide sequence encoding type VII collagen (C7) or its functional group. Type VII colonization, including dystrophic epidermolysis bullosa, including the administration of autologous genetically modified cells containing the mutant form. The present invention relates to compositions and methods for the treatment of antigen deficiency. [Background technology]

[0003] Type VII collagen (C7) is the dermal-epidermal junction (DEJ), which holds all layers of the skin together. It is an important protein in the formation of anchoring fibers in the stingray (Burgeson 1993; Le igh et al. 1988). Dystrophic epidermolysis bullosa (DEB) is characterized by a deficiency of C7. It causes damage to the anchoring fibers that connect the epidermis to the underlying dermis, affecting the skin and organs. It is a genetic disease characterized by a mutation in the COL7A1 gene that encodes C7. Mutations cause C7 deficiency. Patients with DEB suffer from severe blisters. Dominant dystrophic epidermolysis bullosa (DDEB) is characterized by generalized blistering. Transient hydrocele is a form of DDEB that resolves spontaneously during childhood. Genital dystrophic epidermolysis bullosa (RDEB) is a debilitating blistering skin disease. Another form of B, in which blisters form where skin rubs against each other. Without fibers, the skin layers separate and become damaged by normal everyday activities, including rubbing or scratching. Responds to any type of friction, including steroids, resulting in severe blistering, open wounds, and scarring. .

[0004] Currently, in the absence of curative treatment, patients rely primarily on palliative care. Preclinical studies using this strategy have demonstrated that by restoring C7 function in RDEB skin, it is possible to reverse the disease. These findings reveal encouraging results suggesting that the effects of steroids can be corrected. The method relied on transplantation of fragile cultured epidermal tissue, which was difficult to replace. removal of the graft, resulting in scarring (Mavilio et al. 2006; Chen ,Nat.Genet.2002;Siprashvili 2010). Other methods include This includes delivery of corrective C7 by direct viral expression and systemic delivery of recombinant protein. (Remington 2009; Woodley et al. 200 3) All of these potential treatments face several challenges, including cost and biosafety concerns. It is fraught with issues.

[0005] The use of replication-deficient, self-inactivating (SIN) lentiviral vectors (LVs) allows for the transfer of genes This provides multiple advantages to therapy: 1) the ability to transduce both dividing and non-dividing cells; 2) High transduction efficiency, 3) Long-term persistent transgene expression, 4) Triggering an immune response 5) the ability to distribute sequences encoding viral proteins that can be transfected into transcriptionally inactive Improved safety profile due to SIN long terminal repeat (LTR) and 6) large truncated The ability to package transgenes. Clinical trials using LV vectors for gene therapy Stability studies from clinical trials have shown preferential targeting within or near proto-oncogenes or tumor suppressor genes. No integration was demonstrated. 10 billion LV vector-transduced T cells were administered per subject. Patients were followed for an average of four years and showed no signs of leukemia or other adverse events. Chromosome leukodystrophy, Wiskott-Aldrich syndrome (WAS) and adrenoleukodystrophy The studies for the indication of ALD have shown that the duration of treatment is up to 21 months and 32 months, respectively. During the 36-month follow-up, no abnormal clonal proliferation was found (Bi ffi et al.2013;Aiuti et al.2013).

[0006] Treatment with autologous genetically modified human dermal fibroblasts (GM-HDFs) is a treatment that uses the patient's own fibroblasts. provides a promising alternative method based on correcting genetic defects in cells ex vivo (O rtiz-Urda et al.2003;Woodley et al.2003) In supplying C7 to the basement membrane zone (BMZ) of mice bearing RDEB skin grafts, h COL7A1 gene-corrected fibroblasts have been reported to be superior to keratinocytes. Furthermore, injection of fibroblasts has been shown to improve keratinocyte function (Goto et al. 2006). This can be accomplished more easily than transplanting a computer. Summary of the Invention

[0007] The present invention relates to a method for treating a patient suffering from type VII collagen (C7) deficiency. This involves extracting cells, preferably fibroblasts or keratinocytes, from the dermis or epidermis of a patient with C7 deficiency. Obtaining latinocytes, a trait comprising the COL7A1 gene or a functional variant thereof The cells are contacted with transducing lentiviral vector particles to produce cells with a specific vector copy number. The steps of forming autologous gene-modified cells containing the COL7A1 gene or a functional variant thereof are described. In the method, the lentiviral vector particles are transfected in a range of 0.1 to 5.0 copies per cell. and culturing the autologous genetically modified cells to have a transduction vector copy number within the range of 100-1500. and administering the genetically modified cells to a patient with C7 deficiency. In one aspect of the invention, C7 deficiency may be achieved by injection, preferably intradermal injection. Recessive dystrophic epidermolysis bullosa (RDEB) or dominant dystrophic epidermolysis bullosa (DDEB), etc. The subtype of RDEB is dystrophic epidermolysis bullosa (DEB). and as such, Hallopeau-Siemens Hallopeau-Siemens type, non-Hallopeau-Siemens type These include RDEB, atypical RDEB, pretibial RDEB, distal RDEB, and centripetal RDEB. It can be obtained.

[0008] In one embodiment of the present invention, the transducing lentiviral vector particle comprises: (a) a L Modified 5' long terminal repeat within the TR (where the promoter of the modified 5' LTR is cytomegalovirus (b) a COL7A1 gene or a functional variant thereof; (c) a COL7A1 gene or a functional variant thereof; At least one lentivirus central polypurine tract, (d) Hepatitis B virus post-transcriptional regulation (e) modified 3'LTR (the modified 3'LTR is a nucleotide sequence of the wild-type 3'LTR) R) containing a deletion in the transfer lentiviral vector, Here, the COL7A1 gene or a functional variant is incorporated into cells to produce a functional COL7A1 gene. Genetically modified cells are generated that contain a gene or a functional variant thereof.

[0009] In one embodiment, there are at least two lentiviral central polypurine sequence elements. In another embodiment, the PRE is a protein derived from woodchuck hepatitis virus. hepatitis virus post-transcriptional regulatory element (WPRE). Transfer lentiviral vectors used to construct transduction vectors Preferably, the transduction vector is selected from the group consisting of pSMPUW and pFUGW. The antiviral vector particle is INXN-2004 or INXN-2002.

[0010] Administration of autologous genetically modified fibroblasts to patients with C7 deficiency can be by injection, topical, oral, or biologic means. This may be done in any suitable way, including embedding in a compatibility matrix.

[0011] Another aspect of the present invention is a lentiviral vector particle comprising a functional COL7A1 gene. C, such as in RDEB or DDEB patients, that are transduced and express type VII collagen. 7 deficiency patient-derived autologous genetically modified fibroblasts, wherein the lentiviral vector Particles contain transduction vector copy numbers ranging from 0.1 to 5.0 copies per cell .

[0012] Another embodiment includes: (a) a modified 5' long end within the LTR (wherein the modified 5' LTR (b) the COL7A1 gene or (c) at least one lentiviral central polypurine sequence element; (d) Hepatitis B virus post-transcriptional regulatory element (PRE), and (e) modified 3'L Transfer RNA containing the modified 3'LTR (the modified 3'LTR contains a deletion relative to the wild-type 3'LTR) In one embodiment, the invention relates to a self-inactivating lentiviral vector formed from the vector. , the vector comprises at least two lentiviral central polypurine sequence elements; PRE is a virus that causes woodchuck hepatitis The vector of the present invention includes an INIRUS post-transcriptional regulatory element (WPRE). A vector designated XN-2004 and containing the sequence of the IGE308 plasmid; or INXN- There is a vector called 2002.

[0013] Another embodiment of the present invention is INXN-2004, which comprises the sequence of the IGE308 plasmid. or transduced with a lentiviral vector designated INXN-2002. Antiviral vector-transduced lines obtained from RDEB or DDEB patients The present invention relates to pharmaceutical compositions comprising fibroblasts.

[0014] In one embodiment, the present invention provides vectors such as INXN-2002 and INXN-2004. This relates to cells transduced in vitro or ex vivo using the method described above.

[0015] In one embodiment, the present invention provides a method for the preparation of a medicament for the treatment ... These relate to autologous fibroblasts transduced with fibroblasts, which are described in U.S. Pat. No. 8,728,819. It was obtained and grown according to the methods described in the specification and international patent application.

[0016] International Publication No. 2002 / 003004 entitled "Method for culturing minimally passaged fibroblasts and uses thereof" No. 08 / 027984, each of which is incorporated herein by reference.

[0017] In one embodiment, the present invention provides a method for the production of human autologous skin cells, such as fibroblasts and / or keratinocytes. The present invention relates to the use of concentrated lentivirus for the transduction of cells.

[0018] In another embodiment, the present invention provides a method for the preparation of a fibrous lentivirus by centrifugation with the lentivirus. For transduction of autologous human skin cells such as blastocytes and / or keratinocytes.

[0019] In one embodiment, the present invention provides a method for the production of human autologous skin cells, such as fibroblasts and / or keratinocytes. The use of both concentrated lentivirus and centrifugation for transduction of cells.

[0020] In one embodiment, the present invention provides a method for contacting a lentivirus with a cell line in two or more separate transduction steps. The present invention relates to the use of autologous human skin cells, such as fibroblasts and / or keratinocytes, which have been induced to grow in the skin.

[0021] In one embodiment, the present invention provides a method for contacting a lentivirus with a cell line in two or more separate transduction steps. Regarding the use of human autologous skin cells, such as fibroblasts and / or keratinocytes, Here, the cells are cultured and transfected once, twice, or three times before the second or any subsequent transduction step. Subculture.

[0022] The present invention also relates to the treatment of patients suffering from pseudosyndactyly, which comprises administering to the patient a functional COL7A transduced with a lentiviral vector particle containing a gene or a functional variant thereof, and administering to said patient a population of autologous cells derived from said patient that express type VII collagen. Includes top.

[0023] The present invention further provides a compound useful in treating, inhibiting or preventing blistering in dystrophic epidermolysis bullosa (DEB). and a method for alleviating or treating the lesions in RDEB patients, which comprises the use of an autologous gene of the present invention. Administering the genetically modified cells.

[0024] Another aspect of the present invention is a vector comprising a functional COL7A1 gene or a functional variant thereof. particles and expressing type VII collagen, such as DDEB or RDEB. An isolated population of autologous genetically modified fibroblasts from patients with C7 deficiency, as well as the The present invention relates to a method for producing a population of

[0025] The present invention also provides a method for the production of genetically modified human skin fibroblasts or keratinocytes per cell. The present invention also relates to a method for increasing the integrated transgene copy number of the COL7A1 gene. or a functional variant thereof. The vector was contacted with human skin fibroblasts or keratinocytes obtained from a patient with C7 deficiency. The transduction composition is then transferred to a spinocule. The cells were then subjected to spinoculation to transfect human dermal fibroblasts or keratinocytes. The cells are then transduced into human skin fibroblasts or keratinocytes to form keratinocytes. The transgene copy number is higher in the transducing composition than in the non-spinoculated transducing composition. The present invention further includes a supertransduction step, in which the transduced cells are transduced into a second and a transducing lentiviral vector of the present invention to form a second transducing composition. Optionally, the composition is subjected to spinoculation. The viral vector is INXN-2002 or INXN-2004, preferably INXN Transduced human skin fibroblasts or keratinocytes are spinoculated. at least or more per cell compared to a transduction composition that has not been subjected to transduction or supertransduction is approximately 1, 2, 5, 10, 20, 25, 27, 28, 29, 30, 35, 40, 45, or In another embodiment, the present invention provides a method for producing a recombinant vector comprising: at least 0.05, 0.09, 0.41, or 0.74 per cell, or about 0.1 Incorporated transistors in the range of 0.1 to about 5, 0.1 to about 1, 0.4 to about 1, or 0.4 to about 0.75 Expression is achieved by the gene transfer methods described herein. For example, C7 expression has been shown to increase with spinoculation or compared to transduced human skin fibroblasts or keratinocytes that had not been subjected to a second transduction. and demonstrated a 10, 25, 50, 100, 150, or 200-fold increase in do. [Brief explanation of the drawings]

[0026] The present invention will now be described in greater detail with reference to the accompanying drawings, when considered in conjunction with the following detailed description. The embodiments shown in the drawings are intended to illustrate the invention. The above is merely illustrative and should not be construed as limiting the invention to the illustrated embodiments.

[0027] [Figure 1A] Figure 1A depicts type VII collagen (C7) trimers that form anchoring fibrils. The COL7A1 gene encodes a 290 kDa α-chain, with three chains forming a triple helix (trimer). Image from Bruckner-Tuderman L. Molecular Therapy (2008) 17:6-7. [Figure 1B]Figure 1B depicts the overall structure of normal and RDEB skin. C7 anchoring fibers bind to other collagens, extracellular matrix proteins, and Lam332 to mediate the connection between the dermis and epidermis. Absence of anchoring fibers can lead to blister formation. [Figure 2] Figure 2 illustrates a cGMP-scale GM-HDF manufacturing process according to one embodiment of the present invention. The C7 expression cassette was cloned into a self-inactivating lentiviral backbone. Pilot-scale production of a lentiviral vector (INXN-2002) containing the COL7A1 gene (LV-COL7) with a titer of approximately 9 x 10 IU / mL was performed for use in a cGMP-scale manufacturing process. Fibroblasts were isolated from a RDEB patient biopsy, expanded, and then divided into three arms for mock, high-dose, and low-dose LV-COL7 transduction. Fibroblasts from each arm were expanded to 2 x CS10 scale and then cryopreserved (drug substance). For patient treatment, the drug substance vials were thawed, formulated (formulation), and then shipped back to the clinical site for injection into the wound healing site of the patient from whom the cells were derived. [Figure 3A] Figure 3A depicts the LV-COL7 (for INXN-2002) copy number per cell. Drug substance vials were thawed and assayed for LV-COL7 DNA copies per cell using qPCR. Primers were specific for the LV shuttle vector. Results demonstrated dose-dependent levels of copy number per cell, with an average of 0.38 and 0.18 copies from the high-dose and low-dose arms, respectively. [Figure 3B] Figure 3B describes the C7 expression levels produced by RDEB patient fibroblasts transduced with LV-COL7: a bulk drug vial was thawed and cultured for 3 days. Conditioned cell culture supernatants were collected and assayed for C7 levels by ELISA. The results show viral dose-dependent protein expression ranging from 60 to 120 ng / mL (C7) in LV-COL7-transduced cells. [Figure 3C]Figure 3C shows the trimeric form of C7 produced by RDEB patient fibroblasts transduced with LV-COL7. Conditioned cell culture supernatant was also used in the immunoprecipitation assay. Immunoprecipitated C7 was separated on non-denaturing SDS-PAGE and visualized by Western blot. C7 produced by RDEB fibroblasts was predominantly trimeric, with LV-COL7-transduced cells expressing more COL7 than mock-transduced cells (arm C). Several low-molecular-weight species showing immunoreactivity were also observed. [Figure 4A] Figure 4A shows the binding of purified COL7 to Lam332. COL7 was expressed in CHO-DG44 cells, purified by size-exclusion chromatography, and assayed for preferential binding to Lam332 compared to BSA (an increase in OD450 corresponds to increased binding of COL7 to Lam332). [Figure 4B] Figure 4B shows the binding of COL7 to Lam332 in LV-COL7-transduced fibroblasts (transduced with the INXN-2002 vector). Vials of the bulk drug were thawed and cultured for 3 days. Conditioned cell culture supernatants were collected and assayed for binding to Lam332 compared to a BSA control. The results show a dose-dependent binding of virus to Lam332. [Figure 5] Figure 5 shows composite RDEB skin grafts on the back of SCID mice analyzed by immunofluorescence staining with a human COL7-specific antibody after intradermal injection of 1 × 10 GM-HDFs (transduced with the INXN-2002 vector). Representative images are shown. COL7 localization was observed in composite grafts (n = 4) made with RDEB keratinocytes 10 days after intradermal injection of 1 × 10 GM-HDFs. Positive control grafts made from normal keratinocytes and fibroblasts showed intense COL7 staining, while negative control grafts showed no COL7 staining at baseline (arrow at DEJ for negative baseline comparison). [Figure 6] Figure 6 provides a schematic diagram of the pSMPUW lentiviral expression plasmid vector. [Figure 7]Figure 7 compares the characteristics of the pSMPUW lentiviral expression vector to third generation lentiviral expression vectors. [Figure 8] FIG. 8 provides a schematic diagram of the pFUGW lentiviral expression plasmid vector. [Figure 9] FIG. 9 shows an electron micrograph of HIV-1 virus particles. [Figure 10] FIG. 10 describes the immunoprecipitation results of TR8 GM-HDF detecting the formation of C7 trimers. [Figure 11A] FIG. 11A depicts the immunoprecipitation results of TR10 and ER1 GM-HDF detecting the formation of C7 trimers. [Figure 11B] FIG. 11B depicts the immunoprecipitation results of TR9 detecting the formation of C7 trimers. [Figure 12] FIG. 12 shows the results of Lam332 binding by C7 expressed by GM-HDFs. [Figure 13] FIG. 13 provides a schematic diagram of the lentiviral vector plasmid construct encoding the COL7A1 gene. [Figure 14] FIG. 14 provides a schematic diagram of the proviral RNA genome structure of the INXN-2002 lentiviral vector. [Figure 15] FIG. 15 shows a schematic diagram detailing the INXN-2002 viral particle. [Figure 16] FIG. 16 provides a schematic of the in vitro immortalization assay to assess the insertional genotoxic potential of INXN-2002. [Figure 17] FIG. 17 shows the typical cell morphology and structure of FXC-007 in culture. [Figure 18] FIG. 18 provides a graph of C7 expression levels determined by ELISA for particular doses of TR8 GM-HDFs. [Figure 19] FIG. 19 depicts a Western blot showing immunoprecipitation of C7 trimers produced by FCX-007 GM-HDFs against NC1-specific antibodies. [Figure 20]Figure 20 shows the assay readout (optical density at 450 nm (OD450)) for the binding assay to detect the interaction of C7 with laminin 332 or bovine serum albumin (BSA) coated wells. Bound C7 was detected using a C7 NC1-specific antibody and an HRP-conjugated secondary antibody. [Figure 21A] FIG. 21A is a graph showing the amount of cell migration over time of normal fibroblasts and patient fibroblasts transduced with LV-COL7. [Figure 21B] FIG. 21B provides images of cell migration over time of normal fibroblasts and patient fibroblasts transduced with LV-COL7. [Figure 22] FIG. 22 provides a schematic diagram of the cloning of the human COL7A1 gene. [Figure 23] FIG. 23 provides a schematic diagram of the cloning of the COL7A1 gene into the pSMPUW expression vector to create the INXN-2002 lentiviral vector transfer plasmid, IGE230. [Figure 24] Figure 24 provides a schematic diagram of the construction of the INXN-2004 lentiviral vector transfer plasmid (IGE308). [Figure 25A] Figure 25A depicts a schematic of the genetic elements of the IGE308 plasmid. [Figure 25B] Figure 25B depicts a schematic of the genetic elements of the IGE308 plasmid. [Figure 26] FIG. 26 shows immunofluorescence analysis of GM-HDF injected composite grafts. [Figure 27] FIG. 27 provides a graph showing LV-COL7 transcript levels as measured by their fold change values ​​relative to control cells. [Figure 28] FIG. 28 provides representative images of C7 staining of INXN-2002 and LV-HA-COL7 transduced RDEB fibroblasts. [Figure 29] FIG. 29 provides a graph showing C7 protein levels measured in cell supernatants of GM-HDFs transduced with TR8, TR9, T10, and ER1. [Figure 30] FIG. 30 graphically depicts the correction of hyperkinesis of GM-HDF over time for TR8, TR9, T10, and ER1. [Figure 31] Figure 31 shows representative indirect immunofluorescence (IF) images at two magnifications, 20x and 5x, to examine C7 protein expression by GM-HDF cells using a C7-specific antibody (for TR12.1 and TR13, only arm A was tested; only arm A of TR8 is shown for comparison with the previous step). [Figure 32] FIG. 32 depicts the percentage of C7-positive (C7+) cells in GM-HDFs for each training run arm of TR11, TR12.1, and TR13. [Figure 33] Figure 33 provides a graph showing the amount of C7 protein levels expressed for TR8, TR11, TR12.1, and 13. Error bars indicate standard deviation; n=3. For TR8, TR12.1, and TR13, only the results for Arm A are shown. [Figure 34] FIG. 34 provides SDS-PAGE / immunoblot for the detection of C7 trimers (a) TR11, (b) TR12.1 and TR13 GM-HDF. [Figure 35] FIG. 35 shows a graph of GM-HDF expressed C7 binding to laminin 332 (Lam332) for TR11, TR12.1, and TR13. [Figure 36] Figure 36 provides the results of a cell migration assay showing the correction of hypermotility (% migration) over time for GM-HDFs for TR11, TR12.1, and TR13. Hypermotility of normal human dermal fibroblasts (NHDFs) and control fibroblasts derived from recessive dystrophic epidermolysis bullosa is shown. DETAILED DESCRIPTION OF THE INVENTION

[0028] Unless otherwise defined, all technical and scientific terms and any Abbreviations have the same meaning as commonly understood by one of ordinary skill in the art of the present invention. Any compositions, methods, kits, and communications similar or equivalent to those described herein. Although any means can be used to practice the invention, preferred compositions, methods, kits and information are The means of communication are described herein.

[0029] All references cited herein are hereby incorporated by reference to the fullest extent of law. The discussion of such references should be a summary of the arguments made by their authors. If any reference (or part of any reference) is related No admission is made that any of the references cited are prior art. We reserve the right to challenge the accuracy and appropriateness of the documents.

[0030] To facilitate understanding of the present invention, several terms are defined herein. Definitions are provided throughout the detailed description.

[0031] When used in conjunction with the term "comprises" in the claims and / or specification, "one The use of the words "a" or "an" can mean "one," but It also coincides with the meaning of "one or more," "at least one," and "one or more."

[0032] Throughout this application, the term "about" means that a value is within the meaning of the device used to determine that value. to show that it includes the variation of error inherent in the device, method, or variation that exists between test subjects. Typically, the term is used to refer to approximately 1%, 2%, 3%, 4%, or 5%, depending on the context. ,6%,7%,8%,9%,10%,11%,12%,13%,14%,15%,16% , 17%, 18%, 19% or 20% or 1%, 2%, 3%, 4%, 5%, 6%, 7 %, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, It is meant to include variability of less than 18%, 19% or 20%.

[0033] The use of the term "or" in the claims is expressly intended to refer to alternatives only. or used to mean "and / or" unless alternatives are mutually exclusive. Although it may be stated otherwise, the present disclosure supports definitions that refer to alternatives only, and "and / or."

[0034] As used in this specification and claims, "comprises" (as well as "comprises" and "including" and all other forms of "having" (as well as "having" and "having" "including" (and "including" and "including" in any form, including in any form, including but not limited to, or "containing" (as well as "containing" and "containing" The term "any form including, but not limited to, " " is inclusive or open-ended and therefore includes additional This does not exclude any specific, unrecited elements or method steps. Any embodiment can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, the composition of the present invention can be used to achieve the method of the present invention. do.

[0035] Nucleic acids and / or nucleic acid sequences may be derived from a common ancestral nucleic acid or nucleic acid sequence, whether naturally occurring or not. are "homologous" if they are artificially obtained. Proteins and / or protein sequences are The coding DNA is derived naturally or artificially from a common ancestral nucleic acid or nucleic acid sequence. Homologous molecules can be referred to as homologs. As described, any naturally occurring protein can be mutagenized using any available mutagenesis method. When expressed, the mutagenized nucleic acid will be similar to the original nucleic acid. The polypeptide encoded by the gene encoding the polypeptide is homologous to a protein encoded by the gene encoding the polypeptide. , deduced from the sequence identity between two or more nucleic acids or proteins (or their sequences) The exact percentage of identity between sequences that is useful in establishing homology is determined by the appropriate Although the sequence may vary depending on the nucleic acid and protein involved, at least 25 sequences are required to establish homology. % sequence identity is typically used. Additionally, higher levels of sequence identity may be used to establish homology. Column identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% Or 99% or more can be used. Methods for determining sequence identity percentage (e.g., BLASTP and BLASTN using default parameters) are used herein. and is publicly available.

[0036] With respect to two nucleic acid or amino acid sequences of a polypeptide, the terms "identical" or "sequence identical" are used. "Uniformity" is the maximum match possible when aligned for maximum matches over a specified comparison window. As used herein, a "comparison window" refers to residues that are the same within two sequences. , at least about 20, usually about 50 to about 200, more usually about 100 to about 150 refers to a segment of adjacent positions, where, after optimal alignment of two sequences, A sequence can be compared to a reference sequence for the same number of consecutive positions. Alignment methods are known in the art. The paper is based on Smith and Waterman (1981) Adv. Appl. Math. .2:482 by the local homology algorithm; Needleman and Wuns Alignment algorithm of ch(1970) J. Mol. Biol. 48:443 From Pearson and Lipman (1988) Proc. Nat. Acad. .Sci USA85:2444 similarity search method; these algorithms Computer implementations of (including but not limited to) Intelligentics, Mountain View, n View Calif., PC / Gene program CLUSTAL, Wi sconsin Genetics Software Package,Geneti cs Computer Group(GCG),575 Science Dr.,M Adison, Wis., USA GAP, BESTFIT, BLAST, FAS TA, TFASTA, etc.); the CLUSTAL program is iggins and Sharp (1988) Gene 73:237-244 an d Higgins and Sharp (1989) CABIOS 5:151-15 3;Corpet et al. (1988) Nucleic Acids Res.1 6:10881-10890;Huang et al(1992)Computer Applications in the Biosciences 8:155-16 5; and Pearson et al. (1994) Methods in Molec cular Biology 24:307-331. ,Alignment is often also performed by inspection and manual alignment.

[0037] In one class of embodiments, for example, BLASTP (or CLUSTAL, or any other available alignment software) By definition, the polypeptides herein have at least 70% similarity to the reference polypeptide, or a fragment thereof. %, typically at least 75%, optionally at least 80%, 85%, 90%, 98% % or 99% or more identical. Similarly, nucleic acids can be expressed in terms of a starting nucleic acid, For example, these can be generated using, for example, BLASTN (or CL) with default parameters. USTAL, or any other available alignment software), at least 50%, 60%, 70%, 75%, 80%, 85% identical to the reference nucleic acid or fragment thereof; It can be 90%, 98%, 99% or more identical. One molecule can have a specific part identity with a larger molecule. When we say that two molecules have a certain percentage of sequence identity, this is based on optimal alignment of the two molecules. The smaller percentage of residues is the percentage that will result in the two molecules being optimally aligned when the This means that the matching residues in the larger molecule are found according to the order in which they are found.

[0038] The term "substantially identical" as applied to a nucleic acid or amino acid sequence means that the nucleic acid or amino acid sequence using the aforementioned program (preferably BLAST) with standard parameters, see Compared to the sequence, there is at least 90% sequence identity or more, preferably at least Preferably, the sequence has at least 95%, more preferably 98% and most preferably at least 99% sequence identity. For example, the BLASTN program (which searches for nucleotide sequences) In this case, the default is a word length (W) of 11, an expected value (E) of 10, M=5, and N=- 4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses Defaults to a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 score. Ring matrix (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). The percentage of similarity is calculated by comparing two optimally aligned sequences over a comparison window. The portion of the polynucleotide sequence within the comparison window is determined by comparing the The minutes are compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. The percentages may include additions or deletions (i.e., gaps) compared to both sequences. Determine the number of positions where identical nucleic acid bases or amino acid residues exist in the , divide the number of matched positions by the total number of positions in the comparison window, and then multiply the result by 10 It is calculated by multiplying by 0 to get the percentage of sequence identity. Alternatively, substantial identity is more pronounced over a region of sequence at least about 50 residues in length. Preferably, it is present over a region of at least about 100 residues, and most preferably the sequence is In a most preferred embodiment, the sequences are substantially identical over at least about 150 residues. The sequences are substantially identical over the entire length of the coding region.

[0039] A "functional variant" of a protein disclosed herein is, for example, a variant that has at least or about 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, The amino acid sequence of the reference protein containing 18, 19, or 20 conservative amino acid substitutions The phrase "conservative amino acid substitution" or "conservative mutation" refers to a mutation that has a common characteristic. It refers to the substitution of one amino acid for another amino acid that has the common characteristics of individual amino acids. A functional method for determining the The aim of this study was to analyze the normalized frequency of amino acid changes in the genomic DNA (Schulz, GE and S chirmer,RH,Principles of Protein Struc ture, Springer-Verlag, New York (1979). According to the analysis, amino acids within a group preferentially exchange with each other, thus affecting the overall protein structure. It is possible to define groups of amino acids that are most similar to each other in their effects on the body (Sc Hulz, GE and Schirmer, RH (ibid.). Examples of conservative mutations are In addition, amino acid substitutions of amino acids within the aforementioned subgroups, e.g., lysine, methyl ... substitution of arginine with glutamic acid, and vice versa, so that the negative charge can be maintained. substitution of aspartic acid with serine so that the free -OH can be maintained; Substitution of threonine; and aspartate with glutamine so that free -NH2 can be maintained. In one embodiment of the present invention, the COL7A1 gene is characterized in that the mutant is C7 protein or its functional group, provided that it is capable of anchoring fibrils between the epidermis and the dermis. encodes a sexual variant.

[0040] Alternatively, or in addition, functional variants contain at least one non-conservative amino acid substitution. A "non-conservative mutation" may include a mutation in a different group of amino acids from a reference protein. substitution of amino acids between each other, e.g., tryptophan with lysine, or phenylalanine with tryptophan In this case, non-conservative amino acid substitutions may be used to prevent functional variants, such as substitutions of serine. Preferably, non-conservative amino acid substitutions do not alter or inhibit the biological activity of the polypeptide. The biological activity of functional variants is increased relative to the reference sequence, so that the biological activity of functional variants is increased relative to the reference sequence. It can be strong.

[0041] The proteins disclosed herein (including functional portions and functional variants thereof) may be one or more The amino acid sequence may contain synthetic amino acids in place of some of the naturally occurring amino acids. Synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norbornylamino acid, Leucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine trans-3 and trans-4-hydroxyproline, 4-aminophenylalanine , 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenyl Alanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline -2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, N-aminomalonic acid, N-aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N' , N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentasiloxane α-aminocyclohexanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid , α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylguanine One example is lysine.

[0042] The present invention relates to vector INXN-2002; and also referred to herein as INXN-2004. IGE308, which is a vector that is substantially identical and / or homologous thereto, and These functional variants are included.

[0043] The term "patient" or "subject" refers to mammals, including humans and animals.

[0044] The term "treating" refers to the alleviation of symptoms of DEB, including RDEB and / or DDEB. means to alleviate or improve and / or prevent its recurrence and / or progression do.

[0045] The term "autologous cells" refers to cells obtained from and later returned to the same individual. The cells may be taken from the body of a DEB patient, and these cells may then be transfected. They can be genetically modified, grown in culture, and then returned to the patient from whom they were originally taken. can.

[0046] The term "transduction" refers to viral infection rather than transfection. It also refers to gene delivery using retroviral vectors.

[0047] The terms "transducing lentiviral vector" or "transducing lentiviral vector particle" are used interchangeably. The "col7a1" gene is a lentiviral expression / transfection vector containing the COL7a1 gene or a functional variant thereof. Using a far plasmid vector, packaging vector, and envelope vector Infectious lentiviral vectors formed from co-transfection of the same packaging cell line. Lentiviral vectors for transduction are vector particles that are generated after transfection. Suitable packaging cell lines are harvested from the supernatant of producer cell cultures. These are well known in the field, for example, the 293T cell line.

[0048] The term "lentiviral vector" refers to a vector that is primarily derived from a lentivirus and has an LTR external structure. and refers to a vector containing functional genetic elements.

[0049] The term "self-inactivating vector" (SIN) refers to the 3' LTR endogenous vector known as the U3 region. The promoter region blocks viral transcription after the first round of viral replication. refers to a vector that has been modified (e.g., by deletion or substitution) so that the vector The tRNA can infect and integrate into the host genome only once and is not further passaged. SIN vectors cannot be replicated in the 3'LTR U3 region after the first round of replication. modified to block viral transcription, eliminating the ability of the virus to passage This significantly reduces the risk of generating unwanted replication-competent virus.

[0050] The term "pharmaceutically acceptable carrier" means any carrier that, within the scope of sound medical judgment, without affecting the activity of the cells and without being toxic to human and animal tissues. without causing irritation, allergic reactions, or other complications. Suitable for use in contact with genetically modified cells and compatible with a reasonable benefit / risk ratio. The term "autologous genetically modified" is used herein to refer to a pharmaceutically acceptable carrier. It is biocompatible with the target cells and does not reduce their activity or cause their death. It may be an injection solution that is not strained.

[0051] The present invention relates to an autologous gene-modified cell therapy for patients with C7 deficiency. The patient had type VII collagen, which is important for the formation of anchoring fibers at the dermal-epidermal junction (DEJ). Either there is a deficiency of C7 or its production is substantially reduced. This causes dystrophic epidermolysis bullosa (DEB). Collecting DEB patient cells, such as tinocytes, and identifying the functional COL7A1 gene or its function. Genetically modifying the harvested cells to insert functional mutations, propagating the genetically modified cells, and administering the population of genetically modified cells back to the DEB patient.

[0052] Cells are obtained from patients suffering from DEB. Fibroblasts or keratinocytes are preferred. The cells may be obtained by known methods, for example, from a patient's skin sample, scraping, etc. In one embodiment, the cells are obtained from non-blistering skin of a DEB patient. In another embodiment, the cells are obtained from blistered skin of a DEB patient. The cells obtained from the These cells can be cultured using standard cell culture techniques. The collected patient cells are then transfected with functional type VII collagen gene (C7 ) or a functional variant thereof.

[0053] The COL7A1 gene has the nucleic acid sequence encoding the 290 kDa alpha chain, SEQ ID NO: 1, The three chains form a triple helix (trimer) as depicted in Figure 1A. C7 is It has the amino acid sequence of SEQ ID NO: 2 and is involved in the formation of anchoring fibers at the dermal-epidermal junction (DEJ). Mutations in the C7 gene result in the formation of a mooring line that maintains the connection between the epidermis and dermis. This results in the absence or reduction of C7, which makes up the fibers. C7-anchored fibers are mediates the binding of other collagens, extracellular matrix proteins, and the dermis to the epidermis The absence of anchoring fibers leads to blistering of the skin. The present invention also provides vectors containing nucleic acids and polypeptides substantially identical to COL7A1 and C7. In another embodiment, the present invention relates to the use of a mutant as a marker for the formation of a medicament for the treatment ... If the fibers can be anchored, functional variants of the COL7A1 or C7 proteins can be encoded. Another aspect of the present invention includes a vector containing a nucleic acid encoding the COL7A1 gene. This includes the use of only the single reading frame of the C7 protein or a functional variant thereof. In some embodiments, the COL7A1 nucleic acid encodes: Codon-optimized for increased expression of C7 in transduced cells.

[0054] According to the present invention, COL7A1 is a transducible lentivirus that is replication-deficient and self-inactive. The virus vector can be used to transduce harvested DEB cells. Lentiviral vectors are derived from the human immunodeficiency virus (HIV-1) and are used to Pseudotypes containing heterologous VSV-G envelope protein but not -1 envelope protein As previously reported, a 400-bp deletion was introduced into the U3 region of the LTR. This results in a self-inactivating (SIN) vector (Zuffery 1998 The vectors of the present invention are distinguished from second and third generation SIN vectors. These vectors provide increased safety and, as further described herein, facilitate cloning. It has been modified to increase its potency.

[0055] The vectors used to construct the transducing lentiviral vectors of the present invention are The vector is introduced into a packaging cell line by transfection or infection. The guinea pig cell line produces transducing lentiviral vector particles containing the vector genome. Packaging vectors, transfer vectors, and After co-transfection of the vector and the envelope vector, the recombinant virus was recovered from the culture medium. The calcium phosphate is then collected and titrated by standard methods used by those skilled in the art. by sodium transfection, lipofection or electroporation. Typically, a dominant selectable marker (kanamycin, neomycin, DHFR, or glutamine) is used. The packaging construct can be introduced into a human cell line along with a target gene (e.g., a ribosomal proteinase), This is followed by selection in the presence of the appropriate drug and isolation of clones. The gene can be physically linked to the packaging gene in the construct.

[0056] The packaging functions are constructed to be expressed by a suitable packaging cell. Stable cell lines are known, see, for example, U.S. Patent No. 5,629,999, which describes packaging cells. , 686,279; and Ory et al., (1996). Packaging cells with lentiviral vectors integrated into them are used to transfect producer cells. Thus, the producer cells form packaged cells carrying the therapeutic gene of interest. The preferred cell line is a cell or cell line capable of producing or releasing infectious viral particles. An example of a suitable lentiviral vector packaging cell line is 293 cells.

[0057] In one embodiment of the present invention, the transducing lentiviral vector comprises: (a) a modified 5' A lentiviral expression plasmid vector containing flanking long terminal repeats (LTRs) (wherein The promoter of the modified 5'LTR is the cytomegalovirus promoter. (b) COL 7A1 gene; (c) at least one lentiviral central polypurine sequence element; and (d) a modified 3'LTR, wherein the modified 3'LTR is a deletion relative to the wild-type 3'LTR. and contains a deletion of the Hepatitis B virus post-transcriptional regulatory element (PRE) It will be built.

[0058] In another aspect of the invention, the deleted Hepatitis B virus post-transcriptional regulatory element (PRE) is , woodchuck posttranscriptional regulatory element PRE (WPRE).

[0059] In another aspect of the invention, the lentiviral expression plasmid vector comprises two or more lentiviral vectors. Contains the viral central polypurine tract (cPPT) element. Integration of the cPPT element It has been shown to increase gene expression levels.

[0060] In another embodiment of the invention, the 3'LTR is a commonly used standard third generation SIN L The 400 bp fragment (Zuffery 1998) was not a 133 bp deletion of the V vector. The 400 bp deletion prevents read-through transcription. This increases safety by blocking the vector transcripts in the packaging cells. It is believed to be more stable and therefore increases viral titers.

[0061] In another embodiment of the invention, the lentiviral expression plasmid contains a Hepatitis B virus transcript. A post-regulatory element (PRE) (preferably a woodchuck post-transcriptional regulatory element PRE) The WPRE may be incorporated into the vector to increase the viral titer. It has been found that

[0062] The addition of cPPT and the modified 3'LTR deletion were performed on a recombinant plasmid containing the COL7A1 gene of the present invention. Improve the function of human viral vectors to improve gene expression levels, viral titers, and safety. I know it will increase.

[0063] The present invention further provides specific elements common to third generation lentiviral expression vectors. This includes the use of lentiviral delivery vectors that lack the human hinge region. Safety concerns about the use of lentiviral vectors have been raised by the use of third-generation lentiviral vectors. Concerns about generating replication-competent lentiviruses that may arise from recombination between the fragmented genomes (Tareen et al., 2013). The gag sequence and the The ribonucleotide and / or the rev responsive element (RRE) may be deleted from the viral vector. Thus, in one embodiment of the present invention, the gag sequence is absent from the vector. In an embodiment, both the gag sequence and the RRE are absent from the vector.

[0064] The starting material for generating the lentiviral vectors of the present invention is preferably a large C cPPT that can accommodate insertion of the OL7A1 gene (8.89 kbp) or its functional mutants and PRE. Preferably, the present invention The starting material for constructing lentiviral vectors for transduction is the lentiviral expression promoter. Plasmid vector, pSMPUW (Cell Biolabs, Inc., San Diego, CA) ego (CA) and pFUGW (Addgene, Cambridge, MA). will be done.

[0065] In one embodiment of the present invention, pSMPUW is used to construct a lentiviral vector for transduction. Selecting a lentiviral expression plasmid vector. Figure 6 shows the pSMPUW lentivirus. A schematic diagram of the genetic elements in the pSMPUW gene expression plasmid vector is shown. The characteristics of the avian viral expression vectors are depicted in Figure 7, which show the level of gene expression and safety. In order to enhance expression, the vectors have been modified from third-generation lentiviral expression vectors. The MPUW lentiviral expression vector contains a multiple cloning site (MCS) followed by the Woodchuck Hepatitis Virus Transfer The remaining gag (Δgag) and RRE elements encode the posttranscriptional regulatory element (WPRE). This element was removed from the pSMPUW vector construct. The construct contained a 133bp fragment commonly used in standard third generation SIN LV vectors. Rather than deletion, a larger 400 bp deletion was used in the 3'LTR U3 region. The wild-type COL7A1 gene was inserted into the pSMPUW vector.

[0066] In another embodiment of the present invention, the pFUGW vector is used for the construction of a transducing lentiviral vector. An antiviral expression plasmid is selected. Figure 8 shows the gene expression in this plasmid vector. The pFUGW vector features an RRE, two cPPTs, and a nucleotide sequence encoding the RRE. element, and the WPRE element, which are useful for lentivirus production and transfection. This may improve gene expression.

[0067] In another embodiment of the invention, the WPRE element is deleted from the lentiviral vector. are.

[0068] In one embodiment of the present invention, the transducing lentiviral vector particle of the present invention is INX N-2002 (vector transfer plasmid-IGE230) or INXN-20 04 (Vector Transfer Plasmid - IGE308) or and a substantially identical vector containing a functional variant of

[0069] Cells taken from DEB patients are transformed with a functional COL7A1 gene. In embodiments, cells are transduced with a lentiviral vector carrying the COL7A1 gene. In another aspect of the present invention, the lentiviral vector is a self-inactivating vector. The copy number of the integrated transgene can be assessed using any known method. For example, quantitative PCR, multiplex ligation-dependent probe amplification, and fluorescence in situ hybridization fluorescence in situ hybridization (FISH), copy number screening by microarray, and conventional nuclear The copy number of the transgene integrated into each cell may be determined by gene typing. The number of copies may be modulated by the viral dose administered to the cells during production. Integration tons per cell in RDEB harvested cells transduced with L7A1-containing vectors The transgene copy number is dose-dependent.

[0070] Cells taken from DEB patients and transformed with a functional COL7A1 gene contain functional C These cells will have the OL7A1 gene and will exhibit normal cell morphology. They may be grown or expanded in culture using culture techniques.

[0071] Harvested fibroblasts transduced with a COL7A1 transduction vector within the scope of the present invention In this case, morphological characteristics of normal fibroblasts are observed. For example, normal fibroblast morphology is It contains cells that present an elongated spindle or spindle appearance with thin extensions. The stellate nuclei also contain larger, flat stellate cells, which may have a cytoplasmic leading edge. Figure 9 shows the normal cell morphology of fibroblasts transduced with the COL7A1 transduction vector. An example is shown below.

[0072] Produced by harvested DEB patient cells transduced with the COL7A1 transduction vector The production of C7, which is involved in the assembly of tethered fibers, has also been observed. DEB patient cells transduced with the COL7A1 transduction vector It has been found that C7 trimers with the correct structure, size, and function can be formed. There are.

[0073] In one aspect of the present invention, fibroblasts transduced with a COL7A1 transduction vector The expressed C7 was shown to be able to form tethered fibers using immunoprecipitation with anti-C7 specific antibodies. For example, the anti-C7 specific antibody, fNC1, can be confirmed by SDS-PA. For selective capture and enrichment of C7 from culture supernatants for detection by GE / immunoblot For example, Figures 9 and 10 show the transduction of COL7A1 transduction vectors. We demonstrate that C7 produced by transfected fibroblasts was predominantly trimeric.

[0074] In another aspect of the invention, the function of C7 is measured by a laminin binding assay or a cell migration assay. C7 can be evaluated using known methods such as the use of fibronectin, laminin, and the like. Immobilized extracellular matrix proteins including Lam332, COL1, and COL4 It has been shown that it binds to extracellular matrix (ECM) components (Chen, et al., 2014). 02a). The interaction between C7 and Lam332 is mediated by the NH2-terminal NC1 domain of C7. This occurs due to the native conformation of both Lam332 and C7 NC1. (Rousselle, et al., 1997). Binding between C7 and Lam332 This is important for establishing the correct Lam332 structure at the dermal-epidermal junction. This organization is essential for interactions with extracellular ligands and surface receptors, as well as for signal transduction. (Waterman, et al., 2007). To detect the binding of purified Lam332 to C7, we investigated the binding of the C7 NC1 domain. An ELISA using an antibody that binds to C7 / Lam332 has been developed. Although this has been described in the literature (Chen, et al., 2002a), to the knowledge of the present inventors To date, it has never been used to test for C7 present in the supernatant of transduced cells. The results in Figure 12 show that the training and engineering C7 Lam33 expressed by GM-HDFs from (Engineering) orchids 2.

[0075] Additionally, cell migration assays can be used to assess functional C7 activity. RDEB fibroblasts and keratinocytes were found to be significantly more abundant than their normal counterparts when tested in vitro. It was found that the expression of C7 can restore normal motility. It was found that (Chen, et al., 2000; Chen, et al., 2002 b;Cogan,et al.,2014;Baldeschi et al.,200 3) Suitable assays include the use of fluorochromes to measure fibroblast and keratinocyte migration. Colloidal gold migration assay, or wound healing assay, for measuring keratinocyte migration Cells with functional C7 activity exhibited RDE in these assays. They may exhibit lower motility than B cells.

[0076] In one aspect, the present invention relates to a pharmaceutical preparation comprising autologous genetically modified cells derived from a DEB patient. These cells may be present in any amount suitable for delivery to the patient from whom the cells were originally harvested. For example, the above cells are 1.0 to 5.0 × 10 7 cells / mL, 1.0~4.0×10 7 cells / mL, 1.0~3.0×10 7 cells / mL, or 1.0-2.0 x 10 7 cells / m The cells may be present at a cell concentration of 1 L. The cells may be cultured in a medium such as Dulbecco's Modified Eagle's Medium (DMEM). The cells are in a suitable suspension to maintain viability. In particular, the viability of the cells in the formulation is , ≥ 60%, 70%, 75% or 80%. Phosphate-buffered saline solution that can be used to wash cells from thawed vials containing transformed cells. It may be present in a formulation such as saline.

[0077] The autologous genetically modified cells of the present invention have a nucleotide sequence of at least or about 0.05, 0.09, 0.41 or or about 0.74, or about 0.1 to about 6.0, about 0.1 to about 5.5, about 0.1 to about 5.0, about 0.1 to about 4.5, about 0.1 to about 4.0, about 0.1 to about 3.5, about 0.1 to about 3.0, about The range of the transduction vector is 0.1 to about 1, about 0.4 to about 1, or about 0.4 to about 0.75. Preferably, the transduction vector copy number is about 0.1 to about 5. Transduced human skin fibroblasts or keratinocytes are spinoculation at least or more per cell compared to a transduction composition that has not been subjected to transduction or supertransduction. is approximately 1, 2, 5, 10, 20, 25, 27, 28, 29, 30, 35, 40, 45, or The FCX-00 of the present invention has an integrated transgene copy number that is 50 times higher. C7 protein expression values ​​of 7 cells were ≥ 300, 350, 400, 450, 500, and 550 or 600 ng / day / E6 cells. Preferably, the C The C7 protein expression value is ≧500 ng / day / E6 cells. Transduced human dermal fibroblasts or keratinocytes that had not been subjected to spinoculation or supertransduction Increases of 10, 25, 100, 150, or 200 fold were also observed compared to latinocytes. I put it out.

[0078] Multiplicity of infection (MOI) refers to the number of vector particles per cell used for transduction. According to the invention, as shown in the examples below, even at a reduced MOI, the desired transduction vector can be expressed. For example, the present invention provides a method for achieving a maximum copy number of ≦15, ≦14, ≦13, ≦12, ≦1 This relates to an MOI of 1, or ≦10. Preferably, the MOI is about 1-10.

[0079] The formulations of the present invention can be used to treat a variety of disorders in patients with type VII deficiency. In particular, the formulations of the present invention are suitable for treating DEB, including DDEB or RDEB. In one embodiment of the present invention, it is also possible to treat subtypes of RDEB, and Subtypes include, but are not limited to, Hallopeau-Siemens Hallopeau-Siemens type, non-Hallopeau-Siemens type RD These include RDEB, atypical RDEB, pretibial RDEB, distal RDEB, and centripetal RDEB. can be.

[0080] The present invention further provides a method for treating, alleviating, preventing, and / or inhibiting various symptoms resulting from C7 deficiency. For example, DEB is known to cause scarring after blistering, which may be a contributing factor to the Shrinkage, swallowing problems (if the mouth and esophagus are affected), jointed fingers and toes, and mobility Therefore, in one aspect of the present invention, the mitten hand syndrome, also known as In another aspect, the present invention contemplates treating, alleviating, inhibiting, and / or preventing pseudosyndactyly. , e.g., neonatal milia, joint contractures, systemic soft tissue fibrosis, organ fibrosis, corneal lesions, scars It causes scar plaque, cicatricial alopecia, nail dystrophy, tongue adhesions, and increased incidence of dental caries. Treatment, alleviation, and / or treatment of deep fibrosis and / or scarring associated with RDEB and / or DDEB. In another aspect, the present invention relates to the reduction, inhibition, and / or prevention of oral diseases associated with RDEB. In another embodiment, the present invention relates to the treatment, alleviation, prevention, and / or inhibition of mucosal and gastrointestinal lesions. For the treatment, prevention, reduction, and / or inhibition of blisters associated with DEB patients.

[0081] The administration of autologous genetically modified cells can be determined by one skilled in the art based on the needs of the patient. Administration may be at any suitable time. For example, administration may be once, once daily, once monthly, every three months, etc. It may be conducted once or once or twice a year.

[0082] The formulations of the present invention containing autologous genetically modified cells can be administered by, but are not limited to, injection, topical administration, oral administration, or the like. or by implantation in a biocompatible matrix. Injection may be administered, for example, parenterally, intradermally, subcutaneously, intramuscularly, intravenously, intraosseously, intraarterially, intraocularly, or intraocularly. Intraperitoneal and intraperitoneal injections, or by direct injection into specific organs / tissues, e.g., the prostate or liver. For topical administration, the formulation may be administered directly to the affected area, such as at the site of a lesion. Debridement of the affected tissue may be performed before applying the agent directly to the site. The formulation may be encapsulated in a suitable delivery system, such as a suitable polymer capsule, or may be incorporated into a biocompatible matrix. Conjugates or implants, e.g., collagen matrices, hydrogels, skin grafts It may be embedded in a strip or mesh.

[0083] The autologous genetically modified cells of the present invention may be used alone or in combination with other therapeutic agents for DEB patients. Examples of therapeutic agents used to treat DEB include Zorb topical treatments such as Lisa, skin grafts, anti-inflammatory drugs, antibodies, and other promising gene or cell vectors. In another embodiment, the modified cells of the present invention are used in bone marrow transplantation therapy (or in skin or mucosal areas where other lineage cell therapies (such as mesenchymal stem cells) have not provided adequate treatment It may be administered to the C7 expression area of ​​autologous gene-modified cells in combination with other therapeutic agents. Preferably, performance is not impaired.

[0084] The present invention further relates to a genetically modified C7-deficient cell line obtained from a patient with C7 deficiency and transduced according to the present invention. Methods for increasing or enhancing integrated transgene copy number per cell in engineered cells In particular, certain steps during transduction result in the accumulation of transgene copies in these cells. In one aspect of the present invention, it has been found that the number of C7-deficient cells is substantially increased. contacting the cells with a lentiviral vector of the present invention and injecting the composition into spinoculation In this method, the lentiviral vector is transduced into the cells. The addition of spinoculation allows the spinoculation or at levels ≥2-fold compared to non-transduced, genetically modified human dermal fibroblasts However, other methods for increasing copy number have not been reported. To find the target cells, passaging them through first transduction by spinoculation and then optionally passaging by a second transduction by spinoculation. A second transduction step (or supertransduction) was unexpectedly discovered. This supertransduction using spinoculation is referred to as spinoculation or supertransduction. It was found to increase copy number by two-fold compared to the original traditional transduction method that does not contain did.

[0085] In another embodiment, INXN-2002 is converted to INXN-2004 by the lentiviral vector. Traditional transduction without spinoculation or supertransduction by changing the target Increases transgene copy number fourfold in genetically modified human dermal fibroblasts compared to It was discovered that...

[0086] By extensively assessing integrated transgene copy number, spinoculation Addition of spinoculation-mediated supertransduction, and INXN-2002 The cumulative change to INXN-2004 induces spinoculation or hypertransduction. Unexpectedly, we were able to increase copy number by >27-fold compared to traditional transduction methods that do not contain the original gene. (See Example 9.) (Compare TR12.1 and TR3) with TR12.1 (which did not involve supertransduction). Regarding the increased copy number of transduced cells from patients with 7 deficiency.

[0087] To further illustrate the present invention, the following non-limiting examples are set forth. [Example]

[0088] FCX-007 was synthesized using a lentiviral vector (INXN-2002) (see Example 1) either a lentiviral vector (INXN-2004) (as shown in Example 2) The present invention relates to a method for producing a human collagen type 7 protein by using either of the following methods: It is a suspension of viable autologous human dermal fibroblasts.

[0089] Example 1 A. Structure and structure of FCX-007 transduced with lentiviral vector INXN-2002 and characterization FCX-007 cells derived from lentiviral vector INXN-2002 cells were cultured in bovine fetal Iscove's Modified Dulbecco's Medium (IMDM) (50.0%) without fetal serum, Profr eeze-CDM™ (42.5%) and dimethyl sulfoxide (DMSO) (7.5%) The FCX-007 drug substance (DS) is suspended in a cryopreservation medium consisting of The characteristics are the primary structure of autologous human dermal fibroblasts (HDFs) and the transduction and genetic modification of HDF cells. and the structure of the lentiviral vector used to modify the gene.

[0090] B. Lentiviral Vector (INXN-2002) Used to transduce and transfer human collagen 7A1 gene into HDF cells. The NXN-2002 lentiviral vector (LV) encodes the human collagen 7A1 gene. INXN-2002LV is a recombinant lentiviral vector that encodes a heterologous VSV Human immunodeficiency virus type 1 (HIV-1) pseudotyped with the -G envelope protein INXN is a self-inactivating (SIN) lentiviral vector constructed as a base. The viral particle of the -2002 lentiviral vector is approximately 120 nm in diameter and is a single-stranded It consists of two copies of an RNA genome and many proteins. Specific molecular formula and molecular weight , or stereochemistry is not available.

[0091] The structural features of INXN-2002LV are similar to those of the primary structure of the RNA virus genome and the viral The primary structure of the INXN-2002 RNA genome is similar to that of the viral genome. The virus particle structure is determined by determining the complete nucleotide sequence of the VSV-G protein. The structure of HIV-1 particles is estimated from the structure of the HIV-1 virus with added protein pseudotyping. The structure of the child is outlined below.

[0092] C.INXN-2002 RNA virus genome structure INXN-2002LV is a heterologous VSV protein, not an HIV-1 envelope protein. Human immunodeficiency virus type 1 (HIV-1) pseudotyped with the -G envelope protein A self-inactivating (SIN) lentiviral vector constructed as a base. By introducing a p deletion into the U3 region of the LTR, a self-inactivating (SIN) vector was created. For the construction of INXN-2002LV, pSM PUW lentiviral expression plasmid vector (Cell Biolabs, Inc., The pSMPUW lentiviral expression platform was selected. Schematic diagram of the genetic elements of the smid vector.

[0093] Completely remove the coding elements between the 5'LTR and 3'LTR of the HIV-1 virus. The vector contains a multiple cloning site followed by a woodchuck hepatitis virus post-transcriptional site. Regulatory element (Woodchuck Hepatitis Virus Posttr ansscriptional regulatory element)(WPRE) To maximize the cloning capacity of the vector, a multicloning site is included. Digest the vector with BamHI at the 5' end of the 3' left terminal repeat and KpnI at the 5' end of the 3' left terminal repeat. These elements were removed by inserting the COL7A1 gene containing the CMV promoter. The gene expression cassette is cloned into the digested vector by single-strand annealing, Lentiviral vector plasmid encoding the COL7A1 gene as shown in FIG. A construct is generated.

[0094] Lentiviral vector plasmid constructs were transfected with three helper plasmids (pCMV- G, pCMV-Rev2, and pCgp) were co-transfected into HEK293T cells. The pCMV-G plasmid was used to generate INXN-2002 lentiviral vector particles. The smid provides the VSV-G pseudotyped surface protein, and pCMV-Rev2 provides efficient pCg provides HIV-1 Rev protein for RNA transport and packaging p is a structural protein for INXN-2002 lentiviral vector particle production and Figure 14 provides the viral enzyme protein. A schematic diagram of the proviral RNA genome structure of the TATA virus is shown.

[0095] D. INXN-2002 lentiviral vector particle structure The INXN-2002 lentiviral vector is an HIV-1-derived vector backbone. It is constructed using cellulose as a base material and has a virus particle structure similar to that of the HIV-1 virus. 9 shows an electron micrograph of an HIV-1 particle.

[0096] HIV-1 particles are spherical with a diameter of approximately 120 nm and an estimated molecular weight of 277 MDa. (Carlson 2008). The particles are composed of short protruding envelope proteins. The envelope protein is responsible for infection and delivery of the RNA virus to the target cell. interacts with receptors on target cells for delivery of the viral RNA genome. A conical core that houses the two chains can be observed inside the virus particle. The cone-shaped nucleo-core is formed by the viral capsid proteins.

[0097] In the case of INXN-2002, the glycoprotein of VSV-G (vesicular stomatitis virus (VSV -G)) as a substitute for HIV-1 envelope proteins, which enhances vector stability Improved viability, target cell targeting, and transduction efficiency (Cronin 2005) are achieved. .

[0098] During production, INXN-2002 viral particles were assembled and transfected VSV-G protein buds from the surface of HEK293T cells. The vector core and enzyme proteins are supplied by the pCgp helper plasmid. It is supplied by the plasmid and is responsible for efficient RNA genome transport and packaging into viral particles. The Rev protein required for replication is provided by the pCMV-Rev2 plasmid. Other HIV-1 accessory proteins, including u, Vif, Vpr, Nef, and Tat It is noted that all of the following are removed from the INXN-2002 vector.

[0099] After budding from the producer cell surface, proteases are packaged inside the virus particle. The enzyme cleaves the Gag precursor protein into its component proteins (MA, CA, and NC). , converting immature virions into mature, infectious INXN-2002 vector particles. 1A shows a schematic diagram detailing the mature INXN-2002 virus particle.

[0100] The two strands of the INXN-2002 RNA genome are bound by the capsid protein (CA). The nucleocapsid (NC) protein is packaged inside the conical core formed by Forms a stable complex with the RNA genome inside the capsid core. The virus forms a coat on the inner surface of the membrane. The VSV-G envelope protein The HIV-1 virus grows through the spiked cell membrane and forms a lipid envelope. Based on recent 3D analysis of the particle structure (Carlson 2008), Table 1 The components of the INXN-2002 lentiviral vector, as well as the components A brief description of the function of each element is provided below. TIFF2026004289000002.tif130170

[0101] E.INXN-2002 characterization Table 2 shows the characterization of INXN-2002 manufactured by City of Hope. The results of the characterization assays are listed below. 2 Lot number 0786-240-0002-1 is listed on the attached CoA, which , the lot intended for use in the manufacture of the FCX-007 clinical formulation. TIFF2026004289000003.tif105170

[0102] F. Characterization of INXN-2002 by In Vitro Immortalization Assay Insertional genotoxicity of INXN-2002 using in vitro immortalization (IVIM) assay The study was conducted at Cincinnati Children's Hospital ital Medical Center,Division of Experimente tal Hematology & Cancer Biology (CCHMC) carried out.

[0103] The principle of the IVIM test is that normal bone marrow (BM) lineage-negative (Lin-) cells are expressed in vitro. Although they cease proliferation after 3-4 weeks, certain In the presence of vector integration, some clones continued to grow for more than 5 weeks. The number of such immortalized clones represents the oncogenicity of the vector. The mortalized clones were expanded and analyzed for stem cell markers by FACS and vector expression by qPCR. Further analysis of copy number or site of integration by LAM-PCR. or Prdm16, insertions into common integration sites (cis) were generated in IVIM assays. This has been frequently observed in immortalized clones (Calmels et al., 2005; Modlich et al., 2008).

[0104] Bone marrow (BM) lineage-negative (Lin-) cells from C57BL / 6 mice were incubated with lineage-specific antibodies Lin-BM cells were then cultured and isolated from complete BM by magnetic sorting using the BM. The cells were stimulated in the full growth medium for 2 days. On the fourth day, RetroNectin (10 μg / cm 2 In a 48-well plate coated with INXN-2002 vector (Takara) Figure 16 shows the schematic layout of the IVIM assay.

[0105] To increase the transduction efficiency of INXN-2002 in Lin-BM cells, The -2002 vector was concentrated approximately 14-fold. For transduction, 80 μL of concentrated INXN- 2002 with 150 μL of transduction medium. 5 To each well containing cells The plate is centrifuged at 1000 g for 70 minutes at 32°C (spinoculation). The transduction efficiency was further increased by incubating the cells at 37°C in 5% CO2. Incubated overnight in Beta.

[0106] Achieving high INXN-2002 vector copy numbers in transduced Lin-BM cells To achieve this, INXN-2002 transduction was repeated on days 5, 6, and 7. Four successive transductions were performed.

[0107] Expand the transduced Lin-BM cells for 10 days, increasing the cell concentration every 2 days from 2 to 4 x 10. 5 The medium was adjusted to 100 cells / ml and fresh medium was supplied as needed. Cells were harvested and counted. A portion of the cells was then submitted for DNA isolation and qPCR. The remaining cells were then cultured between days 18 and 21 to determine the vector copy number (VCN). The cells were then returned to culture medium for plating in the potential IVIM assay.

[0108] Table 3 shows the results of pilot studies of INXN-2002-transduced Lin-BM cells harvested on day 10. The results of the test run are shown. TIFF2026004289000004.tif53170

[0109] Use of concentrated INXN-2002 vector and four consecutive spinoculation transductions Nevertheless, the vector copy number achieved in Lin-BM cells was significantly higher than that achieved in the IVIM study. The target copy number of 1 to 3 was significantly lower than the expected target copy number. .

[0110] Low vector copy numbers obtained in IVIM assays and RDEB fibroblast transduction The low vector copy number achieved in all of the input runs confirmed this locus of INXN-2002. The kit exhibits intercalated genotoxicity when used to transduce RDEB fibroblasts. This locus of INXN-2002 by IVIM assay is considered to be minimal. Further analysis of the bait was not pursued.

[0111] FCS-007 transduced with G.INXN-2002 The human dermal fibroblasts used to manufacture FCX-007 are derived from living skin biopsies. Biopsies were digested with Iberase® to release dermal fibroblasts. After transduction, cells are expanded using standard cell culture techniques. At the end of the culture expansion, cells were harvested, washed, and then diluted to 1.0–3.0 × 10 7 cells / mL The DS is tested for purity and is >98% by CD90 staining. Ensure that the culture contains 100% fibroblasts and that cell viability is ≥ 85%.

[0112] FCX-007 cells transduced with INXN-2002LV in DS formulation When cultured on a tissue culture surface, the cells exhibit a typical fibroblast morphology. They have a longitudinal spindle or spindle appearance with thin extensions or are They may also have the appearance of larger, flat stellate cells that may have tips. A mixture of cell morphologies can also be observed. Figure 17 shows the typical cell morphology and The structure is shown.

[0113] The cells expressed fibroblast-specific markers, CD90 (Thy-1), and the 35 kDa cell surface glycoprotein. Proteins and extracellular matrix proteins such as various collagens are also present. They express proteins specific to normal fibroblasts.

[0114] H. FCX-007 derived from lentiviral vector INXN-2002: drug substance release profile FCX-007 raw material was extracted at three stages during manufacturing: during manufacturing, bulk harvest, and after cryopreservation. The drug (DS) was characterized. Table 4 shows the release of FCX-007DS produced by PCT. A list of characterization assays and specifications for the finished drug substance is provided. The results are for Training Run 8 Arm A, Arm B, and Arm C ( The non-transduced control is described in the attached CoT. TIFF2026004289000005.tif97170

[0115] I. Further characterization of FCX-007DS derived from INXN-2002 A training manufacturing run of FCX-007 was performed to confirm functional C7 expression. Further characterization will be performed. The representative evaluation shown below is Training Run 8. (TR8), in which cells were either high (3.4 IU / cell) or low ( Transduced with either LV-COL7 (1.7 IU / cell) or mock-transduced These transduction arms are also referred to as arms A, B, and C, respectively.

[0116] J. INXN-2002 transduced FCX-007DS:C7 expression levels An enzyme-linked immunofluorescence assay (ELISA) was used to quantify C7 expression by FCX-007. For this assay, TR8 drug substance vials (high dose, low dose, and After thawing and culturing for 3 days, the conditioned cell culture supernatant was collected and transferred to C7. The results in Figure 18 show viral dose-dependent protein expression, which , 60–120 ng / mL of C7 in LV-COL7-transduced cells.

[0117] K. INXN-2002-transduced FCX-007:C7 trimer formation Tethering fibrils are formed from the assembly of C7 trimers. Immunoprecipitation of C7 followed by non-denaturing SDS PAGE / immunoblot analysis confirmed the proper expression and expression of C7 trimers by FCX-007. In Figure 19, the formation of NC1-specific antibodies was detected in the 1st and 2nd passages after freezing. C7 was immunoprecipitated from FCX-007 cell culture supernatant and analyzed by non-denaturing SDS-PAGE. The proteins were isolated by HPLC and visualized by Western blot. The C7 expressed was mainly a trimer (red arrow, approximately 870 kDa), and was not expressed in mock-transduced cells. LV-COL7 transduced cells expressing more C7 than LV-COL7 transduced cells (arm C, asterisk). It contained two amino acid sequences (arms A and B) in monomeric (290 kDa) and dimeric (580 kDa) forms. Assay controls included immunoprecipitation of purified C7 (Pur COL7) and antibody or included immunoprecipitations without test samples (IP controls).

[0118] FCX-007 transduced with L.INXN-2002: C7 binds to Lam332 C7 interacts with laminin 332 at the dermal / epidermal junction (DEJ). Lammin332 interactions are important for the functionality of anchoring fibers (Chen 2002, Rousselle 1997, Waterman 2007). The binding assay for detecting the activity was developed at Intrexon. The cells (high dose, low dose, and mock transduction) were thawed and cultured for 2 days. The conditioned cell culture supernatant was collected. The cells were collected and placed in wells coated with Lam332 or bovine serum albumin (BSA). The cells were incubated and bound C7 was detected using a C7 NC1 specific antibody and an HRP-conjugated secondary antibody. The assay readout was the optical density at 450 nm (OD450). The results show virus dose-dependent binding to Lam332 compared to the BSA control.

[0119] Migration evaluation of FCX-007 derived from M.INXN-2002 To assess the function of C7, a migration assay was developed. Chen 2000 reported that R Skin cells from DEB patients were found to be more abundant than normal skin cells in artificial wound margins created on tissue culture vessels. These results demonstrate that C7 application can restore migration speed. Normal human dermal fibroblasts (NHDF; Lonza) and FCX-007 bulk vials These cells were thawed and cultured in small strips on culture dishes to prevent cell adhesion. The cells were seeded into a culture dish with an insert. The strip was then removed and the open area The speed at which cells migrated into the septum was monitored by microscopy and analyzed using ImageJ software. The irregular shape representation plugin was used to quantify the results. Figures 21A-21B show the results of this assay. Both the migration rate (A) and migration images (B) are shown for the mock-transfected mice. fibroblasts from RDEB patients migrated faster into the open area than NHDFs, and LV-COL We show that transduction with 7 restores patient cells to a migration rate similar to that of NHDF. These results are consistent with those described by Chen 2000.

[0120] Example 2: FCX-007 transduced with INXN-2004 lentiviral vector FCX-007 induces the expression of human collagen 7 protein (C7) in INXN -2004 Autologous fiber genetically modified with lentiviral vector (LV-COL7) FCX-007 is a blast cell product used to manufacture DS / INXN-2004. The materials used for INXN-2002 production were IGE-230 LV-COL7 vector The procedure was the same as that described in Example 1 above, except that the plasmid used was IGE-3. 08 INXN-2004 vector was created using the LV-COL7 vector plasmid The same helper plasmids, pCMV-G, pCMV-Rev2, and pCgp, were used. The 293WCB cell line was co-transfected with the vector.

[0121] INXN-2004 lentiviral vector IGE308 LV-COL7 vector transfer plasmid The IGE308 plasmid was constructed using standard molecular cloning methods. The method involves cloning the human COL7A1 gene and then regenerating the cloned COL7A1 gene. By introducing it into the IFN-γ virus vector (SIN) backbone, pFUGW, E-308 involved generating the LV-COL7 vector transfer plasmid .

[0122] Cloning of the human COL7A1 gene IGE308 cloned into the LV-COL7 vector transfer plasmid The COL7A1 gene was transfected with INXN-2002 (IGE230 vector transfer plasmid) This is the same COL7A1 gene that was cloned into the COL7A1 gene (smid).

[0123] To amplify the COL7A1 gene from human genomic cDNA, Takara Protease Primers were designed and prepared using ImeScript reverse transcriptase. Four primer pairs were designed to amplify approximately 2 kb of the COL7A1 gene. Calculated. TIFF2026004289000006.tif78170

[0124] Human genomic cDNA with 5' overhangs for cloning into expression vectors 2 designed to amplify the 5'-end 3790 base pairs (bp) of COL7A1 from NA Amplification with two primer pairs (CollF2 / CollR4 and CollF4 / CollR1) The two 5' PCR products (2127 bp and 2993 bp) were amplified by overlapping extension. The fragments were joined by extension PCR to generate a final product of 3790 bp.

[0125] To clone the remaining gene, a fragment of DNA (341 bp, CollA1- 3, Table 6 were synthesized, which consisted of PCR-amplified 5'3790 bp gene fragment and cloning It contains the final 322 bp of the COL7A1 gene, including the overlap with the vector. TIFF2026004289000007.tif64170

[0126] NheI and ClaI clones were cloned using the In-Fusion HD Cloning Kit. The inducible expression vector (VVN-257673) was pre-digested with 2 CO The L7A1 gene fragment (3790 bp and CollA1-3) was assembled. Bacterial clones were identified by PCR using primers specific for the vector and COL7A1 sequences. The clones were screened to determine candidates for sequencing. Positive clones were confirmed by DNA sequencing. The resulting plasmid was designated VVN-431. It was named 1835.

[0127] VVN-4311835 and COL7A1 expression plasmids purchased from Origene (SC300011) was digested with BstZ17I and SapI. COL7A1 gene A 6276 bp fragment of SC300011 was cloned into VVN-4311835. The resulting plasmid, VVN-4311835 (the same plasmid number was used) was used to The COL7A1 sequence was sequenced to confirm that it was complete and mutation-free.

[0128] The VVN-4311835 plasmid was engineered to contain a site just outside the COL7A1 coding sequence. The excised COL7A1 gene was digested with two restriction enzymes, NheI and ClaI. The vector was cloned into a constitutive expression vector, VVN-257231, which also contains NheI and Digestion with ClaI yielded VVN-4319958. It expresses full-length human COL7A1 under the control of a constitutive CMV promoter.

[0129] FIG. 22 provides a schematic diagram of the cloning of the human COL7A1 gene.

[0130] Introduction of cloned COL7A1 gene into lentiviral vector (SIN) First, the pSMPUW lentiviral expression vector (VPK-211, Cell Biology) was used. olabs, Inc., San Diego, CA) for its enhanced security and large Based on the efficient cloning capability, the INXN-2002 gene encoding the COL7A1 gene was identified. was selected for the construction of antiviral vectors.

[0131] Figure 7 compares the pSMPUW vector with the standard third generation SIN LV vector. The pSMPUW vector encodes a multiple cloning site (MCS) into which Woodchuck Hepatitis Virus Posttranscription The remaining gag (Δgag) and RRE elements are then The pSMPUW vector construct was further constructed using standard rather than the commonly used 133 bp deletion in typical third generation SIN LV vectors. A larger 400 bp deletion within the LTR U3 region was used.

[0132] pSMPUW lentivirus for construction of INXN-2002 lentiviral vector Details of cloning the COL7A1 gene into the expression vector are described in Example 1 above. and are further explained below.

[0133] For the introduction of the entire coding sequence, a CMV promoter followed by a Kozak sequence was used. An insert containing a truncated version of the COL7A1 gene with the BclI and SapI restriction sites This insert was synthesized into two fragments, CColG and ColG2 (IDT). These two fragments were cloned into the In-Fusion HD Cloning Kit (Table 8). The plasmid backbone (6 3968-3R), and primers specific to the COL7A1 gene (ColS14). Colony PCR with different primers was used to find positive clones. The plasmid from this clone was purified and sequence verified to generate IGE228. 8 was digested with FspI and BamHI.

[0134] First attempt to clone the COL7A1 gene using the BclI and SapI sites The PCR product was used as a linker to bypass the BclI restriction site. An alternative strategy was devised. Primers EPF5 and CollR3 were used to clone the plasmid template. A PCR product was generated from plate VVN-4319958. This product was used to identify COL7A1 BamHI, which cuts 48 bp upstream from the 5' end of COL7A1, and The 5' linker was generated by digesting the fragment with FspI, which cuts at 1630 bp.

[0135] The wild-type COL7A1 gene was digested with FspI and SapI from VVN-4319958. This fragment was ligated to digested IGE228 and a 5' linker to generate COL7A1, VV A lentiviral construct for expression of N-4580853 (also called IGE230) was prepared. The COL7A1-specific primer ColS13 and the lentiviral backbone were used. Positive clones were found using 63968-3R, which is specific for the CMV promoter. The plasmid was sequenced from the 3′ end of COL7A1 to the 3′ end of COL7A1.

[0136] Table 7 below shows the results of introducing the COL7A1 gene into the pSMPUW lentiviral expression vector. The synthetic gene sequence and primers used to make the gene are shown. TIFF2026004289000008.tif225170

[0137] Figure 23 shows the COL7A1 gene cloned into the pSMPUW expression vector and expressed in vitro. Generate the XN-2002 lentiviral vector transfer plasmid, IGE230 A schematic diagram is provided.

[0138] As a result of ongoing development, deletion of the RRE element and the pSMPUW vector construct The use of a large 400 bp deletion within the 3'LTR U3 region of the large COL7A1 gene Combined with the requirement to package the 8.8 kbp fragment, the LV-COL7 vector The results were compared with those of the RDEB fibroblasts in terms of the C7 protein expression after transduction and subsequent infection (infectious titer). It was found that this has an adverse effect on

[0139] Third-generation GEN SIN vector, pFUGW (FUGW, plasmid #14883; Select the second-generation LV-COL7 vector, IN INXN-2004 was constructed to improve vector copy number. Figure 8 shows a schematic diagram of the genetic elements of the pFUGW lentiviral expression plasmid vector. Provide a diagram.

[0140] The pFUGW vector was developed for lentiviral vector generation and improved transgene expression. To achieve this, RRE, two cPPT elements, and WPRE (woodchuck hepatitis virus Contains woodchuck hepatitis virus post-transcriptional regulatory elements In order to accommodate the large COL7A1 gene (8.8 kbp) insertion, To maximize gene cloning capacity, the IGE308 lentiviral vector was used. A transfer plasmid was constructed.

[0141] Digestion of the vector with PacI and XhoI removed the WPRE element, hUBC promoter, and The motor and GFP reporter gene were removed. The 5' end of the CMV promoter, followed by CO An insert containing a small fragment of the 3' end of the L7A1 gene was generated. Gib (1CN) was synthesized at IDT as a G-Block fragment (sequence shown in Table 8). The fragment was assembled with the digested pFUGW vector using the son cloning derivative method. Primers specific to the plasmid backbone (FugwQCF and FugwQCR) ) and colony PCR were used to identify positive clones. The mide was purified and sequence confirmed to generate IGE301.

[0142] Next, IGE301 was digested with BaeI. IGE230 was digested with AseI and SapI. The 3' end of the CMV promoter, the 5'UTR, and most of the COL7A1 gene were isolated. This fragment was cloned into Ba using the Gibson cloning derivative method. The primers specific for COL7A1, ColS, were inserted into the eI-digested IGE301 vector. 13 and FugwQCR specific for the lentiviral backbone. The plasmid derived from the positive clone was purified and sequenced to identify IGE308. It was made.

[0143] Table 8 below shows the introduction of the COL7A1 gene into the pFUGW lentiviral expression vector. The present invention provides synthetic gene sequences and primers for use in the

[0144] Plasmids were purified using the Qiagen MaxiPrep Kit according to the manufacturer's protocol. It was maxi-prepped. TIFF2026004289000009.tif111170

[0145] Figure 24 shows the INXN-2004 lentiviral transfer plasmid (IGE30 8) provides a schematic diagram of the construction.

[0146] Production of IGE-308 plasmid Five steps: transformation, glycerol stock preparation, scale-up, capture, and dialysis The IGE308 plasmid was produced using a process consisting of filtration and formulation.

[0147] Transformation: The seed stock of IGE308 plasmid was transformed into competent E. coli (E. coli). The transformation promoter was provided to Aldevron for transformation into DH10B. The plates were stored at 34 °C, which is standard for lentiviral constructs.

[0148] Glycerol Stock Preparation - Picking Isolated Colonies from Transformation Plates Seed cultures were prepared by the following method. Each culture was miniprepped and analyzed by agarose gel. The best cultures, as determined, were used to make standard glycerol stocks. To compare media, the same 500 mL culture was grown in each of the following media types: Rapid Growth Medium and Maximum Yield Medium ield) medium (for a total of two 500 mL cultures). Cellulose stocks were used to inoculate cultures and a full panel of QC assays was performed on each prep. This allowed Aldevron to assess the stability of the plasmid in each media type. became possible to test.

[0149] Based on the initial QC results, the transformed E. coli (E. coli) was cultured in shaker flasks at 34°C. A fast-growing medium was selected for the growth of li) A total of 8 L of culture was prepared.

[0150] Capture - After lysing 8 L of E. coli culture, the DMAE anion was first The plasmid was purified using exchange chromatography, yielding a total of 833.8 mg. HIC (hydrophobic interaction chromatography) chromatography on OS resin An additional purification step was performed using PEG. This step yielded 663 mg of Purified DNA was obtained.

[0151] Concentration and formulation - Purified plasmid is adjusted to final buffer and concentration by continuous diafiltration The final buffer used was TE.

[0152] A total of 200 mg of IGE308 plasmid was shipped at a concentration of 1.4 mg / mL. Prior to further production of N-2004, the IGE308 plasmid was analyzed.

[0153] Analysis of IGE308 plasmid sequence The IGE308 plasmid used to produce INXN-2004 contains a 4-fold bidirectionally sequenced GLP using primer walking and oligonucleotide synthesis to obtain the desired results The IGE308 plasmid was fully sequenced using SeqWright under the conditions described above. The sequence is based on the IGE308 structure provided by SeqWright. The gene elements of the IGE308 plasmid showed 100% identity to the reference sequence. The results are shown in Figures 25A-25B. Table 9 shows the genetic elements with their corresponding functions. Provide a list of: TIFF2026004289000010.tif148170 TIFF2026004289000011.tif200170 TIFF2026004289000012.tif138170

[0154] The COL7A1 gene sequence of the IGE308 plasmid (without the stop codon TGA) was Bank Homo sapiens collagen, type VII, α1( COL7A1) sequence (NM_000094.3). The sequence alignment is shown in the Appendix. The three silent point mutations are listed in Table 10 below. No sequence gaps were found. Three silent point mutations were identified. It does not affect the C7 protein that is loaded. TIFF2026004289000013.tif51170

[0155] Helper plasmids, pCMV-G, pCMV-Rev2, and pCgp Three helper plasmids, pCM, used in the production of INXN-2004 LV vectors VG, pCMV-Rev2, and pCgp were provided by CoH.

[0156] 293T Working Cell Bank (WCB) 293TWCB used for the production of INXN-2004 LV vector was provided by CoH. was done.

[0157] Other starting materials for the production of INXN-2004 Other starting materials for INXN-2004 LV vector production were also provided by CoH. Ta.

[0158] Biopsy tissue Using standard aseptic procedures, three 3-4 mm punch skin biopsies (dermal and epidermal layers) were taken. RDEB biopsies are collected from non-blistering areas of the subject's body. Biopsies are collected by the treating physician and Introduced in cold, sterile phosphate-buffered saline (PBS) and stored in a refrigerated infectious transport container. Shipped next day. This container is not suitable for transporting potentially biohazardous tissue. It is designed to maintain a temperature of 2 to 8 degrees Celsius.

[0159] a. Reagents, solvents, and auxiliary materials i. Reagents and solvents The FCX-007 drug substance manufacturing process involves two animal-source reagents: fetal bovine serum (FBS) and A lipsin-EDTA solution was used. Complete growth medium.

[0160] Complete Growth Medium (CGM) for all cell culture expansion steps in FCX-007 production Using sterile transfer, mix 18 L of IMDM with 2 L of FBS in a 20 L bag. The CGM is prepared by storing the formulated CGM bag in a refrigerator at 5±3°C. Save.

[0161] Starting growth medium After biopsy digestion in T-75 cell culture flasks in the presence of antibodies, FCX-007 production was performed. Use Initiation Growth Medium (IGM) for the initial inoculation of fibroblasts. 58.2 mL of CGM in a syringe was added to 0.6 mL of 100x concentrated antibiotic solution in an ISO5 BSC. Prepare the initial growth medium by adding GA solution (sterile). The final concentration is: 0.4 mg / mL gentamicin, 0.295 μg / mL amphotericin B. Additionally, GSH solution (1.2 mL of a 50x solution) is also added to the 500 mL square medium bottle. Seal the bottle and mix by inverting 3-5 times. IGM should be mixed at least 10 times before direct use. Incubate in a 37°C incubator for 60 minutes.

[0162] The starting growth medium is prepared fresh immediately before use, so an expiration date does not apply and There is no quality control outgoing inspection.

[0163] b. Transduction medium Transduction medium (TM) was used for transduction of fibroblasts with INXN-2004. The transduction medium is prepared by dissolving 40 mL of I in a 250 mL centrifuge tube containing 10 mL of CGM. In ISO5 BSCs, MDM was added and the FBS concentration was diluted to 2% in the medium. Mix the 250 mL tube and place it inside a 37.0°C incubator. Place the cells in a cool, dry place and incubate until needed for INXN-2004 transduction.

[0164] The transduction medium is prepared fresh immediately before use, so an expiration date does not apply. There is no quality control outgoing inspection.

[0165] c.GSH solution GSH is added to the initiation growth medium (IGM) and CGM, which increases cell proliferation. Used for early T-75 and T-175 fibroblast cultures to prepare 1 L of IMD Prepare 50x GSH suspension in an ISO5001 BSC by dissolving 51.1 g of GSH in 100 mL of PBS. Prepare a stock solution. Filter the solution through a 0.22 μm filter. Discard the filtrate in a 50 mL centrifuge tube. Aliquot into tubes at 20 mL per tube and freeze at -80°C for later use. Saved.

[0166] d.RetroNectin(TM) solution To enhance the transduction efficiency of INXN-2004 in fibroblasts, RetroNe ctin (trademark) is used in the FCX-007 manufacturing process. 5 mL is stored in an ISO5 BSC. Using a syringe and 18g needle, 2.5mL of clinical grade WFI was injected. Sterile transfer into a Nectin™ vial and administer RetroNe at 1.0 mg / mL Reconstitute RetroNectin™ by gently swirling. Thoroughly dissolve the RetroNectin™. The sample is collected using the attached 5 mL syringe. Aseptically attach the syringe filter to the syringe and fill it with reconstituted RetroNectin (commercially available). Sterile filter the 100ml (standard) into a 250ml centrifuge tube. Reconstitute RetroNec in 122.5 mL of PBS in a 250 mL centrifuge tube. tin™ and dilute RetroNectin™ to 20 μg / mL. Mix thoroughly with a pipette. Add 0.3 mL of diluted RetroNectin™ solution to each of the T-25 flasks. 5μg / cm 2 The surface is coated with RetroNectin™. For round INXN-2004 transduction, prepare 5 x to 10 x T-25 flasks For INXN-2004 supertransduction, prepare 18 x T-25 flasks.

[0167] RetroNectin™ solution is prepared fresh immediately before use and should be used within the expiration date. No limits apply and no quality control outgoing inspections are performed.

[0168] Cryopreservation medium The cryopreservation medium is a 2x concentrated solution, which is collected and stored during the FCX-007 manufacturing process. Addition of Fibroblasts to the Washed Cell Seed Stock and FC for Storage in Liquid Nitrogen X-007 drug substance is formulated. Cryopreservation medium is 0.85 volumes ProFreeze (trademark) )-CDM (2x) mixed with 0.15 volumes DMSO to obtain 1 volume cryopreservation medium It is prepared by:

[0169] Cryopreservation media is prepared fresh immediately before use, so expiration dates do not apply and There is no quality control outgoing inspection.

[0170] Example 3: Training Runs 8, 9, and 10 and Enhancement of Biopsy Enzyme Digestion Training Run 8 and Biopsy Enzyme Digestion Enhancement In the aforementioned TR, the biopsy digestion method achieved effective digestion of biopsy tissue of 3-4 mm size. In TR8, in order to improve overall digestive efficiency, The application of additional shear stress during the digestion process was incorporated into this process. The digestion process was modified as follows: Change: Pulse vortex the centrifuge tubes at the maximum setting for 5 seconds every 15 ± 2 minutes during the digestion process. vortex the tubes, and return them to the orbital shaker after each vortex. At the end of the 0 minute incubation, pulse the centrifuge tube at the maximum setting for 10 seconds. Tex it.

[0171] Biopsies from RDEB donors were processed and digested using an enhanced digestion method. GSH The cells from the digest were inoculated into a T-75 flask using the supplemented medium. The cells showed robust growth and reached 90% confluence on day 14 for passaging. To evaluate the inoculation conditions, cells were harvested from T-75 flasks and placed in control, arm A, and 3 x T-175 flasks were seeded as arm B. Cells from all three arms were expanded and Transduced with 10L pilot LV-COL7 vector in 1-CS (control arm was transduced), further expanded in 2 × 10 CS, and then harvested for cryopreservation. . TIFF2026004289000014.tif201170

[0172] The total number of cells harvested from 10-CS will be used for further manufacturing of the FCX-007 drug for injection. The collected cells were analyzed for COL7A1 gene copy number (transduction The results were tested for cell viability, cell purity, and cell viability. Analysis of cells collected from all three arms is provided. TIFF2026004289000015.tif78170

[0173] COL7A1 gene copy number and C7 protein expression are multiplicity of infection (MOI) dose-dependent Increased efficacy was observed. Details of INXN-2002 transduction development and optimization are described below. The low INXN-2002 IU titer was due to the low transduction MOI and subsequent COL7A1 gene expression. Although the gene copy number was restricted, C7 protein expression was confirmed by in vitro and in vivo results. As will be seen, biologically relevant findings were observed in a preliminary clinical setting. These results suggest that RDEB fibroblasts were treated with INXN-2002 and excess functional C7 protein. It has been shown that transduction can be achieved with proteins.

[0174] Arm A cell products from TR8 are being developed as proof-of-concept trials. It was used for experimental testing as well as toxicology and biodistribution studies.

[0175] Training Run 9 Biopsies from RDEB donors were processed and digested using an enhanced digestion method. GSH Cells from the digest were inoculated into T-75 flasks using supplemented medium. The cells showed good growth and reached 90% confluence on day 19 for passage. To evaluate the OL7 transduction conditions, cells were harvested from T-75 flasks and cultured as control, anti- Three T-175 flasks were inoculated as arm A and arm B. The cells in all three arms were Cells were expanded and transfected with the LV-HA-COL7 vector (containing an HA tag in the construct) in 1-CS. After transduction (the control arm was mock transduced) and further expansion in 2 × 10 CS The collected cells were analyzed for COL7A1 gene copy number (transduction efficiency), C7 transcription factor (CTF), and IL-1 expression. Protein expression and cell purity were tested. Table 13 shows cell growth in TR9. . TIFF2026004289000016.tif222170

[0176] Table 14 provides the results of an analysis of the harvested cells from all three arms. TIFF2026004289000017.tif82170

[0177] Again, the COL7A1 gene copy number and C7 protein expression were MOI-dose dependent. These results also indicate that RDEB fibroblasts are favorably proliferated and L Transduced with V-HA-COL7 vector to express C7 protein This makes clear that:

[0178] Scaling up to 10 training runs and six 10-CS According to the proposed clinical protocol (see Module 5), 4 x10 8 FCX-007 drug cells are required, and repeated administration is possible. To meet the needs of FCX-007 drug, the cell yield achieved with TR8 and TR9 Based on this, it may be necessary to scale up the final cell expansion to six 10-CS.

[0179] Biopsies from RDEB donors were processed and digested using an enhanced digestion method. GSH The cells from the digest were inoculated into a T-75 flask using the supplemented medium. The cells showed robust growth and reached 90% confluence on day 19 for passage. The cells were seeded into 2 × T-175 flasks, one for INXN-2002 transduction. and one for the cell substrate control. Cells from one flask were expanded and cultured in 1-CS. were transduced with 1000 INXN-2002 and further expanded in 6 × 10 CS before harvesting. Table 15 shows cell proliferation in TR10. TIFF2026004289000018.tif159170

[0180] Compared to TR8 and TR9 cells, TR10 cells grow slower and require fewer passaging steps. The number of cells obtained in each case was also small, but this may be due to the variability between different RDEB donors. This shows the possibility.

[0181] The total number of cells harvested from 6 × 10 CS was 100% of the total number of cells obtained from a single dose of the injectable FCX-007 drug. It is considered suitable for the production of

[0182] Table 16 provides the results of the analysis of the harvested cells. TIFF2026004289000019.tif60170

[0183] Example 4 INXN-2002 transduction development and optimization Lentiviral transduction of skin fibroblasts was first developed and a model lentiviral GF 96 wk together with P (GeneCopoeia, LP-EGFP-LV105-0205) Normal human dermal fibroblasts (NHDF; Lonza, CC-251) cultured in well plates 1) was used for optimization.

[0184] Initial optimization using GeneCopoeia's LV transduction protocol as a starting point The cell density at the time of transduction, the volume of the transduction culture, and the amount of RetroNectin™ Use, superinfection (retransduction of cells with virus on two consecutive days), time of cell plating ( (time of transduction relative to the day before transduction), as well as serum content in the transduction medium. The conditions for achieving this were evaluated. The optimal transduction procedure was then carried out at a GMP manufacturing site. Details of the study are provided below.

[0185] A. Cell seeding conditions for LV transduction and RetroNectin™ coating The effect of

[0186] NHDFs in the exponential growth phase were harvested and inoculated at different inoculation densities on two different days. One set of non-tissue culture treated 96-well plates was seeded according to the manufacturer's recommendations. Following recommendations, the cells were coated with RetroNectin™ (RetroNectin Tin™ coating density is 20 μg / cm 2 One day after LV transduction For cells seeded before (day -1), remove spent medium and set aside for transduction on day 0. Add 100 μL of fresh medium containing the LV vector to the wells. On day 0, cells and LV vectors in 100 μL of fresh medium were added simultaneously to the wells. The MOI used was 2000 vp / cell for all conditions. After incubation, the medium was removed and 100 μL of fresh medium was added to the cells for further incubation. After 96 hours of transduction, the F of GFP signal for BD LSRII was For transduction efficiency analysis by ACS analysis, cells were harvested and analyzed using FlowJo software. (v.10). Table 17 lists the LV transduction efficiency under various conditions. . TIFF2026004289000020.tif66170

[0187] These results demonstrate that it is not necessary to pre-seed the cells the day before LV transduction. Cells and LV vectors can be added simultaneously at the time of transduction, which is This is convenient for MP manufacturing procedures. Coating the culture surface significantly increased LV transduction efficiency. The lower the number of cells, the lower the LV transduction efficiency. Therefore, during the LV transduction process, approximately 1 × 10 4 cell / cm 2 A relatively high cell seeding density of 1000 μg / ml is required.

[0188] B. Effect of MOI and supertransduction on LV transduction efficiency In this study, we investigated the effects of MOI and supertransduction on LV transduction of fibroblasts. Ta.

[0189] Prior to use for transduction, one set of non-tissue culture treated 96-well plates was retrofluorinated. ectin(trademark)(20μg / cm 2 ) at the time of transduction. 3000 cells containing LV vector in 1 μL were added to the wells. After overnight incubation, After incubation, remove the medium and resupply the cells with 100 μL of fresh medium for further incubation. For supertransduction, one day (approximately 24 hours) after the initial transduction, the medium was removed and The same amount of LV vector in 100 μL of fresh medium was added to the wells. Afterwards, the medium was removed and the cells were fed with 100 μL of fresh medium for further cultivation. 96 hours after transduction, transduction was characterized by FACS analysis of GFP signal on a BD LSRII. For transfection efficiency analysis, cells were harvested and analyzed using FlowJo software (v.10). Table 18 lists the LV transduction efficiency under various conditions. TIFF2026004289000021.tif63170

[0190] The higher the MOI, the higher the transduction efficiency, regardless of whether supertransduction was performed or not. Supertransduction resulted in a further increase in LV transduction efficiency, however the increase was not found to be significant. This was not done and was not achieved in a GMP manufacturing site.

[0191] C. LV-transduced culture volume and FBS concentration Lentiviral vector particles are first transduced into fibroblasts in culture to achieve transduction. Like other viral particles, LV particles in solution are required to contact cells. Due to the motility of the cells, productive transduction is generally a random event. The use of small or spinocal transduction enhances viral vector transduction of target cells. It has been shown that fibroblast L The effects of transduction dose / culture depth and medium FBS concentration on V transduction were evaluated.

[0192] One set of non-tissue culture treated 96-well plates was retrofluorinated prior to use for transduction. ectin(trademark)(20μg / cm 2 ) at the time of transduction. 3000 cells in 100 μL or 50 μL of medium containing 10% FBS LV vector The same transduction conditions were repeated in a medium containing 2% FBS. After incubation, remove the medium and add 100 μL of 10% FBS for further incubation. The MOI used was 2000 vp / mL for all conditions. 96 hours after transduction, FAC of GFP signal on BD LSRII Cells were harvested for transduction efficiency analysis by S analysis and analyzed using FlowJo software. Table 19 lists the LV transduction efficiency under different conditions. TIFF2026004289000022.tif43170

[0193] As expected, a decrease in the transduction medium volume (depth) resulted in a significant increase in LV transduction efficiency. Furthermore, the results showed that the use of 2% FBS transduction medium was beneficial for LV transduction. Based on these results, we recommend using the minimum amount of transduction medium, i.e., 1-CS The 2% FBS transduction medium was incorporated into the GMP manufacturing process, keeping the volume at approximately 60 mL. The GMP manufacturing process for CX-007 requires a surface area of ​​636 cm 2 1 layer CellSTACK( ) is used for LV transduction of fibroblasts. During the 3-hour transduction, cells Transduction of 60 mL (0.9 mm deep) was achieved without adverse effects (e.g., desiccation). It has been found feasible to use the input amount.

[0194] D. Evaluation of RetroNectin™ Coatings and Culture Surface Types RetroNectin™ coating on culture surfaces enhances the LV phenotype of fibroblasts. The RetroNectin™ manufacturer recommends a dose of 4 μg / cm 2 ~2 0 μg / cm 2 We recommend a coating amount of RetroNectin™ in the range of RetroNe without any negative effect on LV transduction of fibroblasts. To minimize the amount of citrin used in the coating, The amount of Nectin™ was evaluated.

[0195] Additionally, the RetroNectin™ manufacturer reserves the right to modify, reverse engineer, or otherwise alter the RetroNectin™ product. The use of non-tissue culture treated plastic surfaces for coating is also recommended. The culture flasks and CellSTACK® used for fibroblast culture are all Tissue culture-treated and non-tissue culture-treated 96-well plates Both were coated with different amounts of RetroNectin™ and fibroblast L V transduction was evaluated.

[0196] Both non-tissue culture treated and tissue culture treated 96-well plates were treated with different amounts of Re. troNectin (trademark) (20 μg / cm 2 , 10 μg / cm 2 , and 5 μg / cm 2 ) and then used for transfection. Add 3000 cells in 50 μL of transduction medium to each well along with the LV vector. After 3 hours of transduction, the medium was removed and the cells were transferred to 100 ml of ... All cells were fed with 100 μL of fresh medium. The MOI used was 100 μL for all conditions. 2000vp / cell. 96 hours after transduction, GFP signaling on the BD LSRII was observed. Cells were harvested for transduction efficiency analysis by FACS analysis of the signal and analyzed using FlowJo software. The results were analyzed using software. Table 20 lists the LV transduction efficiency under different conditions. TIFF2026004289000023.tif51170

[0197] All three doses of RetroNectin™ used in the coating were Comparable LV transduction efficiencies of fibroblasts were observed. 2 Re troNectin™ coating density for use in the FCX-007 manufacturing process Lower LV transduction efficiency was observed on tissue culture-treated surfaces, but this difference , tissue culture treatment in the INXN-2002 transduction step of the FCX-007 manufacturing process The results were not found to be significant enough to preclude the use of thoracic flasks.

[0198] Overview of the development and optimization of fibroblast LV transduction The results of the aforementioned studies using the LV-GFP model vector for fibroblast transduction Based on these results, we performed the following studies on INXN-2002 transduction of fibroblasts derived from RDEB donors. The LV transduction protocol below was chosen.

[0199] 5 μg / cm 2 Pre-coating the culture surface with RetroNectin™ For RetroNectin™ coating, tissue culture treated flasks were It can be used.

[0200] Approximately 1×10 4 cells / cm 2 At a cell seeding density of 100 μg / ml, cells and LV vectors were simultaneously transduced. Add occasionally.

[0201] High transduction efficiency (10 mL of INXN-2002LV-Col7 vector per transduction) To achieve this, use a high MOI that does not cause toxic effects to the cells.

[0202] After transduction for 3 hours at 37°C in transduction medium containing 2% FBS, The medium is then replaced or fed to the cells.

[0203] For high transduction efficiency, a low volume of transduction medium is required, i.e., in the case of a 96-well plate. 50 μL / well for single-layer CellSTACK®, or 60 mL for single-layer CellSTACK® Use.

[0204] No supertransduction is required.

[0205] F. INXN-2002 LV transduction of fibroblasts Based on the LV transduction protocol developed above, we used INXN-2002 to transduce 96 Normal human dermal fibroblasts (NHDF, Lonza, CC-2511) were cultured in well plates. Given the relatively low potency of INXN-2002, three doses of INXN-2002 were administered. 2002 was used for transduction: 12.5 μL (1:4 dilution), 3.1 μL (1: 0.8 μL (1:16 dilution), and 0.8 μL (1:64 dilution). LV-COL7 to transduce cell genomes To ensure stable integration of the vector, the transduced cells were passaged three times. Genomic DNA was extracted from the cells and amplified using primers specific to the LV-COL7 vector sequence. The transduction efficiency was quantified as gene copy number per cell. Table 21 lists the transduction efficiency, measured as gene copy number per cell. . TIFF2026004289000024.tif90170

[0206] At the highest dose of INXN-2002 vector, a 1 / 4 dilution (MOI=40), cells A significant toxic effect on the cells was observed: most of the cells did not recover from the transduction step. At the end of cell expansion, the lowest gene copy number per cell was obtained. The MOI was approximately 10 IU / cell, which resulted in a gene copy number of 0.86. was obtained.

[0207] INXN-2002 LV transduction in training runs LV transduction protocol (section

[0260] ) and INXN-2002 transduction Following the MOI results from the 96-well vector administration / in vitro assay, RDEB fibroblasts were cultured in vitro. XN-2002 transduction was performed on a large scale in TR8, TR9, and TR10. The single-layer CellSTACK® used for NXN-2002 transduction contained 5 μg / cm 2 At the time of transduction, the cells were pre-coated with RetroNectin™. One layer of CellSTACK® was filled with 60 mL of transduction medium containing 2% FBS. Cells and INXN-2002 were added simultaneously. After 3 hours in a 37°C incubator, The cells were fed with 130 mL of complete growth medium containing 10% FBS. That is, first one 10-layer CellSTACK®, then two or six The cells were then passaged onto a 10-layer CellSTACK® and harvested. The gene copy numbers of the 1000 genes were analyzed by qPCR. Table 22 shows the results from the training runs performed. The results of INXN-2002 transduction are described. TIFF2026004289000025.tif115170

[0208] FCX following the LV transduction protocol developed using small-scale 96-well plates Successful INXN-2002 transduction of RDEB fibroblasts was achieved at the -007 manufacturing scale. The gene copy number in transduced cells increased with increasing MOI of transduction. The low potency of NXN-2002 may result in the loss of gene copies of the transduced and harvested cell product. The number of copies of FCX-007 cells containing high gene copy numbers was relatively low. For the purpose of generating a cellular product, minimal toxic effects were exerted on the cells during the transduction process. From this, 10 mL of INXN-2002 was selected for GMP manufacturing transduction.

[0209] Engineering Run Preparation for GMP manufacturing of FCX-007 and manufacturing master batch record (Mas Upon completion of the Manufacturing Batch Record (MBR), the approved Manufacturing Batch Record Engineering runs were performed using biopsies from RDEB donors along with the GM P-grade INXN-2002 LV vector was used for transduction. Table 23 shows Cell proliferation figures from engineering runs are provided. TIFF2026004289000026.tif116170

[0210] Cell proliferation and yield from ER were comparable to those of TR8 and TR9, and better than TR10. The rate of cell proliferation was also significantly higher, indicating variability in cell proliferation among different RDEB donors. The harvested cells were packed and cryopreserved as shown in Table 24. TIFF2026004289000027.tif85170

[0211] The total number of cells harvested from 6 × 10 CS was calculated using two doses of the injectable FCX-007 drug. It is considered to be sufficient for construction.

[0212] The harvested cells were tested according to the proposed FCX-007 drug substance specifications. Table 25 shows: The analytical results of the collected cells are described. TIFF2026004289000028.tif122170

[0213] Example 5 - Description of manufacturing process development to improve LV-Col7 transduction efficiency FCX-007 cell formulations manufactured using the aforementioned process were used for cell transduction. The INXN-2002 vector allows for the expression of C7 through a low gene copy number and C7 expression level. As shown by the LV- To increase Col7 transduction efficiency, two approaches were implemented: 1) superior vector selection; Provide a titer and express a stable transgene (collagen V) in transduced fibroblasts. II) A novel LV-Col7 lentiviral vector (INXN-2004) with improved expression ) development, and 2) further optimization of the LV-Col7 transduction process for fibroblasts. Details of the development of the novel INXN-2004 vector are described in Section 3.2.S.2.3 Further optimization of the LV-Col7 transduction process and cell expansion studies are ongoing. The procedure is explained below.

[0214] Spin transduction (spinoculation) Spin transduction involves centrifugation of viral vectors against target cells under gravity. Spin transduction to enhance retroviral vector transduction efficiency in target cells Spinoculation was performed as described in J Virol Methods. s.1995 Aug;54(2-3):131-43.Centrifugal en hancement of retroviral mediated gene tr ansfer.Bahnson AB1,Dunigan JT,Baysal BE, Mohney T, Atchison RW, Nimgaonkar MT, Ball ED,Barranger JA.and Hum Gene Ther.1994 J an;5(1):19-28.Improved methods of retrov iral vector transduction and production for gene therapy.Kotani H1, Newton PB 3rd ,Zhang S,Chiang YL,Otto E,Weaver L,Blaes e RM, Anderson WF, McGarrity GJ.).

[0215] RDEB fibroblasts from the previous TR8 culture were cultured. Cells in the exponential growth phase were harvested. A previous INXN-2002 vector lot was used for transduction. For spin transduction, different cells were placed in 50 μL of transduction medium (IMDM + 2% FBS). MOI INXN-2002 includes 1 x 10 3 The cells were treated with RetroNectin ( standard)(5μg / cm 2 ) of a 96-well tissue culture-coated plate pre-coated with The plate containing the cells and INXN-2002 vector was then incubated for 130 min at RT. The plate was centrifuged at 0g for 1.5 hours at 4°C. After centrifugation, the plate was placed in an incubator at 37°C. The cells were then placed in 100 μL of complete medium for 3 hours to recover and grow. The medium (IMDM + 10% FBS + GSH) was added to each well. The plate was then placed in an incubator. The cells were then transferred back to the normal non-spin transduction medium and further cultured. Standard non-spin transduction as described above was used as a control. After transduction, cells were passaged three times, with 3–5 days of culture between each cell passage. The culture supernatant was collected to assess C7 protein levels and the gene coding per cell. Cells were harvested for cell count analysis. Table 26 shows the Col7 expression levels under different transduction conditions. Show the bell. TIFF2026004289000029.tif73170

[0216] Table 27 lists the vector copy numbers per cell for the harvested cells. TIFF2026004289000030.tif82170

[0217] The results were particularly striking at the lower MOI and INXN-2002 vector dilutions tested. , Col7 production levels and vector copy numbers per cell using the spin transduction method There is a slight increase in both.

[0218] Optimization of spin transduction parameters To further increase the transduction efficiency of INXN-2002, we have been adjusting the centrifugation time, speed, and Spin transduction parameters including the degree and temperature were evaluated. The same cell culture conditions were used as in the previous study. In the initial experiments, the centrifugation temperature was investigated. The 96-well plates containing the NXN-2002 vector were incubated at 1300g, 4°C or 25°C. The cells were then centrifuged for 1.5 hours at either room temperature or room temperature. To examine the effect of centrifugation speed on transfection efficiency, cells and INXN-2002 vectors were The 96-well plate containing the vector was heated at either 1300 g high speed or 300 g low speed. Separate experiments were performed in which the mixture was centrifuged at 4°C or 25°C (RT) for 1 or 2 hours. In both experiments, the cells were incubated at 37°C for post-centrifugation incubation. After 3 hours, 100 μL of complete medium was added to each well. The cells were subcultured three times. After culturing, Col7 expression in the culture supernatant was analyzed, and the number of vector copies per cell in the harvested cells was counted. - Collected for numerical analysis.

[0219] Table 28 shows spin phenotypes relative to Col7 expression levels and vector copy number per cell. The effect of transfection temperature is noted. Standard non-spin transfection was included as a control.

[0220] Again, this is indicated by higher C7 protein expression and vector copy numbers per cell. As shown, INXN- 2002 demonstrated enhanced transduction. The spin transduction temperature had no significant effect on overall transduction efficiency. showed no effect.

[0221] Table 29 below shows the effect of centrifugation speed and temperature on INXN-2002 transduction efficiency. The effect of time is described. TIFF2026004289000031.tif71170 TIFF2026004289000032.tif81170

[0222] The results showed that when spin transduction was performed at RT and 1300 g for 1 hour, C7 protein expression was significantly increased. Moderate INXN-2002 transduction efficiency as indicated by expression and vector copy number The improvement is shown in Fig. 1. The significance of the improvement was unknown, so the spin transduction parameters developed initially were used. The meter (centrifugation at 1300 g for 1.5 hours at 4°C) was selected for further testing. did.

[0223] Supertransduction to further enhance INXN-2002 transduction efficiency In the early development of the INXN-2002 transduction described above, supertransduction (second transduction) ) was evaluated. To further increase the transduction efficiency of INXN-2002 in fibroblasts, Next, we evaluated supertransduction again in combination with spintransduction. In the current study, supertransduction was performed on cells one passage after the initial spin transduction. Passaging allows cells to recover from the initial spin transduction and prepare them for a second spin transduction. It is expected that acceptance will be even higher.

[0224] Similarly, RDEB fibroblasts from TR8 cells in the previous exponential growth phase were harvested and analyzed using this The optimized spin transfection method described above was used for the study, at 1300 g and 4°C for 1.5 hours. Centrifugation was used for INXN-2002 transduction. 72 min after initial spin transduction After ~96 hours, the transduced cells were passaged once. 72-96 hours after the second passage, Cells were harvested and re-transduced using the same spin transduction conditions. After the cells were passaged three more times, Col7 expression was analyzed in the culture supernatant and in the harvested cells. For this study, INXN-200 was collected for vector copy number analysis. Table 30 shows the effect of ultracentrifugation on INXN-2002 transduction efficiency. The effect of transduction is described. TIFF2026004289000033.tif51170

[0225] Ultraspin transduction improves INXN-2002 transduction efficiency in RDEB fibroblasts C7 protein expression increased approximately 1.7-fold, resulting in a significant increase in vectors per cell. The number of target copies increased approximately threefold.

[0226] Data show that additional transduction resulted in a 40% increase in Col7 expression and 100 copies per cell. This increase led to the addition of superinfection to the production protocol.

[0227] Transduction with INXN-2004 vector Concurrent with the development of improved transduction methods, different lentiviral backbones (i.e. A novel LV-Col7 vector (called "INXN-2004") based on pFUGW was developed. They also developed a system that can be easily found.

[0228] INXN-2004 single spin transduction RDEB fibroblasts from TR8 cells harvested from previous exponential growth phase were used for this study. In the first study, a pilot lot of the INXN-2004 vector was used to A single round of spin transduction was performed as described above. Standard non-spin transduction was used as a control. After three passages of the cells, Col7 expression in the culture supernatant was analyzed, and The harvested cells were collected for vector copy number analysis per cell. Table 31 shows the INXN Col7 expression levels and vector counts per cell using -2004 vector transduction The target copy number should be stated. TIFF2026004289000034.tif67170

[0229] INXN in combination with spin transduction method compared to INXN-2002 vector A significant improvement in transduction efficiency was achieved using INXN-2004. The 2004 vector exhibited approximately 10-fold higher C7 protein expression and vector copies per cell. Even under non-spin transduction conditions, RDEB fibroblasts These results suggest that the novel INXN This paper supports the use of the pFUGW base for the construction of the -2004 vector.

[0230] Consistent with what was reported above, the relatively low C7 expression level and vector expression at an MOI of 17 Spin transduction was more efficient at higher MOI conditions than fibroblasts, as indicated by the target copy number. This is thought to exacerbate the toxic effects of the INXN-2004 vector on the cells. The use of a relatively low MOI has proven effective in the manufacture of the FCX-007. No impact.

[0231] INXN-2004 superspin transduction In the second study, we compared superspin transduction (as described above) with INXN-2004 transduction. The scale of transduction was increased from 96-well plates to T-25 flasks. The delivery parameters were 5 μg / cm2 Same RetroNectin™ coating density, and 1×10 4 cells / cm 2 The same cell density at transduction of The transduction volume in the T-25 flask was 4.2 mL. The T-25 flask containing the cells and virus was centrifuged at 1300 g for 1.5 hours at 4°C. The cells were returned to the incubator at 37°C and incubated for 3 hours. After incubation, 4.2 mL of complete medium was added to the flask for further culture. After three passages, Col7 expression in the culture supernatant was analyzed, and the vector count per cell in the harvested cells was Table 32 shows the INXN-2004 vector transduction efficiency. The effect of superspin transduction on the rate of gliomas seroconversion is described. For this study, INXN-2004 A pilot lot of vector was used. TIFF2026004289000035.tif41170

[0232] As previously observed with the INXN-2002 vector, low MOI transduction conditions Under these conditions, superspin transduction resulted in a relatively mild increase in Col7 protein expression levels. Regardless, there was a significant (approximately 5-fold) increase in vector copy number per cell in transduced cells. This resulted in an increase.

[0233] Transduction Development and Optimization Overview Both the previous INXN-2002 and the new INXN-2004 vectors were used. In this case, ongoing process development will involve transducing LV-Col7 into RDEB fibroblasts. Significant improvements in efficacy were achieved. These improvements led to the development of biologically potent FCX- Based on the results of research into manufacturing process development, it should be possible to manufacture 007 cell formulations. For the production of FCX-007 using the INXN-2004 vector for transduction, the following steps were performed: The transduction procedure was selected.

[0234] 5 μg / cm 2 Pre-coat the culture surface with RetroNectin. Use tissue culture treated flasks for troNectin™ coating .

[0235] At the time of transduction, cells and INXN-2004 vector are added simultaneously. The cell seeding density is , about 1×10 4 cells / cm 2 is.

[0236] The cells and INXN-2004 vector were transduced in a 4°C transduction vessel at 1300 g for 1. Centrifuge for 5 hours.

[0237] After transduction in transduction medium containing 2% FBS, cells were incubated at 37°C for 1.5–2.0 h. After incubation, the medium was replaced with complete medium containing 10% FBS or Supply to cells.

[0238] Low transduction medium volume (50 μL / well for 96-well plates, or 0.35 μL / well) mL / cm 2 ) to use.

[0239] Supertransduction is performed on cells one passage after the initial transduction.

[0240] Example 6 - RDEB Genetically Modified Human Dermal Fibroblasts Transduced with INXN-2002 Cell analysis For the following purposes, we have developed the RDEB gene derived from the INXN-2002 lentiviral vector: Genome-modified human dermal fibroblasts (GM-HDFs) were analyzed:

[0241] Establishing composite human skin grafts (porcine dermis) composed of RDEB keratinocytes by ID injection Evaluate the localization of COL7 expression in the oocytes (prepared above).

[0242] Composite RDEB-human skin grafted onto SCID mice treated with RDEB GM-HDFs To evaluate the carcinogenic / tumorigenic potential of (prepared on porcine dermis).

[0243] GM-HDF (INXN) was cultured by seeding fibroblasts on the reticular side of the composite explant culture. Early attempts to evaluate the effects of GM-HDF on devitalized porcine dermis were likely due to the lack of GM-HDF in the dermis. This may be due to inadequate administration or insufficient time for proper diffusion and attachment of rC7 on day 19. Using an alternative approach, we investigated the effects of the previously reported phenotype on six pre-test animals. The function of the existing grafts was evaluated by direct ID injection. The grafts proved more robust than expected, allowing direct injection into established RDEB skin grafts. This approach more closely mimics the disease and clinical route of administration.

[0244] This study reports on the initial evaluation of ID injection of GM-HDF into established RDEB skin grafts. This study provides additional data to achieve long-term goals for in vivo pharmacology. Plan the test.

[0245] A. Test Materials A.1 Test Substances Test materials were prepared and tested to API release specifications prior to shipping. The test materials were prepared one day before each scheduled treatment. GM-HDFs were thawed and washed. The GM-HDFs were then resuspended in DMEM and evaluated according to shipping regulations. The uncorrected RDEB HDF (mock-transformed) was administered within 48 hours. (transduced) were isolated and expanded from the same patient as GM-HDF.

[0246] Test materials and cells used in the preparation of composite skin grafts were shipped frozen. The cells used to prepare the strips can be thawed and cultured for testing.

[0247] Lot number TR8 (non-transduced control) was used as the RDEB-HDF negative control. Unit number TR8 Arm A (GM-HDF-LV-COL7) was used as the GM-HDF test material. and used it.

[0248] Test material analysis was performed at the manufacturing facility prior to shipping. Remaining test material was stored at -70°C for one year. Storage: Test substances are stored in Dulbecco's Modified Eagle's Medium (DMEM).

[0249] A.2. Other Chemicals and Materials A list of other ingredients is provided in Table 33. TIFF2026004289000036.tif61170

[0250] Group 1 (negative group) cells: non-transduced cells derived from a donor from training run 8 RDEB cells were thawed and passaged once before transduction. Two days after passage, cells were harvested. and liquid-based transfection of COL7 containing the plasmid VVN4317513 Using the reagent Transfex (ATCC), 1.2 x 10 6 cells in a 50ml conical Low serum growth in tubes (ATCC PCS-201-030 and PCS-201-04 1) Transfected in culture medium. Incubate with complexed Transfex for 10 minutes. After the incubation, DNA and Optimen (LifeTech), 1 × 10 6 Transformers Transfer the infected cells to a T150 flask and incubate at 2 x 10 5 cells were introduced into T25 cells. The cells were then placed in a 37°C 5% CO2 incubator for 16 hours. At this temperature, the medium was removed and the cells were completely Complete growth medium (IMDM (Sigma), 920 mg / mL of reduced L-glutathione (S igma) 10% heat-inactivated FBS (Atlantic Biologicals) and 1× Glutamax (LifeTech). The cells were returned to a 5% CO2 incubator at ℃. At the time of shipping, regardless of the orientation of the flask, Fill all cell flasks to their maximum capacity so that all parts of the flask are in contact with the medium. Filled.

[0251] B. Study Design B.1. Randomization Due to the high failure rate of RDEB human skin grafts, mice in these animal groups were randomly assigned to Do not randomize.

[0252] B.2. Justification of species and numbers for testing NOD.CB.17-PRKDC scid / scid mice demonstrate pre-concept clinical efficacy / virulence Evaluation using human xenograft models, the standard species used to demonstrate biological research This test is the rodent species of choice for the evaluation of test substances. The study was designed to minimize the number of test animals that could provide data.

[0253] B.3. Route of Administration Fibroblasts can be administered by intradermal implantation of normal human skin grafts. The cells may be administered by inoculation prior to explantation.

[0254] B.4. Dose Administration Lentiviral transduction (COL7A1 corrected) RDEB GM-HDF(INXN-2 Intradermal cell injections of 002 and negative controls (vehicle or RDEB-HDF) were performed at 30 The injection may be performed with a gauge needle. The injection is first pierced through the skin and then directed upwards back to the surface. and the needle may be directed as far as possible toward the surface.

[0255] Composite grafts were seeded with cells prior to implantation according to the protocol outlined in the in vitro studies. However, the composite culture is not elevated to the air-fluid interface prior to transfer.

[0256] B.4.1. Preparation of fibroblasts for injection (composite graft) Remove fibroblasts from liquid nitrogen storage one week before injection and store in a cool, dry place for 3 min until small ice crystals remain. The vials were thawed in water at 7°C, sterilized, and then transferred to the BSC. The supernatant was discarded and the cells were resuspended in DMSO and spun at 1000 RPM for 10 minutes. Wash in EM + 10% FBS 1x Antibody Medium and then spun again at 1000 RPM for 10 minutes. The pellet was resuspended in an appropriate volume and plated onto a 15 cm TC dish. The medium was changed and the grafts were passaged at 80% confluence until they were injectable. Cells were trypsinized, neutralized in DMEM + 10% FBS medium, and spun at 10,000 RPM. After vortexing for 10 minutes, the cell pellet was resuspended and an aliquot of cells was taken and triturated. Viability was determined by counting cells using Pan blue staining. 50 uL volume Medium 1.0×106 Draw up 100 cells into the syringe. Attach a 30g needle and Place the mice at 4°C until ready to inject. TIFF2026004289000037.tif114170TIFF2026004289000038.tif64170 TIFF2026004289000039.tif125170

[0257] C. Skin graft C.1 RDEB human skin graft C.1.1 Fibroblasts INXN-20, along with normal fibroblasts and unmodified RDEB fibroblasts as controls. 02 Genetic modifications produced by lentiviral gene transfer, such as lentiviral vectors Human dermal fibroblasts (RDEB GM-HDF) were initially used to inject the dermis. The cells are cultured on devitalized dermis to form a skin equivalent approximately 2 cm square. The regenerated skin composite is a layer of human keratinocytes cultured on top of fibroblast-injected human devitalized dermis. It consists of sites.

[0258] Isolation and culture of primary keratinocytes Wild-type keratinocytes were isolated from neonatal foreskin, and primary RDEB keratinocytes were isolated from patient The latter was isolated from patient skin samples in 15 mL centrifuge tubes transported on wet ice. A 6 mm pan immersed in 15 mL of keratinocyte growth medium 50 / 50V in a separation tube The sample should be taken as a biopsy. There should be no air bubbles in the sample being transported. 50V is a mixture of HKGS (0.2% bovine fetal pituitary extract [BPE], bovine insulin [5 mg / mL], hydrocortisone [0.18 mg / mL], bovine transferrin [5 m 50% medium 154 (I) supplemented with 1000 mg / mL of human epidermal growth factor (HGF) [0.2 mg / mL]. Invitrogen) and 50% keratinosa supplemented with recombinant human EGF1-53BPE Contains KSFM (Invitrogen) to reduce bacterial contamination. , and add the antibiotic AV100 (amikacin / vancomycin).

[0259] 25 U / mL dispase (Becton Dickinson) overnight at 4°C The dermis and epidermis are separated by this treatment. Next, the epidermis is peeled from the dermis and 5 mL of Place the epidermal "peelings" in a 15 mL centrifuge tube containing TrypLE 10x. Incubate for 15-30 min in a 37 °C water bath with gentle agitation every 5 min. TrypLE is prepared by adding an equal volume of DTI (defined trypsin inhibitor) or DMEM + 10% FBS. Neutralize. Cells are pelleted at 1200 rpm and transferred to a T75 Corning® Purecoat™ Collagen 1 12 mL 50 / 50 V flat on a 1-ml flask If PureCoat™ flasks are not available from the manufacturer, a substitute is available. Instead, 1 ml of collagen per 10 cm plastic dish was added for 15 min at 37°C. Collagen 1 coating can be prepared using Vitrogen 100 collagen (1 ml), HEPES (2 m), BSA (100 μL of 100 mg / ml) and 1x HBSS (100 ml) in a 0.2 µM filtered solution. Aspirate excess collagen coating. To ensure homogenous distribution of cells, Swirl the container and incubate at 37°C in 5% CO2 for at least overnight before any manipulation or observation. Place the cells in a centrifuge tube. Allow the cells to adapt to tissue culture conditions for 3 days. After adaptation, resuspend the cells every 2 days or as needed. When the cells reach approximately 70% confluence, the medium is changed as needed. Passage the chemokines.

[0260] In 50 / 50V medium, keratinocytes grow well for 5 passages. Use keratinocytes within the next passage.

[0261] Preparation of devitalized porcine dermis A sheet of split-thickness porcine skin is obtained. This is made from dermatome-harvested porcine skin. It can be prepared in the same manner as the method for devitalizing human dermis (as described above). in sterile PBS containing cyclosporin / streptomycin / amphotericin / gentamicin The porcine dermis was washed three times with PBS and then diluted with 1M NaCl and 2x antibiotics in PBS for 3 min. Incubate at 7°C for 72 hours. Separate and discard the epidermis and transfer to 1x PBS containing antibiotics. Wash three times with PBS to remove excess salt and gentamicin in the solution.

[0262] A piece of porcine dermis was cultured in 50 / 50V or KGM for 4 days to ensure a sterile environment. The dermis can be stored in the antibody solution at 4°C with weekly changes of PBS. The dermis should not be stored for more than a few months before use.

[0263] Fibroblast cells for organotypic culture inoculation Devitalized dermis is cut into 2 cm square pieces and placed basement membrane side down in a 6-well culture plate. Place the dermis in place and allow the dermis to dry slightly.

[0264] Fibroblasts were seeded into each well and centrifuged at 1200 rpm for 5 minutes in a humidified atmosphere of 5% CO2. The cells were then returned to 37°C for 3-4 days, with the medium replaced daily. During this period, the fibroblasts attached to the dermis They attach to the cells and begin to migrate into the tissue.

[0265] Inoculation of keratinocytes After the fibroblasts had migrated through the dermis, the dermis was carefully peeled off and placed on a circular dermal support (A Add a thin layer of Matrigel to seal and fix the dermis in place. KGM is added to the interstitial compartment of ADS. KGM consists of FBS (10%), Adenosine monophosphate (ADP), and ATP. Insulin (5%), thiazolinone (1.8 × 10 M), hydrocortisone (0.4 μg / mL), and insulin (5%) μg / mL), cholera toxin (1 × 10 to 10 M), EGF (10 ng / mL), trans Feline (5 μg / mL), and triiodo-L-thyronine (1.36 ng / mL) were added. A total of 100 μL of DMEM:Ham's F12 was added to the ADS. Keratinocytes are trypsinized, counted, and seeded in L of KGM. Return the assembly to the incubator. Do not stir the ADS for at least 24 hours. The keratinocytes in the lower chamber must be maintained at the air-liquid interface. The medium is changed daily. Organotypic culture continues for 1-2 weeks.

[0266] Transplantation of organotypic cultures The organotypic cultures are ready for transplantation after 7-10 days. Skin grafting is performed as previously described. Bandages and sutures are removed after 7-10 days. The dressing is then replaced with a new one for another week, after which the graft is allowed to air out and mature.

[0267] Graft harvesting and cutting The mice are humanely sacrificed and the grafts are carefully harvested. The grafts are bisected and then separated into two equal parts. The sections were further cut into 1 mm squares for immunoelectron microscopy (IE). The other half is frozen in OCT on dry ice with the center border marked. Cutting begins at this edge. Cut one half for immediate analysis and the other half for later analysis. Save for analysis. Cut ten 8 μm sections starting from the central edge and label sequentially. Discard the next 40 sections. Repeat until the end of the graft is reached. Allow to air dry and then Fix in ice-cold 50 / 50 acetone / methanol.

[0268] Implantation of established RDEB composite grafts After the graft is established, fibroblasts are injected intradermally directly into the skin. The injection is 50 μL. The quantity is as follows:

[0269] Mice previously implanted with composite grafts as described herein were injected with various Devitalized porcine dermis was inoculated by centrifugation with a suitable dose of RDEB GM-HDF. The dermis was then flipped over and 1 million cells were added to the dermis and grown in tissue culture for 1 week. RDEB keratinocytes were inoculated and grown in culture for over a week. After a 2-week tissue culture period, the grafts were placed in SCID mice and sutured into place. The bandages were applied for 13 days, at which point the bandages were removed and the grafts were then placed in a 50-well tube at 38-41 days after transplantation. μL of fibroblasts were injected.

[0270] D. Experimental Procedure D.1. COL7 localization Immunofluorescence microscopy was used to assess the expression of type VII collagen. For immunofluorescence (IF) analysis of skin cultures using the specific antibody NP185.

[0271] D.1.2. NP 185 Antibody Immunofluorescence Microscopy Method Immediately after CO2 euthanasia, composite grafts were harvested from the SCID mice. The tissue was frozen in a Leica at -21°C. Cut at 8 μM thickness on a CM1850 cryostat and freeze in ice-cold 50% methanol / 50 The slides were fixed in 1% acetone. The slides were rehydrated, washed three times with 1x PBS, and then incubated with the primary antibody (N Incubate in P185 10 μg / ml (mouse anti-human collagen VII) for 1 hour at room temperature. The slides were washed and then bated with Alexa 488-tagged goat anti-mouse IgG antibody. (1:400, 1 hour, room temperature, Life Technologies) and nuclear Hoesc Incubated in ht 33342 counterstain (Life technologies) The samples were washed three times with 1x PBS and then fluoromounted prior to imaging. Images were taken with a Zeiss Observer.Z1 fluorescence microscope at 20x magnification. Obtained in a mirror.

[0272] Grafts harvested on day 19 were added as positive and negative control arms. Injections were given on days 8, 39, or 41 and harvested on days 48, 49, or 51 (Table 37). TIFF2026004289000040.tif100170

[0273] E. Data Analysis IF images were generated and provided to the PI in a blinded manner. After the data were analyzed and interpreted by the PI, , and unblinding was performed.

[0274] F. Results Representative results from IF analysis of harvested explant tissues are shown in FIG.

[0275] Negative control (image "RDEB uncorrected" - arrowhead at DEJ for negative baseline comparison) C7 staining at the DEJ was visualized in all NP185 staining conditions except for (see arrowhead). Regarding the positive control, the DEJ of normal keratinocyte / fibroblast-inoculated grafts showed a strong DEJ. Staining was observed in small sections that may represent pre-seeded corrected fibroblasts. Small foci of positive staining were observed in Group 1 injected with blastocytes (Image 1b), but both The remaining uncorrected fibroblast-injected grafts in the mice were found to be less robust (Figure 1). (As shown in Figure 1a). Representative images of four mouse grafts injected with corrected fibroblasts. In Figures 4a-d, C7 staining at the DEJ was observed even just 10 days after transplantation. It was.

[0276] No tumors were observed in any of the grafts during the course of the study.

[0277] G. Conclusion RDEB GM-HDF TR8 injected intradermally at approximately the proposed clinical dose was shown to inhibit inactivated Bcl-1. In composite grafts of RDEB keratinocytes on the dermis, RDEB localized to the DEJ in vivo. Furthermore, the C7 obtained from the inoculation of GM-HDF into the composite graft before transplantation The results show that GM-HDF can sustain expression of C7 localized to the DEJ. Using the data, we predict the clinical dose and confirm the similar localization, durability, and persistence of the effects of GM-HDF. Sex and phenotypic correction can be characterized.

[0278] The results of the study support the use of subcutaneous injections of autologous GM-HDF as an effective treatment for RDEB in patients. show.

[0279] Example 7 - Non-GLP in vitro GM-HDF cell characterization and proof-of-concept evaluation The objectives of this study were to: (a) identify the expected regulatory pathways to be used in the treatment of patients with RDEB; (b) characterizing GM-HDF produced using a GMP manufacturing process; To evaluate the copy number and expression level of C7 in LV; and (c) by GM-HDF. The objective is to confirm the functionality of the expressed C7.

[0280] A. Test Materials A.1. Test Substances GM-HDF from training runs 8, 9, and 10, and engineering runs GM-HDFs obtained from (as previously described) were characterized. TIFF2026004289000041.tif164170

[0281] B. Primer and probe sequences used in qPCR analysis TIFF2026004289000042.tif123170

[0282] C. Experimental Procedure C.1 LV-COL7 copy number Nucleic acid isolation was performed using Qiagen's AllPrep kit according to the manufacturer's instructions. 3 x 10 5 gDNA isolated from GM-HDF cells was standardized to 12.5 ng / μl. 8 μl of normalized gDNA was added to 20 μL assay (10 μL Taqman (registered trademark) Gene Express, 1.8 μL of nuclease-free water, 0.06 μL of 100 μM forward primer, 0.06 μL of 100 μM reverse primer, 0. 0.4 μL of 100 μM Taqman® probe) was used. Serially dilute linearized C7 lentiviral shuttle vector (1e6 copies / reaction to 5 copies) 4 μL of human gDNA (1.5e4 cells / reaction) was also added to the 20 μL assay described above. The Taqman® assay used was PR13843. In addition, a 20 μL assay (10 μL Taqman® Gene Ex) Press, add 1.0 L of nuclease-free water, and 1 μL of 20x ACTB primer / probe. Commercially available human gDNA (Clontech) (1.5e4 cells / reaction) A separate standard curve was also performed using 8 μL of 10 ... Electrophoresis was performed on an ABI7900 using cycle parameters: 50°C for 2 minutes, 95°C for 1 minute 0 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min.

[0283] C.2 COL7A1 RT-qPCR A vial of GM-HDF cells was thawed and cultured on Qiagen's AllPrep™ kit. gDNA and RNA were isolated using a kit. RNA (800 μg) was used for Quan cDNA was generated using TA's qScript™ according to the manufacturer's instructions. Next, as previously described, Taqman® assay PR13653 was used. , cDNA, and gDNA were tested. Data were compared with the housekeeping gene ACTB (dCT ) and compared with the control data (ddCT). The obtained ddCT was calculated using the formula 2^- (ddCT) was used to convert to fold change.

[0284] C.3 C7 immunofluorescence For immunofluorescence analysis, 1.2 x 10 4 GM-HDFs were cultured in a 24-well plate. After overnight attachment and fixation on DL / Lamin-coated coverslips, the cells were then washed with methanol / acetone. The coverslips were permeabilized with a 50% / 50% mixture. After washing the coverslips three times with 1x PBS, Blocking was performed with 10% goat serum in PBS for 30 minutes at room temperature. After three additional washes with PBS, , coverslips with 1.25 μg / mL fNC1 antibody in 1% goat serum / PBS. Incubation was followed by three more washes with PBS and then 1% goat serum in PBS. 5 μg / mL Alexa Fluor® 555-conjugated goat anti-rabbit IgG They were incubated together at room temperature for 1 hour. NucBlue® Live Cel l Stain the coverslip with Stain Ready Probes Reagent. The images were then mounted on slides. A 290 ms exposure time was used, and the images were then taken on a Zeiss microscope at 20x magnification. Images were acquired using an Axio Observer microscope. After permeabilization, NucBlueLive (registered trademark) was used to visualize the nuclei (blue). Cell Stain™ or fNCl antibody to visualize C7 expression (red). and Alexa Fluor® 555-conjugated goat anti-rabbit IgG (5 μg / mL). Images were acquired at 20x magnification using an exposure time of 290 ms.

[0285] C.4 C7 protein ELISA Briefly, a standard curve of purified His-NC1 fragments (9.8–625 ng / mL) or C Collect supernatant containing 7 proteins (derived from GM-HDFs cultured for 3–5 days) and use Nunc M The samples were immobilized on an axiSorp® 96-well plate overnight at 4°C. The pull was tested in the same sample matrix (20% RDEB fibroblast-conditioned medium). After washing the coated wells with PBST, they were incubated with 3% BSA / PBS at 37°C. Blocked for 1 hour. Polyclonal anti-NC1 Ab (fNC1, 0.5 μg / mL) was used. , followed by the secondary antibody donkey anti-rabbit IgG HRP (Jackson ImmunoRes Detection was achieved by incubation with 0.08 μg / mL of T Bound antibodies were detected by colorimetric development with MB substrate solution. The reaction was quenched. Afterwards, SpectraMax® Plus 384 (Molecular Dev The absorbance was measured at 450 nm using a fluorimeter.

[0286] C.5 Immunoprecipitation of C7 trimers First, the magnetic protein G beads were washed with 1x PBS-T. In all subsequent steps, the beads were allowed to bind to the magnet for at least 2 minutes. The beads were coated with 5 μg of anti-C7fNC1 and then rotated (Glas-Col, room temperature) The mixture was incubated for 10 minutes at a speed of 14 rpm (30-14 rpm). The supernatant was collected and washed with Ab binding / washing buffer. The supernatant was collected from the M-HDFs. The C7-containing supernatant was added to the beads / C7 supernatant mixture and spun. The beads were incubated overnight at 4°C with stirring (setting 30-14 rpm). The next day, the beads were placed on a magnetic The target antigen was bound to the gel and washed three times with washing buffer. 10 μl of 4× loading dye (50 mM glycine, pH 2.8) The samples were denatured at 70°C for 10 minutes. A total of 30 μL of denatured DNA was eluted in 10 μL of denatured DNA. 12 μl of the sample (the remaining sample was stored at 4°C) was added to 12 wells of 3-8% Trisodium EDTA. Load (per well) onto a 1000 volt acetate gel and run for 4 hours at 150 volts. The gel was removed from the cassette and placed in transfer buffer containing 10% methanol. The gel was then wet transferred to a nitrocellulose membrane at 15 volts and 4°C overnight. The next day, the voltage was increased to 50 volts for 30 minutes on ice. After transfer was complete, TBS - 5% milk in T, stirred (Labnet ProBlot™ Rocker Block the blot for 2 hours at room temperature, then agitate for 2 hours at room temperature (25, 60 rpm). Incubate with commercial anti-C7LH7.2 (0.25 μg / mL) while stirring. The blot was washed three times (5 min each) with 1x TBS-T with agitation, and then incubated at room temperature for 1 hour. The plates were then probed with HRP-conjugated goat anti-mouse IgG (0.1 μg / mL) while stirring for 1 minute. Lumiglo Ultra™ Chemiluminescent Substrate and Fuj The blots were developed using the ifilm LAS 3000 Imaging System. I opened it.

[0287] C.6 Lam332 combination A 96-well MaxiSorp™ plate was filled with 100 mM carbonate buffer (pH 9 The plates were coated with 1 μg of Lam332 or BSA in .3) at 4°C overnight. The tissue was rinsed five times with PBS-T and then blocked with 1% BSA in PBS-T for 1 hour at room temperature. The covered wells were rinsed five times with PBS-T and then incubated overnight at 4°C with the supernatant from the transduced cells. After three more washes with PBS-T, the bound C7 was incubated with the fNC1 antibody. (0.5 μg / mL, PBS-T) for 3 hours at room temperature, followed by incubation with HRP -conjugated donkey anti-rabbit IgG (final concentration 0.8 μg / mL in PBS-T) at room temperature The C7-binding antibody was detected by a colorimetric reaction using TMB. SpectraMax® Plus 384 plate reader (Molecule The absorbance of the product was measured at 450 nm using a 300 nm chromatograph (300 nm chromatograph). All incubations were performed on an orbital shaker (GeneMate) at speed setting 2. ) was used.

[0288] C.7 Cell migration assay CytoSelect™ 24-well from Cell Biolabs, Inc. A wound healing assay kit was used for the cell migration assay. GM-HDFs (0.8 × 10 5 ) were inoculated into 24-well plates in the presence of wound inserts and allowed to grow for 48 hours. The wound inserts were collected and placed in a low concentration ( The cells were further cultured in 1% serum medium for 8 hours. After 4, 6, and 8 hours, the cells were then imaged using Celigo® Imaging Cytometer. Brightfield images of the wound were acquired using a Cyntellect System. Using imageJ (National Institutes of Health) The surface area of ​​the wound was measured at each time point. Data were expressed as the percentage of migration across the wound area (% migration). and record it.

[0289] C.7. Data Analysis qPCR was performed using an ABI 7900 instrument using SDS 2.3 software. The experimental data was displayed and exported to SDS 2.4 software. AgeJ software was used to quantify the percentage of cell migration in the assay described above. ageJ is a program of the National Institutes of Health (NIH). Developed by the National Institute of Health, it is an open-source imaging platform designed for scientific multidimensional imaging. It is an image processing program.

[0290] D. Results D.1. Characterization of C7 Expression D.1.1. LV-COL7 Copy Number and Transcript Levels Taqman® assays target various regions of the viral shuttle vector. Of the Taqman® assays tested, the C7 coding sequence was The targeted design exhibited unacceptable levels of background amplification. The aqman® assay, PR13843, was described by Greenberg et al. (2006) targeted the sequence found at the 5' end of the LV backbone. It was decided.

[0291] Obtain GM-HDF training run cells and wash with dPBS, then RLT buffer. Each training sample was resuspended in 100 ml of PBS using Qiagen's AllPrep kit. DNA (for copy number) and RNA (mRNA) were extracted from drug substance vials from each arm of Glan. The isolated DNA and RNA were then analyzed by the following methods: qPCR and RT-qPCR assays as described in sections C.1 and C.2 above. Table 40 shows the results for training runs 8, 9, and 10 (TR8, TR9, and TR10). 10) as well as copy numbers / cell from the engineering run (ER1). The number of copies of the transgene integrated into each cell is determined by the number of viruses administered to the cells during generation. It can be modulated by the dose, which is clearly less than 1 copy / cell. Similarly, the LV-COL7 transcript levels shown in Figure 40 also varied with virus dose and and copy number, expressing high levels (3-5 LOG) of lentiviral C7 sequences compared to control cells. Correlates with Arms A and B from the training run shown. TIFF2026004289000043.tif155170

[0292] D.1.2.C7 Protein Expression To examine C7 protein expression by GM-HDF cells using a C7-specific antibody Indirect immunofluorescence (IF) was used to identify the fibroblasts. Normal human dermal fibroblasts (NHDF) were used in this assay. The control arm of the training run was used as a negative control. In the representative image shown in Figure 28 below, a small percentage (<10%) of INX N-2002 and LV-HA-COL7-transduced RDEB fibroblasts exhibit C7 staining This is consistent with the number of copies of the integrated LV (Table 18). The signal from DF is brighter than that from NHDF, and is produced by GM-HDF. This shows a higher amount of C7.

[0293] To measure C7 secreted by GM-HDFs into the cell culture medium, Intrexon A direct ELISA was developed by [the authors]. This assay is described in section C.4 above. The purpose of this study was to use NC1 region-specific polyclonal antibodies to detect the NC1 region. ELISA assay results of GM-HDF from the training run showed that C7 expression was LV dose-dependent. The expression level was in the range of 45 to 90 ng / mL (cell supernatant). Figure 29).

[0294] D.2. Functional Evaluation of C7 Expressed by GM-HDF9.2.1. Formation of C7 Trimers The formation of C7 trimers is important for the assembly of anchoring fibers. To confirm that C7 expressed in the IL-16 receptor can form tethered fibers, selective capture assays were performed. Immunoprecipitation (IP) with anti-C7 specific antibody (fNC1) was used to capture the expressed C Whether 7 forms a trimer or not, then, for detection by SDS-PAGE / immunoblot. The concentration of C7 from the culture supernatant was evaluated.

[0295] The optimal conditions for immunoprecipitation were determined by testing the commercially available C7 antibody and fNC1C7 antibody. The coating of protein G beads with fNC1 was established by commercial antibodies. The coatings used in this assay yielded higher levels of C7 binding compared to the non-coated coatings. Controls were determined by nonspecific binding of C7 to beads, as well as C7 isolated from GM-HDF culture supernatants. Along with purified C7 spiked into the conditioned medium as a control for C7 specificity, An antibody control was run to exclude antibody-coated beads incubated with and without it. Does not include cuts.

[0296] The immunoprecipitation results for TR8, TR10, and ER1 showed that the GM-HDF-induced The C7 formed was found to be mainly a trimer (Fig. 10 and Fig. 11, respectively). Several immunoreactive lower molecular weight species were also observed, which were identified as dimers and The anti-C7 antibody (left panel, Figure 11B) and the C7 protein and antibodies specific for the HA tag incorporated into the protein (right panel, Figure 11B). IP of C7 from TR9 GM-HDFs was detected at low levels on immunoblots.

[0297] D.2.2. Lam332 Binding by C7 Expressed from GM-HDFs C7 binds fibronectin, laminin 332 (Lam332), COL1, and COL It has been shown to bind to immobilized extracellular matrix (ECM) components, including 4. The interaction between C7 and Lam332 is The NH2-terminal NC1 domain of C7NC1 mediates the native binding of Lam332 and C7NC1. The C7 and L Am332 binding is important for establishing the correct Lam332 structure at the dermal-epidermal junction This organization is mediated by interactions with extracellular ligands and cell surface receptors, as well as by cellular It is important for cellular signaling (Waterman, et al., 2007). To detect the binding of C7 to purified Lam332, ntrexon was used to identify the C7NC1 domain. An ELISA was developed using antibodies against C7 / Lam332. This has already been reported in the literature (Chen, et al., 2002a), but to our knowledge This has not been used to test for C7 present in the supernatant of transduced cells. The results in Figure 12 show the GM-H performance from the training and engineering runs. 1 shows dose-dependent binding of Lam332 by C7 expressed by DF.

[0298] D.2.3. Cell Migration Assay Previous studies have shown that RDEB fibroblasts and keratinocytes behave differently than their normal counterparts. We demonstrated that C7 expression can restore normal motility. (Chen, et al., 2000; Chen, et al., 200 2b;Cogan,et al.,2014;Baldeschi,et al.,20 03) Most of these tests measure the migration of fibroblasts and keratinocytes. gold salt colloid migration assay, or wound healing assay to measure keratinocyte migration Here, we used either CytoSelect™ 24 NH was measured using a wound healing assay kit (Cell Biolabs, Inc.). The motility of DFs and GM-HDFs was measured.

[0299] Immunofluorescence staining of C7 showed that only a small percentage (<10%) of transduced cells were highly transduced. We found that a small number of C7-producing cells did not express the C7 protein, and that cell migration The question arose as to whether the presence of C7 in the medium could have a global effect on To determine which treatments were sufficient to reverse the RDEB hyperactivity phenotype, three training Wound healing assays were performed on GM-HDFs from the NHDFs. was used as a positive control and a mock-transduced control (RDEB) arm as a negative control.

[0300] Control fibroblasts from TR8, TR10, and ER1 are reversed by expression of C7 However, control fibroblasts from TR9 showed significant It was difficult to examine the functionality of HA-tagged C7 in this assay because it did not exhibit a hyperkinetic phenotype. It's making it difficult.

[0301] conclusion The number of integrated transgene copies per cell in GM-HDFs varied with the viral dose. The transgene counts were gene-dependent and ranged from 0.009 to 0.03 transgene copies per cell.

[0302] Dose-dependent expression of GM-HDF cells was detected by qPCR, immunofluorescence staining, and ELISA. Targeted C7 expression was confirmed.

[0303] The structure of C7 expressed by GM-HDF cells was similar to that of TR8, TR10, and ER1. Immunoprecipitation / SDS-PAGE / immunoblot analysis of the ATP-binding protein revealed that it was primarily a trimer. TR9 showed minimal trimer formation.

[0304] C7 produced from TR8 GM-HDF cells was shown to be highly potent in in vitro binding assays. Binding to minin-332 and motility of control RDEB cells in an in vitro migration assay Correction of the overexpression phenotype demonstrated functionality. TR9 binds to laminin 332. Although the binding of LV-HA-COL7 to COL7 was demonstrated, the results of the migration assay were equivocal. The TR9 HA-GM-HDFs generated from transduction contained low levels of the trimer, Lam33 2 and a clear lack of hyperkinesis correction.

[0305] Confirmation of in vitro functionality of HA-C7 expressed by TR9 HA-GM-HDFs Therefore, a hybrid in vitro and in vivo pharmacological / toxicological evaluation of TR 8 was selected. This resulted in the RDEB complex prepared in normal or devitalized human skin. This precluded the ability to assess expression, localization, and persistence in the graft. Composite grafts prepared with human dermis were used in these studies. Immunostaining was used to distinguish rC7 from native C7 in this approach.

[0306] Example 8 - Formulation The FCX-007 formulation consists of a sterile suspension of each patient's own live autologous fibroblasts. These fibroblasts were cultured using INXN-2004LV-COL7 and human wild-type CO2 A phenol red-free Darbec-based steroid steroid, genetically modified to encode the L7A1 gene, It is formulated in Ducco's Modified Eagle's Medium (DMEM).

[0307] Formulation Development FCX-007 formulation and cell dose development will be conducted by azficel-T (LAVIV) Based on the manufacturing experience of Fibrocell, a viable autologous fibroblast preparation for injection, Fibroblasts were cultured at 0.5 to 3.0 × 10 cells per well without any viscosity issues. 7 Cell concentration range in cells / mL The data were formulated in DMEM at 4°C (azficel-T BLA#125348). , the cell concentration is 5.8 × 10 7 At cells / mL, viscosity becomes an issue for formulation injections As a result, the ratio of 1.0 to 3.0 × 10 7 Cell concentration in cells / mL DM EM was selected as the formulation for FCX-007DP.

[0308] surplus amount Up to 10 vials of FCX-007DP will be shipped to clinical sites as single doses for therapeutic injection. When administering, gently invert the DP vial three times and then inject with a 21-gauge needle. Aseptically draw 1 mL from each vial into a sterile 1 mL syringe. Then, use a 21-gauge syringe. The needle was replaced with a 30-gauge needle for intradermal administration. Repeat this method.

[0309] FCX-007DP vials are filled with 1.2 mL of fibroblast suspension and 1.0 mL The recoverable volume of the solution is introduced into a 2.0 mL vial. An excess volume of 0.2 mL is used for clinical The intent is to ensure that 1.0 mL can be obtained from the vial at the time of administration at the bedside. This is what is done.

[0310] a. Physicochemical and biological properties i.Physicochemical properties FCX-007DP is a 2 mL cryovial filled with 1.2 mL of phenobarbital. It is provided as a genetically modified autologous fibroblast suspension in DMEM without red. The cells are demonstrated to be viable cultures by assay. Table 41 shows the FCX The physicochemical properties of -007DP are shown below. TIFF2026004289000044.tif42170

[0311] biological properties FCX-007 is genetically engineered to express human collagen 7 protein (C7). It is a sterile autologous fibroblast preparation. INXN-2004 vector copies per cell The number and expression of C7 protein are unique biological properties of FCX-007DP. Wash thawed FCX-007DS cells with PBS and DMEM buffer for X-007DP FCX-007DS is a viable cell suspension preparation produced from FCX-007DS by Before shipping for manufacturing of 007DP, the I per cell of FCX-007DS was NXN-2004 vector copy number and C7 protein expression are analyzed. Table 42 shows the F The biological properties of CX-007DP are shown. TIFF2026004289000045.tif68170

[0312] Manufacturing recipe To manufacture the FCX-007 formulation, FCX-007 drug substance vials are frozen in liquid nitrogen. Remove from the existing gas phase, thaw and pool in an ISO5BSC. The cells were washed with water (PBS) and subsequently cultured in Dulbecco's modified PBS without phenol red. After washing with DMEM, 1.0–3.0 × 10 7 to the target concentration of cells / mL The formulated cells are then suspended in 1.2 mL of phenol red-free DMEM. Fill into 2 mL cryovials at 100 mL / vial. A formulation is defined as a batch. The proposed treatment batch size is 1.2 mL of cell suspension. Up to ten 2 mL vials containing

[0313] Table 43 provides a list of all ingredients used in the drug manufacturing process. TIFF2026004289000046.tif112170

[0314] Manufacturing process and process control explanation b. Manufacturing process flow chart The FCX-007 formulation vial was removed from liquid nitrogen storage and mixed in a ThermoMixer ( Once thawed using ISO5 BS, the FCX-007 formulation begins production. C, 250 ml of phosphate-buffered saline (PBS) containing >5x bulk volume of wash buffer. Pool the thawed FCX-007DS cells in a mL centrifuge tube for resuspension. The cells were centrifuged at 1000 rpm for 10 minutes at 5±3°C and washed with PBS washing buffer. Then wash with >5x bulk volume of Dulbecco's Modified Eagle Medium (DMEM). This is followed by resuspension and centrifugation at 1000 rpm for 10 minutes at 5±3°C. Remove the DMEM wash buffer. The washed cells were collected at 2.0 x 10 7 Cells / mL target Resuspend in phenol red-free DMEM to a concentration of 1000 mM in ISO 5001. Add the cell suspension to a volume of 1.2 mL per vial into sterile 2.0 mL cryovials. Manually fill the FCX-007DP vials. Store at 2-8°C until release by QA at the Cube™ shipper for delivery to the clinical site. Save.

[0315] Manufacturing process description FCX-007 formulation is prepared as previously described. It is a synonym for 7.

[0316] Step 1: Removal of FCX-007 drug substance vials from liquid nitrogen storage and lot verification FCX-007 drug substance stored in a liquid nitrogen freezer was Subsequently, FCX-007 will be requested from the FCX-007 manufacturing batch. 07 The bulk drug vial is removed and moved to the clean room.

[0317] Step 2: Thawing the cryovial To remove from the cleanroom for processing applications, SOP-0099 GMP Prod uction Line Clearance / GTP Line Cleaning Therefore, the line clearance of the clean room is carried out by QA. Before processing in the BSC and non-vial EM sampling, (In Process Environmental and Personnel Indoor, employee, and BSC Environmental Monitoring (EM) in accordance with Start.

[0318] Disinfect the FCX-007 DS cryovial and allow the cell suspension to cool, leaving only a few ice crystals. Thaw in a ThermoMixer® equilibrated to 37°C until almost completely thawed. Thaw vials in the BSC.

[0319] Step 3: PBS wash In a BSC, pipette approximately 150 mL of PBS (>5 volumes of FCX-007DS Transfer the contents of the thawed cryovial (cell suspension) into a 250 mL centrifuge tube. Transfer the mixture into a 250 mL centrifuge tube. Immediately, using a pipette, add 3.0 mL of fresh PBS to each cryovial as a rinse. Transfer the rinse solution to a 250 mL centrifuge tube. Gently swirl the tube. Transfer the tube to a centrifuge and centrifuge at 1000 RPM. Centrifuge the cells for 10 minutes at 5±3°C. After centrifugation, transfer the PBS wash supernatant to a tube. Collected from.

[0320] i. Step 4: DMEM Wash Using a pipette, add approximately 200 mL of DMEM (>5 volumes) without phenol red. Add FCX-007DS cell suspension) to the tube and resuspend the cell pellet. Gently swirl the tube to mix the cell suspension. and centrifuge the cells at 1000 RPM for 10 minutes at 5±3°C. Using a pipette, transfer 0.9 mL of supernatant from the centrifuge bottle to the "Lot, QC-Sterile" Transfer to each of two separate 2 mL cryovials (1.8 mL total) labeled Keep the sterile, labeled cryovials in the BSC. Wash the remaining DMEM. Aspirate the supernatant from the centrifuge tube.

[0321] Step 5: Cell pellet resuspension in DMEM The volume of DMEM used to resuspend the cell pellet is calculated as follows:

[0322] Divide the volume of thawed FCX-007DS cell suspension by 1.5

[0323] After resuspension, 1.0-3.0 x 10 7 To achieve a cell concentration within the specified range of cells / mL To do this, a 1 / 1.5 volume factor is used to compensate for possible cell loss during the washing steps.

[0324] Using a pipette, transfer the calculated amount of DMEM to the cell pellet in the centrifuge tube. Resuspend. Using a pipette, add 0.1 mL of cell suspension to the "lot, QC-count" For cell counting, transfer to a 2 mL cryovial labeled "Cell Viability" Submit the cryovial to QC. Using a pipette, measure the total volume of the remaining cell suspension. The cell suspension is then placed in a 2-8 °C refrigerator for later end use.

[0325] Step 5a: Re-dilution (if necessary) QC cell concentration is 3.0 × 10 7 If the number of cells / mL is higher, use fresh DMEM. 2.0×10 7 Further dilutions are performed to reach the target cell concentration of cells / mL. Submit the sample again to QC for cell counting to confirm the final cell concentration.

[0326] Step 6: Final filling Remove the cells from the refrigerator and transfer them to the BSC for final filling. Manually fill the thoroughly mixed cell suspension into 2 mL cryovials in the following order:

[0327] Fill 0.1 mL of cell suspension into the first sterile sample vial (reserved from step 4). (added to the sample to be maintained).

[0328] Fill formulation vials at 1.2 mL per vial.

[0329] Fill a second sterile sample vial with 0.1 mL of cell suspension (reserved from step 4). (added to the sample to be maintained).

[0330] Fill QC vials at ≦1.8 mL per vial.

[0331] Sterile and QC vials are submitted for testing.

[0332] Step 7: Preserving DP vials for shipping and dispatch DP vials are kept at 5±3° C. for shipping and dispatch to clinical sites.

[0333] Explanation of in-process control environmental management All cell culture operations are performed using aseptic techniques in a certified IS within an ISO 7 environmental background. Perform this in an O5 biological safety cabinet (BSC). Vial containers are purchased pre-sterilized from Corning. A perator is someone who is competent in aseptic filling operations.

[0334] In-process control and testing The process controls and tests performed for release of FCX-007 formulations are described below.

[0335] Step 1 The correct FCX-007 drug substance lot was verified by QA before use in FCX-007DP manufacturing. Check that.

[0336] Step 2 ThermoMixer used to thaw FCX-007 drug substance vials The temperature of the vial is controlled at 37°C. A small piece of ice is still left in the vial to prevent overheating. Thaw the contents of each vial to a sufficient extent.

[0337] Step 3 To ensure adequate washing of cells, use PBS wash volumes of ≥ 5 volumes of FCX-007DS Control the cell pool. Centrifugation conditions should be adjusted to 100% to ensure adequate cell pelleting. The temperature is controlled at 1000 rpm, 10 minutes, and 5±3°C.

[0338] Step 4 Similar to the PBS wash step, adjust the DMEM wash volume to ensure adequate washing of the cells. The centrifugation conditions were adjusted to a volume of ≥ 5 volumes of FCX-007DS cell pool. To ensure the mixture is thawed, the temperature is controlled at 1000 rpm for 10 minutes at 5±3°C. Two 900 μL aliquots of the DMEM wash supernatant were taken for use in sterility testing of the formulation. Take.

[0339] Steps 5 and 5(a) (if necessary) The resuspended cell concentration is 1.0-3.0 x 10 7 cells / mL, and viability ≥ 70% do.

[0340] Step 6 The filling volume of the FCX-007DP vials is controlled at 1.2 mL per vial. Cell counts and viability determinations, as well as Gram staining, are performed on filled QC vials. The sterile vials are submitted for sterility testing. The results of this sterility testing are not available until after the drug is shipped. Release of DP is based on negative Gram staining. The use of rapid microbial tests is covered in the Guidance for FDA Reviewers. and Sponsors “Content and Review of Che Mistry,Manufacturing,and Control(CMC)Inf ormation for Human Somatic Cell Therapy Investigation New Drug Applications (INDs) )” was instructed in April 2008.

[0341] ii. Materials Management During the FCX-007 formulation manufacturing process, single-use, disposable products (e.g., (e.g., pipettes, centrifuge tubes, and cryovials) to Excludes cleanup and carryover. Raw materials selected for use in the manufacturing process must comply with USP V All disposable items are gamma irradiated and are acceptable for chemical treatment as per JIS I. PBS and DMEM wash buffers are purchased pre-sterilized from the supplier. The process steps are purchased sterile from the manufacturer. It has parameters.

[0342] Example 9 - In vitro characterization of optimized GM-HDF This example demonstrates an enhanced LV transduction procedure with the LV-COL7 construct INXN-2004. We describe the characterization of GM-HDFs generated using three training runs (TRs). 11, TR12.1, and TR13) were performed to prepare cells for GMP-scale analysis. TR11 was transduced with the LV-COL7 vector INXN-2002 and expressed in a second Centrifugation enhancement (spinoculation) with and without a transduction step (supertransduction) (option) was used to evaluate the LV transduction procedure. Using a modified transduction procedure, TR12.1 and TR13 were transduced into the LV-COL7 vector INX. Transduced with N-2004.

[0343] The copy number of integrated LV-COL7, C7 protein expression, and expression by GM-HDF Each training run was evaluated using assays to assess outcomes such as expressed C7 functionality. GM-HDFs from the mice were characterized.

[0344] LV integration and C7 expression levels were first increased by adding a supertransduction step, and then L The use of V-COL7 INXN-2004 further increased the C7 expressed from GM-HDFs from the germination run assembled into a trimeric form and GM-HDF bound to minin-332, which is important for the formation of anchoring fibers. Expression of C7 was able to reverse the hypermotility observed in RDEB fibroblasts. GM-HD from TR12.1 and TR13 for use in in vivo GLP toxicology studies I chose F.

[0345] The objectives of this study were to: (a) 1) demonstrate an enhanced transduction procedure incorporated into a large-scale GMP manufacturing process; and 2) integrated LV copies by employing the INXN-2004 LV-COL7 construct. (b) demonstrating improved number and expression of C7 expressed by GM-HDFs The purpose was to confirm the functionality of the C7.

[0346] 1. Test Materials 1.1. Test Substances GM-HDFs from training runs (TR) 11, 12.1, and 13 were characterized. . TIFF2026004289000047.tif109170

[0347] 1.2. Primer and probe sequences TIFF2026004289000048.tif68170

[0348] 2. Experimental Procedure 2.1.LV-COL7 copy number Nucleic acid isolation was performed using Qiagen's AllPrep kit according to the manufacturer's instructions. 6.7×10 5 Isolate gDNA from GM-HDF cells and add 8 μl of gDNA. 20 μL assay (10 μL Taqman® Gene Express, 1 0.8 μL nuclease-free water, 0.06 μL 100 μM forward primer, 0 0.06 μL of 100 μM reverse primer, 0.04 μL of 100 μM Taqman ® probe). Serially diluted linearized LV-COL7 Standard curve of avian virus shuttle vector (1e6 copies / reaction to 5 copies / reaction), plus 4 μL of human gDNA (1.5e4 cells / reaction) was also assayed in the 20 μL assay described above. The Taqman® assay used was PR13843. 0 μL assay (10 μL Taqman® Gene Express, 1. 0 µL of nuclease-free water, 1 µL of 20x ACTB primer / probe set) Commercially available human gDNA (Clontech) (1.5e4 cells / reaction ~ 2e2 cells / reaction) A separate standard curve of 8 μL of the 1000 μL reaction was also performed. All samples were cycled using the following parameters: The gel was run in triplicate on an ABI 7900HT using a 50°C column: 2 min at 50°C, 1 min at 95°C. 0 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min.

[0349] 2.2.C7 immunofluorescence For immunofluorescence analysis, 1.2 x 10 4 GM-HDFs were cultured in a 24-well plate. After overnight attachment and fixation on DL / lamin-coated coverslips, the cells were then resuspended in methanol / acetone. The coverslips were then permeabilized with a 50% / 50% mixture of 1x PBS. The sections were then blocked with 10% goat serum in PBS for 30 minutes at room temperature and washed three times with PBS. Afterwards, the coverslips were stained with 1.25 μg / mL of polyclonal fN in 1% goat serum / PBS. Incubation with C1 antibody (α-fNC1) followed by three additional washes with PBS Then, 5 μg / mL Alexa Fluor® in 1% goat serum / PBS was added. Incubated with 555-conjugated goat anti-rabbit IgG for 1 hour at room temperature. ue(registered trademark) Live Cell Stain Ready Probes Rea The coverslips were stained with gent and then mounted on slides. Exposure time: 290 ms. Images were acquired using a Zeiss Axio Observer microscope at 20x magnification. After fixing and permeabilizing NHDFs or GM-HDFs, nuclei (blue) were visualized. Then, cells were stained with NucBlue® Live Cell Stain or C7 expression (red To visualize the chromatin (color), fNCl antibody and Alexa Fluor® 555 were used. The cells were stained with IgG-conjugated goat anti-rabbit IgG (5 μg / mL). An exposure time of 290 ms was used. , Images were acquired at magnifications of 20x and 5x.

[0350] 2.3.C7 Flow Cytometry GM-HDFs (approximately 500,000 cells) were placed in each well of a 96-well V-bottom plate. The supernatant was collected in a tube and concentrated by centrifugation (1500 rpm, 5 min, room temperature). The cells were then resuspended in 200 μL of CytoFix and plated in a 96-well plate. The cells were transferred to a V-bottom plate and incubated at 4°C for 30 minutes. After fixation, the cells were centrifuged (15 Remove the fixative by centrifugation (00 rpm, 5 min, 4°C) and add 200 μL of ice-cold 100% methanol. The cells were permeabilized with PBS for 30 minutes on ice. Then, the cells were kept at -20°C before the staining procedure. For staining, the permeabilized cells were placed in 200 μL of incubation buffer (100 ml). Wash twice with 0.5 g BSA in 1 L of PBS, centrifuging (300 rpm) between washes. The buffer was removed by centrifugation (5 min at room temperature). The washed cells were diluted 1:400. Resuspend the chamber in 100 μl of incubation buffer containing α-fNC1. The cells were then washed twice as described above and then diluted with 1:20 100 ml containing AlexaFluor 555-rabbit IgG secondary antibody diluted to 0.05% Resuspend in 1 μL of incubation buffer and incubate for 30 min at room temperature. The cells were then washed twice as described above and resuspended in 100 μL of PBS. The following parameters are included: FSC voltage = 484, SSC voltage = 209, PE = 500 Flow cytometry was performed using SRII and BD FACSDiva Software (V6.2). C7-expressing cells were counted by cytometry.

[0351] 2.4.C7 Protein ELISA Briefly, a standard curve of purified His-NC1 fragments (9.8–625 ng / mL) or C Collect supernatant containing 7 proteins (derived from GM-HDFs cultured for 3–5 days) and use Nunc M The samples were immobilized on an axiSorp® 96-well plate overnight at 4°C. The pull was tested in the same sample matrix (10% RDEB fibroblast-conditioned medium). After washing the coated wells with PBST, they were incubated with 3% BSA / PBS at 37°C. The cells were blocked with α-fNC1 Ab (0.5 μg / mL) for 1 hour, followed by secondary antibody. Donkey anti-rabbit IgG HRP (Jackson ImmunoResearch, 0.0 Detection was achieved by incubation with 8 μg / mL of TMB substrate solution. Bound antibody was detected by colorimetric development. After quenching the reaction, Spectra Max® Plus 384 (Molecular Devices) The absorbance was measured at 50 nm.

[0352] 2.5.Immunoprecipitation of C7 trimers First, the magnetic protein G beads were washed with 1x PBS-T. In all subsequent steps, the beads were allowed to bind to the magnet for at least 2 minutes. The beads were coated with 5 μg of α-fNC1 and then rotated (Glas-Col, at room temperature). The beads were then incubated for 10 minutes at 30-14 rpm. The supernatant was collected and washed with Ab binding / washing buffer. - The supernatant was collected from the HDFs. The C7-containing supernatant was added to the beads / C7 supernatant mixture and spun ( The beads were incubated overnight at 4°C with a rotating speed of 30-14 rpm. The next day, the beads were placed on a magnet. The target antigen was bound to the antibody and washed three times with washing buffer. - (50 mM glycine, pH 2.8, and 10 μl of 4× loading dye The sample was denatured at 70°C for 10 minutes. A total of 30 μl was used. 12 μl of the sample (the remaining sample was stored at 4°C) was added to 12 wells of 3-8% Tris Load onto acetate gel (per well) and run at 150 volts for 4 hours The gel was removed from the cassette and placed in transfer buffer containing 10% methanol. The gel was then soaked for 20 minutes. Wet transfer of the gel to a nitrocellulose membrane was performed overnight at 15 volts and 4°C. The next day, the voltage was increased to 50 volts for 30 minutes on ice. 5% milk in T was used, and agitation (Labnet ProBlot™ Rocker 2 Block the blot for 2 hours at room temperature, then agitate for 2 hours at room temperature (5, set at 60 rpm). While the cells were incubated with commercial anti-C7LH7.2 (0.25 μg / mL), The blot was washed three times (5 min each) with 1x TBS-T with agitation, and then incubated at room temperature for 1 hour. The mixture was probed with HRP-conjugated goat anti-mouse IgG (0.1 μg / mL) while stirring. umiglo Ultra™ Chemiluminescent Substrate and Fuji Blots were developed using the film LAS 3000 Imaging System. made him do so.

[0353] 2.6.Lam332 join A 96-well MaxiSorp™ plate was filled with 100 mM carbonate buffer (pH 9 The plates were coated with 1 μg of Lam332 or BSA in .3) at 4°C overnight. The tissue was rinsed five times with PBS-T and then blocked with 1% BSA in PBS-T for 1 hour at room temperature. The coated wells were rinsed five times with PBS-T and then incubated with the supernatant from the transduced cells overnight at 4°C. After three more washes with PBS-T, the bound C7 was transferred to α-fNC After incubation with antibody 1 (0.5 μg / mL, PBS-T) for 3 hours at room temperature, together with HRP-conjugated donkey anti-rabbit IgG (final concentration 0.8 μg / mL in PBS-T). The mixture was incubated at room temperature for 2 hours. C7-binding antibodies were detected by a colorimetric reaction using TMB. and analyzed using a SpectraMax® Plus 384 plate reader (Mole by reading the absorbance of the product at 450 nm on a chromatographic device (Central Devices). All incubations were performed on an orbital shaker (GeneM) at speed setting 2. This was carried out using a methacrylate (methacrylate ester).

[0354] 2.7.Cell Migration Assay CytoSelect™ 24-well from Cell Biolabs, Inc. A wound healing assay kit was used for the cell migration assay. GM-HDFs (0.8 × 10 5 ) were inoculated into 24-well plates in the presence of wound inserts and allowed to grow for 48 hours. The wound inserts were collected and placed in a low concentration ( The cells were further cultured in 1% serum medium for 8 hours. After 4, 6, and 8 hours, the cells were then imaged using Celigo® Imaging Cytometer. Brightfield images of the wound were acquired using a Cyntellect System. Using imageJ (National Institutes of Health) The surface area of ​​the wound was measured at each time point. Data were expressed as the percentage of migration across the wound area (% migration). and record it.

[0355] 3. Data Analysis qPCR was performed using an ABI 7900 instrument using SDS2.3 software. The experimental data was displayed and exported to SDS 2.4 software. Flow cytometry data were analyzed using wJo V10 software. Using geJ software, calculate the cell migration rate (%) of the assay described in section 2.7. ImageJ was licensed by the National Institutes of Health. Developed by the American Academy of Health (AHA) and designed for scientific multidimensional imaging It is an open source image processing program.

[0356] 4.Results 4.1. Characterization of C7 Expression The overall goal of this evaluation is to compare transformation methods and LV vectors to determine copy number and The specific objectives are to: 1. To assess the effect of supertransduction on integrated copy number 2. To evaluate the effect of spinoculation on integrated copy number 3. Supertransduction and Spinoculation of INXN-2002 and INXN- Comparing the 2004 copy number values It was.

[0357] For these studies, two GMP lots of the LV-COL7 construct were used: XN-2002 and the construct INXN-2004 described in this example.

[0358] 4.1.1.LV-COL7 copy number Obtain GM-HDF training run cells and culture as described in section 2.1 above. Table 46 shows the results of the training run using the new transformation method. For initial optimization, the transduction step ("spinoculation") During the incubation, an additional centrifugation step ("spinoculation") was performed. In the past, INXN-2002 was used to obtain a composition in the range of 0.013 to 0.025. The copy number of the spinocule was obtained for transduction with INXN-2002. The addition of the additional processing step alone resulted in a significant improvement in the embedded control performance compared to the TR8 results in previous testing. The number of cells increased by ≧2-fold to 0.05 per cell (TR11 arm B, Table 46).

[0359] To further increase the integrated copy number, a second transduction (" The addition of supertransduction, which also uses spinoculation, Therefore, the integrated copy number of INXN-2002 increased approximately twofold (TR11 arm A, Table 46).

[0360] A small research trial (not shown) demonstrated the efficacy of another third-generation autoinactivating LV backbone. The second generation INXN-2004 LV-COL7 construct using the INXN-2004 LV-COL7 gene was Higher integrated copies while using a lower multiplicity of infection (MOI) than that used in 02 It has been shown that it has the potential to achieve a higher number of targets and result in a higher expression level. used on a GMP-scale by an optimized transduction procedure (spinoculation including supertransduction) When combined, INXN-2004 showed approximately 4-8 times higher integration than the INXN-2002 construct. The LV copy number was further improved to 0.41 to 0.74 (TR12.1 and T R13, Table 46) average copy number was achieved.

[0361] Of note, TR12.1 and arm B of TR13 were passaged throughout the entire manufacturing process. The copy number was not tested because the exact copy number was not available (Table 44). The evaluation of LV episomal copies, which may artificially inflate the measured copy number, is also important. Cells should be passaged several times after transduction to ensure that no residual markers or residual markers remain. TIFF2026004289000049.tif125170

[0362] 4.1.2.C7 Protein Expression To examine C7 protein expression by GM-HDFs using a C7-specific antibody, Indirect immunofluorescence (IF) was used. TR11 arms A and B, and TR12.1 and Only arm A of TR13 and TR14 were analyzed by IF. Normal human dermal fibroblasts (NHDF) was used as a positive control, and the control arm of TR11 was used as a negative control. Representative images at 20x and 5x are shown in Figure 31 below. IF images of cells from TR8 arm A with integrated LV-COL7 copy number are also shown in the original step. Consistent with the integrated LV copy numbers shown in Table 46 above, Only TR11 GM-HDFs were visualized with the C7 antibody, but still not TR8. Furthermore, the number of C7-positive cells in TR12.1 and TR13GM-HDFs was significantly higher than that in the previous study. The relatively high copy number measured from the serogroup correlated with the relatively high number of cells positive for C7 detection. Of note, the signal from GM-HDFs, which were C7-positive, was significantly higher than that from NHDFs. The signal produced by GM-HDF per cell is brighter than that produced by GM-HDF. This suggests a large amount of C7.

[0363] Next, a flow cytometry assay was used to determine the specific number of cells expressing C7. As described in section 2.3, this assay was performed using NC1 region-specific polyclonal antibodies. For this experiment, we used the TR12.1 and TR13 arm B cells. In addition, a single transduction procedure (Arm B) and a second-generation LV-COL7 construct (INXN The supertransduction procedure (Arm A) using the GFP-2004 gene was compared with that using the GFP-2004 gene (Arm B). The percentage of C7 positive (C7+) cells for each training run arm was Consistent with the qualitative number of C7-expressing cells as detected by IF (Fig. 32), the measured copies The C7+ in Arm A and Arm B of TR12.1 and TR13 correlated with the number of C7+ in Arm A and Arm B of TR12.1 and TR13 (Table 46). Comparison of cells confirmed that the addition of a second generation nearly doubled the expected expression levels will be done.

[0364] Direct ELISA to measure C7 secreted by GM-HDF into cell culture medium This assay was performed using NC1 region-specific antibodies as described in section 2.4 above. The GM-H antibody from three training runs was used. DF ELISA assay results showed that C7 expression was dependent on the level of integrated LV copy number. This indicates that the expression level reaches approximately 2300 ng / day / 1e6 cells (Figure 33). Representative results from a previous study (RDEB-ADSO-2) compared with TR8 Arm A This results in a >200-fold increase in C7 expression (Figure 33).

[0365] 4.2. Functional evaluation of C7 expressed by GM-HDFs 4.2.1.C7 trimer formation The formation of C7 trimers is important in the assembly of tethered fibers (Bruckner -Tuderman, 1999). C7 expressed by GM-HDF forms anchoring fibers. To confirm that this can be achieved, we used an anti-C7 specific antibody (fN) for selective capture. Immunoprecipitation (IP) with C1) was used to analyze the formation of C7 trimers, followed by SDS-PAGE / immunoprecipitation. The concentration of C7 from the culture supernatant was assessed for detection by immunoblotting. Purified recombinant C7 was used as a positive control, and a capture step without C7-specific antibody was used as a negative control The IP results for the three training runs showed that the C produced by GM-HDF 7 is primarily a trimer (Fig. 34), with several low molecular weight species (possibly It was found that the C7 dimer and monomer were contained in the ATP-dependent ATPase.

[0366] 4.2.2. Lam332 binding by C7 expressed from GM-HDFs C7 binds fibronectin, laminin 332 (Lam332), COL1, and COL It has been shown to bind to immobilized extracellular matrix (ECM) components, including 4. The interaction between C7 and Lam332 is The NH2-terminal NC1 domain of C7NC1 is involved in the transcription of both Lam332 and C7NC1. It is dependent on the native conformation (Rousselle, et al., 1997). The binding of 7 to Lam332 plays a key role in establishing the correct Lam332 structure at the dermal-epidermal junction. Such organization is important in the regulation of the interaction of extracellular ligands with cell surface receptors, as well as in the regulation of the cellular endothelium. are important for cell signaling (Waterman, et al., 2007) Intrexon uses C7NC1 to detect the binding of C7 to purified Lam332. An ELISA was developed using antibodies against the domain. The results of two experiments are shown in Figure 35. , Lam33 by C7 expressed by GM-HDFs from three training runs 2. TR11 control cells were used as a negative control in both experiments. was used.

[0367] 4.2.3. Cell Migration Assay Previous studies have shown that RDEB fibroblasts and keratinocytes behave differently than their normal counterparts. It was revealed that the expression of C7 can restore normal motility. (Chen et al., 2000; Chen et al., 2002b ;Cogan,et al.,2014;Baldeschi,et al.,2003 ) Most of these studies involve the use of gold nanoparticles to measure fibroblast and keratinocyte migration. Salt colloid migration assay, or wound healing assay for measuring keratinocyte migration In this example, either a CytoSelect™ 24-well wound NHDF and G were analyzed using a healing assay kit (Cell Biolabs, Inc.). The motility of M-HDFs was measured.

[0368] To determine whether the presence of C7 in the culture medium is sufficient to reverse the RDEB hypermotility phenotype, To determine the effect of GM-HDFs on wound healing, a wound healing assay was performed on GM-HDFs from the three training runs. The experiment was carried out using NHDF as a positive control and mock-transduced control (RDEB) as a control. Control fibroblasts from TR11 arm C (RDEB) were used as a negative control. The mice exhibited a hyperkinetic phenotype that was reversed by the expression of C7 (Fig. 36). The level of phenotypic reversal was independent of the integration copy number / C7 expression level, which suggests that exercise This suggests that even low levels of C7 expression are sufficient to reverse the hyper-expression phenotype.

[0369] 5. Conclusion The number of integrated transgene copies per cell in GM-HDFs was increased by the enhanced transduction method. Addition of methods (spinoculation and hypertransduction) and LV-COL7 construct INXN- Both modifications to 2004 improved the expression level to a high level of 0.74 copies per cell. The magnification improvements achieved by each process change are as follows: Spinoculation addition: ≥ 2x Addition of supertransduction (including spinoculation): Approximately 2x Change from INXN-2002 to INXN-2004: >4 times Total cumulative change from original process: average >27-fold (TR8 compared to TR8 used in initial testing) 12.1 and TR13)

[0370] Immunofluorescence staining and flow cytometry of integrated copy number-dependent C7 expression in GM-HDF cells Confirmed by ELISA and ELISA. IND Submission 16582 Serial 00 A >200-fold improvement in C7 expression was achieved relative to the TR8 results shown in .

[0371] The structure of C7 expressed by GM-HDF cells was analyzed by immunoprecipitation / SDS-PAGE / immunoprecipitation. Blot analysis confirmed that it was primarily a trimer.

[0372] Furthermore, C7 produced by GM-HDF cells interacted with laminin-332 in vitro. Binding and hypermotile phenotype of control RDEB cells in in vitro migration assays Correction confirmed that it was functional.

[0373] Appendix 4 TIFF2026004289000050.tif220170 TIFF2026004289000051.tif234170 TIFF2026004289000052.tif231170 TIFF2026004289000053.tif230170 TIFF2026004289000054.tif233170 TIFF2026004289000055.tif224170 TIFF2026004289000056.tif235170 TIFF2026004289000057.tif231170 TIFF2026004289000058.tif235170 TIFF2026004289000059.tif116170

[0374] The following is 60010 for IGE308_REFSEQUENCE (SEQ ID NO: 34): Comparison of 743-ITX-00001_CONTIG SEQUENCE (SEQ ID NO: 34) It is an analysis. TIFF2026004289000060.tif211170 TIFF2026004289000061.tif208170 TIFF2026004289000062.tif208170 TIFF2026004289000063.tif208170 TIFF2026004289000064.tif207170 TIFF2026004289000065.tif209170 TIFF2026004289000066.tif211170 TIFF2026004289000067.tif209170 TIFF2026004289000068.tif208170 TIFF2026004289000069.tif209170 TIFF2026004289000070.tif210170 TIFF2026004289000071.tif208170 TIFF2026004289000072.tif209170 TIFF2026004289000073.tif210170 TIFF2026004289000074.tif206170 TIFF2026004289000075.tif208170 TIFF2026004289000076.tif209170 TIFF2026004289000077.tif208170 TIFF2026004289000078.tif208170 TIFF2026004289000079.tif210170 TIFF2026004289000080.tif209170 TIFF2026004289000081.tif208170 TIFF2026004289000082.tif208170 TIFF2026004289000083.tif208170 TIFF2026004289000084.tif209170 TIFF2026004289000085.tif208170

[0375] References a. Aiuti et al. Lentiviral hematopoietic s tem cell gene therapy benefits metachrom atic leukodystrophy. Science, 2013. August; 341:6148. Baldeschi C, Gache Y, Rattenholl a, Bouille P, Danos O, Ortonne JP, Bruckner-Tuderman L, Meneguzzi G. 2003. Genetic correction of canine dystrophic epidermolysis bullosa mediated by retroviral vectors. Human Mo Molecular Genetics 12:1897-905. a. Biffi et al. Lentiviral hematopoietic s tem cell gene therapy benefits metachrom atic leukodystrophy. Science, 2013. August; 341:6148. Bruckner-Tuderman L, Hopfner B, Hammami-Ha uasli N. 1999. Biology of anchoring fibril s: lessons from dystrophic epidermolysis bullosa.Matirx Biology 18:43-54. Burgeson REType VII collagen, anchoring fibrils, and epidermolysis bullosa.J.Inv est. Dermatol. 1993;101:252-255. Chen M, O'Toole EA, Muellenhoff M, Medina E , Kasahara N, Woodley DT. 2000. Development and characterization of a recombinant tr uncategorized type VII collagen "minigene". Imp lication for gene therapy of dystrophic epidermolysis bullosa.J Biol Chem 275:24 429-35. Chen M, Costa FK, Lindvay CR, Han YP, Woodle y DT.2002a.The recombinant expression of full-length type VII collagen and chara cterization of molecular mechanisms unde rlying dystrophic epidermolysis bullosa. J Biol Chem 277:2118-24. Chen M, Kasahara N, Keene DR, Chan L, Hoeffl er WK, Finlay D, Barcova M, Cannon PM, Mazur ek C, Woodley DT. 2002b. Restoration of typ e VII collagen expression and function i n dystrophic epidermolysis bullosa.Nature e Genetics 32:670-5. Cogan J,Weinstein J,Wang X,Hou Y,Martin S,South AP,Woodley DT,Chen M.2014.Aminog lycosides restore full-length type VII c ollagen by overcoming premature terminat ion codons:therapeutic implications for dystrophic epidermolysis bullosa.Molecul ar Therapy 22:1741-52. Deshpande P,Ralston DR,and S McNeil.The use of allodermis prepared from Euro ski n bank to prepare autologous tissue engi neered skin for clinical use.Burns.2013. 39(6):1170-1177. Goto,M.,et al.,Fibroblasts show more pot ential as target cells than keratinocyte s in COL7a1 gene therapy of dystrophic e pidermolysis bullosa.J Invest Dermatol,2 006.126(4):p.766-72. Greenberg KP,Edwin SL,Schaffer DV,Flanne ry JG.2006.Gene Delivery to the Retina U sing Lentiviral Vectors.Advances in Expe rimental Medicine and Biology 572:255-66 Kim YH,Woodley DT,Wynn KC,Giomi W,Bauer EA.Recessive Dystrophic Epidermylosis Bu llosa Phenotype is Preserved in Xenograf ts Using SCID Mice:Development of an Exp erimental In Vivo Model.J Invest Dermato l.1992 Feb;98(2):191- 7. Keene DR,Sakai LY,Lunstrum GP,Morris NP, Burgeson RE.Type VII Collagen Forms an E xtended Network of Anchoring Fibrils.J C ell Biol.1987 Mar;104(3):611-21. Leigh I.M.Eady R.a.Heagerty a.H.Purkis P .E.Whitehead P.a.Burgeson R.E.Type VII c ollagen is a normal component of epiderm al basement membrane,which shows altered expression in recessive dystrophic epid ermolysis bullosa.J.Invest.Dermatol.1988 ;90:639-642. Mavilio F.Pellegrini G.Ferrari S.Di Nunz io F.Di Iorio E.Recchia a.Maruggi G.Ferr are G.Probasi E.Bonini C.Capurro S.Conti a.Magnoni C.Giannetti a.De Luca M.Corre ction of junctional epidermolysis bullae a by transplantation of genetically modified fied epidermal stem cells.Nat.Med.2006;1 2:1397–1402. Ortiz-Urda S.Lin Q.Green CLKeene DRM Arinkovich MPKCoverage PaInjection of genetically engineered fibroblasts run CTS Regenerated Human Epidermolysis Bull osa skin tissue.J.Clin.Invest.2003;111:2 51-255. Remington J.Wang X.Hou Y.Zhou H.Burnett J.Muirhead T.Uitto J.Keene DRWoodley D .T.Chen M.Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophy c epidermolysis bullosa.Mol.Ther.2009;17 :26-3 Rousselle P,Keene DR,Ruggiero F,Champlia ud MF, Rest M, Burgeson RE binds the NC-1 domain of type VII collagen in.J Cell Bio,138:719-28. Siprashvili Z,Ngon T.Nguyen,Maria Y.Bezc hinsky,M.Peter Marinkovich,Alfred T.Lane ,and Paul A.Khavari.Long-term type VII c ollagen restoration to human epidermis is bullous skin tissue.Hum Gene Ther.Oct 2010;21(10):1299–1310. Tareen S, Nicolai C, Campbell D, Flynn P, Sl ough M,Vin C,Kelly-Clarke b,Odegard J,Ro bbins S.2013.a Rev-Independent gag / pol E liminates Detectable psi-gag Recombinant on in Lentiviral Vectors.Biores Open Acc ess.2013 Dec 1;2(6):421-430. 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Claims

1. 1. A method of treating a patient suffering from type VII collagen (C7) deficiency, comprising: Obtaining cells from the dermis or epidermis of the C7-deficient patient; A transduction vector containing a nucleotide sequence encoding COL7A1 or a functional variant thereof. contacting the cells with antiviral vector particles to produce cells with a specific vector copy number; In the step of forming a gene-modified cell, the lentiviral vector is having a transduction vector copy number in the range of 0.1 to 5.0 copies per vector; Culturing the autologous genetically modified cells; administering an effective amount of the genetically modified cells to the patient with C7 deficiency; A method comprising:

2. 10. The method of claim 1, wherein the cells are selected from fibroblasts and keratinocytes. A method characterized by:

3. 3. The method of claim 2, wherein the cells are fibroblasts. 。

4. 2. The method of claim 1, wherein the C7 deficiency is dystrophic epidermolysis bullosa. A method characterized by:

5. 5. The method of claim 4, wherein the dystrophic epidermolysis bullosa is recessive dystrophic epidermolysis bullosa. and characterized in that the disease is selected from the group consisting of dominant dystrophic epidermolysis bullosa (RDEB) and dominant dystrophic epidermolysis bullosa (DDEB). How to do this.

6. The method of claim 5, wherein the C7 deficiency is RDEB. method.

7. 7. The method of claim 6, wherein the RDEB is a Hallo-Siemens peau-Siemens type, non-Hallopeau-Siemens -Siemens) type RDEB, atypical RDEB, pretibial RDEB, distal RDEV, and and centripetal RDEB.

8. 6. The method of claim 5, wherein the C7 deficiency is DDEB. method.

9. 10. The method of claim 1, wherein the transducing lentiviral vector is a lentiviral vector. The method is characterized in that the virus vector is in the form of a virus vector particle.

10. 10. The method of claim 9, wherein the transducing lentiviral vector particles comprise: (a) a modified 5' long terminal repeat within the LTR (the promoter of said modified 5' LTR is (b) a functional COL7A1 gene; and (c) at least (d) a modified 3' LTR (the modified 3' 3'LTR contains a deletion relative to the wild-type 3'LTR) and a transfer lentivirus containing It was constructed from a viral vector and lacked the hepatitis virus post-transcriptional regulatory element (PRE). The COL7A1 gene or a functional variant thereof is incorporated into the cell to produce a functional A method for producing a genetically modified cell carrying a COL7A1 gene.

11. 11. The method of claim 10, wherein at least two lentiviral central polypurine sequences are A method characterized in that there is a column element.

12. 11. The method of claim 10, wherein the deleted PRE is a PRE from a woodchuck hepatitis virus ( Woodchuck hepatitis virus post-transcriptional regulatory element (WPR) E).

13. 10. The method of claim 9, wherein the transducing lentiviral vector particle is pSMP a lentiviral expression plasmid vector selected from the group consisting of pFUGW and pFUGW; A method characterized in that the method is constructed from

14. 14. The method of claim 13, wherein the lentiviral expression plasmid vector is p A method characterized in that the method is FUGW.

15. 10. The method of claim 9, wherein the transducing lentiviral vector particles are XN-2004.

16. 10. The method of claim 9, wherein the transducing lentiviral vector particles are XN-2002.

17. 10. The method of claim 1, wherein the genetically modified fibroblasts are administered by injection, topical administration, oral administration, or the like. The method is characterized in that the compound is administered to the patient by being embedded in a biocompatible matrix. Law.

18. 18. The method of claim 17, wherein the genetically modified fibroblasts are administered by injection. A method characterized by:

19. 19. The method of claim 18, wherein the genetically modified cells are injected intradermally into the patient. A method characterized by:

20. 18. The method of claim 17, wherein the genetically modified fibroblasts are encapsulated in a polymer. The method of claim 1, wherein the compound is encapsulated in a

21. 18. The method of claim 17, wherein the genetically modified fibroblasts are cultured in a collagen matrix. The method of claim 1, wherein the substrate is embedded in a glass tube.

22. 18. The method of claim 17, wherein the genetically modified fibroblasts are in a hydrogel or a membrane. The method of claim 1, wherein the first and second electrodes are embedded in a mesh.

23. Lentiviral vector particles containing a functional COL7A1 gene or a functional mutant thereof Transduced and type VII collagen-expressing collagen VII-derived from a patient with type VII collagen deficiency In autologous genetically modified fibroblasts, the lentiviral vector particles are delivered at 0 . Autologous vectors characterized by having a transduction vector copy number ranging from 1 to 5.0 copies. Genetically modified fibroblasts.

24. Lentiviral vector particles containing a functional COL7A1 gene or a functional mutant thereof Transduced and type VII collagen-expressing recessive epidermolysis bullosa patient-derived In autologous gene-modified fibroblasts, the lentiviral vector particles are A method for producing a transduction vector comprising the steps of: House genetically modified fibroblasts.

25. Lentiviral vector particles containing a functional COL7A1 gene or a functional mutant thereof Transduced and type VII collagen-expressing cells from a patient with dominant dystrophic epidermolysis bullosa In autologous gene-modified fibroblasts, the lentiviral vector particles are A method for producing a transduction vector comprising the steps of: House genetically modified fibroblasts.

26. (a) a modified 5' long end within the LTR (the promoter of the modified 5' LTR is (b) the COL7A1 gene or a functional variant thereof; variants; (c) at least one lentiviral central polypurine sequence element; and (d ) a modified 3' LTR, wherein the modified 3' LTR comprises a deletion relative to the wild-type 3' LTR. and a self-inactivating lentiviral vector formed from a transfer vector comprising the The present invention is characterized in that the hepatitis virus post-transcriptional regulatory element (PRE) is deleted in the A self-inactivating lentiviral vector.

27. 27. The vector of claim 26, wherein the vector comprises at least two lentiviruses. A vector comprising a central polypurine sequence element.

28. 27. The vector of claim 26, wherein the deleted PRE is a PRE of woodchuck hepatitis virus. Woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE).

29. 27. The vector of claim 26, wherein the vector comprises: (a) a modification in the LTR. 5' long end (the promoter of the modified 5'LTR is a cytomegalovirus promoter) (b) the COL7A1 gene; and (c) two lentiviral central polypurines. (d) a sequence element, and (e) a modified 3'LTR (the modified 3'LTR R contains a deletion relative to the wild-type 3'LTR).

30. 27. The vector of claim 26, wherein the vector is designated INXN-2004. A vector characterized by:

31. 27. The vector of claim 26, wherein the vector is designated INXN-2002. A vector characterized by:

32. A transfer vector characterized by being called IGE-308.

33. A lentiviral vector particle characterized by being designated INXN-2004.

34. A lentiviral vector particle characterized by being designated INXN-2002.

35. A stable vector characterized by producing INXN-2002 lentiviral vector particles Virus packaging cell lines.

36. A stable vector characterized by producing INXN-2004 lentiviral vector particles Virus packaging cell lines.

37. A lentiviral vector called INXN-2004 carrying the sequence of IGE308. A pharmaceutical composition comprising transduced fibroblasts obtained from a patient with RDEB.

38. RDEB patients transduced with a lentiviral vector called INXN-2002 A pharmaceutical composition comprising fibroblasts obtained from the

39. A lentiviral vector called INXN-2004 carrying the sequence of IGE308. A pharmaceutical composition comprising transduced fibroblasts obtained from a patient with DDEB.

40. DDEB patients transduced with a lentiviral vector called INXN-2002 A pharmaceutical composition comprising fibroblasts obtained from the

41. Transduced in vitro or ex vivo using the vector of claim 33. A cell characterized by:

42. Transduced in vitro or ex vivo using the vector of claim 34. A cell characterized by:

43. A method for treating a patient suffering from pseudosyndactyly, comprising administering to the patient a functional COL7A1 gene or its Transduced with lentiviral vector particles containing functional mutations and type VII collagen administering to said patient a population of autologous cells derived from said patient, said cells expressing a gene encoding a fusion protein. wherein the lentiviral vector particles are transfected at a concentration ranging from 0.1 to 5.0 copies per cell. A method characterized by having a transduction vector copy number.

44. 44. The method of claim 43, wherein the autologous cell population comprises keratinocytes and fibroblasts. The method of claim 1, wherein the cell is selected from the group consisting of:

45. 44. The method of claim 43, wherein the autologous cell population is fibroblasts. How to do this.

46. 44. The method of claim 43, wherein the cells are administered to the patient by injection, topical, oral, or is achieved by embedding in a biocompatible matrix.

47. Methods for treating, inhibiting, or reducing blister formation in patients with dystrophic epidermolysis bullosa (DEB) a lentiviral vector comprising a functional COL7A1 gene or a functional mutant thereof; a patient-derived autologous cell population transduced with particles and expressing type VII collagen; to the patient, wherein the lentiviral vector particles , and have a transduction vector copy number ranging from 0.1 to 5.0 copies per cell. The method characterized.

48. 48. The method of claim 47, wherein the autologous cell population comprises keratinocytes and fibroblasts. The method of claim 1, wherein the cell is selected from the group consisting of:

49. 49. The method of claim 48, wherein the autologous cell population is fibroblasts. How to do this.

50. 48. The method of claim 47, wherein the cells are administered to the patient by injection, topical, oral, or is achieved by embedding in a biocompatible matrix.

51. 48. The method of claim 47, wherein the patient is afflicted with recessive dystrophic epidermolysis bullosa. A method characterized in that

52. 48. The method of claim 47, wherein the patient is afflicted with dominant dystrophic epidermolysis bullosa. A method characterized in that

53. A method for treating lesions in patients with recessive dystrophic epidermolysis bullosa, comprising administering to the patient a functional COL7A1 gene. transduced with a lentiviral vector particle containing a gene or a functional variant thereof, and administering to said patient a population of autologous cells derived from said patient which express type I collagen. wherein the lentiviral vector particles are 0.1 to 5.0 particles per cell. The method is characterized in that the transduction vector copy number is in the range of 1000.

54. 54. The method of claim 53, wherein the autologous cell population comprises keratinocytes and fibroblasts. The method of claim 1, wherein the cell is selected from the group consisting of:

55. 54. The method of claim 53, wherein the autologous cell population is fibroblasts. How to do this.

56. 54. The method of claim 53, wherein the cells are administered to the patient by injection, topical, oral, or is achieved by embedding in a biocompatible matrix.

57. 54. The method of claim 53, wherein the lesion is located on the oral mucosa. method.

58. 54. The method of claim 53, wherein the lesion is in the gastrointestinal tract. Law.

59. Lentiviral vector particles containing a functional COL7A1 gene or a functional mutant thereof Transduced and expressing type VII collagen, autologous cells of patients with type VII collagen deficiency In an isolated population of genetically modified fibroblasts, the lentiviral vector particles are , and have a transduction vector copy number ranging from 0.1 to 5.0 copies per cell. Characteristic groups.

60. 60. The collection of claim 59, wherein the lentiviral vector particles are INXN-2 A population characterized by being 002.

61. 60. The collection of claim 59, wherein the lentiviral vector particles are INXN-2 A population characterized by being 004.

62. Lentiviral vector particles containing a functional COL7A1 gene or a functional mutant thereof Transduced and type VII collagen-expressing autologous cells from recessive dystrophic epidermolysis bullosa patients In an isolated population of genetically modified fibroblasts, the lentiviral vector particles are , and have a transduction vector copy number ranging from 0.1 to 5.0 copies per cell. Characteristic groups.

63. 63. The collection of claim 62, wherein the lentiviral vector particles are INXN-2 A population characterized by being 002.

64. 63. The collection of claim 62, wherein the lentiviral vector particles are INXN-2 A population characterized by being 004.

65. Lentiviral vector particles containing a functional COL7A1 gene or a functional mutant thereof Transduced and type VII collagen-expressing autologous cells from patients with dominant dystrophic epidermolysis bullosa In an isolated population of genetically modified fibroblasts, the lentiviral vector particles are , and have a transduction vector copy number ranging from 0.1 to 5.0 copies per cell. Characteristic groups.

66. 66. The collection of claim 65, wherein the lentiviral vector particles are INXN-2 A population characterized by being 002.

67. 66. The collection of claim 65, wherein the lentiviral vector particles are INXN-2 A population characterized by being 004.

68. Isolation of autologous genetically modified cells expressing type VII collagen (C7) from patients with C7 deficiency a method for producing a C7-deficient cell population, the method comprising collecting cells from the dermis or epidermis of a patient with C7 deficiency; and a transducing lentivirus containing the COL7A1 gene or a functional mutant thereof. contacting the cells with vector particles to produce COL7A vectors having a specific vector copy number; In the step of forming autologous genetically modified cells containing the lentiviral vector, The vector particles induce transduction vector copy numbers ranging from 0.1 to 5.0 copies per cell. and culturing the autologous genetically modified cells to produce genetically modified cells expressing C7. and obtaining an isolated population of said cells.

69. 69. The method of claim 68, wherein the cells are selected from fibroblasts and keratinocytes. A method characterized in that:

70. 70. The method of claim 69, wherein the cells are fibroblasts. Law.

71. 69. The method of claim 68, wherein the C7 deficiency is dystrophic epidermolysis bullosa. and a method characterized by:

72. 72. The method of claim 71, wherein the dystrophic epidermolysis bullosa is recessive dystrophic epidermolysis bullosa. Epidermolysis bullosa (RDEB) and dominant dystrophic epidermolysis bullosa (DDEB). How to signify.

73. 72. The method of claim 71, wherein the C7 deficiency is RDEB. method.

74. 74. The method of claim 73, wherein the RDEB is a Hall Halopeau-Siemens type, non-Halopeau-Siemens u-Siemens) type RDEB, atypical RDEB, pretibial RDEB, distal RDEV, and centripetal RDEB.

75. 72. The method of claim 71, wherein the C7 deficiency is DDEB. How to do it.

76. 69. The method of claim 68, wherein the transducing lentiviral vector particle is NXN-2002.

77. 69. The method of claim 68, wherein the transducing lentiviral vector particle is NXN-2004.

78. Type VII collagen (C), characterized in that it is produced by the method according to claim 68. 7) An isolated population of genetically modified cells expressing

79. Type VII collagen (C), characterized in that it is produced by the method according to claim 76. 7) An isolated population of genetically modified cells expressing

80. Type VII collagen (C), characterized in that it is produced by the method according to claim 77. 7) An isolated population of genetically modified cells expressing

81. 79. A pharmaceutical preparation comprising the isolated population of claim 78.

82. 80. A pharmaceutical preparation comprising the isolated population of claim 79.

83. 81. A pharmaceutical preparation comprising the isolated population of claim 80.

84. Integrated transcriptome per cell in genetically modified human skin fibroblasts or keratinocytes A method for increasing gene copy number, comprising: A transducing lentiviral vector containing a nucleotide sequence encoding the gene is administered to a patient with C7 deficiency. and contacting the cells with human skin fibroblasts or keratinocytes obtained from a subject to form a transduction composition. and subjecting the transduction composition to spinoculation to form transduced human skin. In a method comprising the step of generating skin fibroblasts or keratinocytes, said transducing The integrated transgene copy number in human skin fibroblasts or keratinocytes is The method is characterized in that the transduction composition is more than that which has not been subjected to ration.

85. 85. The method of claim 84, wherein the transduced human dermal fibroblasts are subjected to a second transduction. and further contacting the lentiviral vector to form a second transduction composition. How to signify.

86. 86. The method of claim 85, wherein the second transduction composition is administered by spinoculation. A method characterized by subjecting

87. 85. The method of claim 84, wherein the transduced human dermal fibroblasts are spinocules. At least one-fold more integration per cell compared to the untreated transducing composition. A method characterized by having a transgene copy number.

88. 85. The method of claim 84, wherein the transduced human dermal fibroblasts are spinocules. At least two times more integration per cell compared to the untreated transducing composition. A method characterized by having a transgene copy number.

88. 87. The method of claim 86, wherein the transduced human dermal fibroblasts are spinocules. less per cell than the transduction composition that has not been subjected to a second transduction step. and wherein both have 5 times more integrated transgene copy number.

88. 87. The method of claim 86, wherein the transduced human dermal fibroblasts are spinocules. less per cell than the transduction composition that has not been subjected to a second transduction step. and wherein both have 10 times more integrated transgene copy number.

88. 87. The method of claim 86, wherein the transduced human dermal fibroblasts are spinocules. less per cell than the transduction composition that has not been subjected to a second transduction step. and wherein both have 20-fold higher integrated transgene copy number.

88. 87. The method of claim 86, wherein the transduced human dermal fibroblasts are spinocules. less per cell than the transduction composition that has not been subjected to a second transduction step. and wherein both have 27 times more integrated transgene copy number.

89. 85. The method of claim 84, wherein the transduced human dermal fibroblasts have a low density per cell.

2. A method according to claim 1, wherein the vector has an integrated transgene copy number of at least 0.

05.

90. 85. The method of claim 84, wherein the transduced human dermal fibroblasts have a low density per cell.

2. A method according to claim 1, wherein the vector has an integrated transgene copy number of at least 0.

09.

91. 86. The method of claim 85, wherein the transduced human dermal fibroblasts have a low density per cell.

2. A method according to claim 1, wherein the vector has an integrated transgene copy number of at least 0.

41.

92. 87. The method of claim 86, wherein the transduced human dermal fibroblasts have a low density per cell.

2. A method according to claim 1, wherein the vector has an integrated transgene copy number of at least 0.

74.

93. 85. The method of claim 84, wherein the transduced human dermal fibroblasts are at about 100% transgenic per cell. having an integrated transgene copy number of 0.1 to about 5.

94. 85. The method of claim 84, wherein the transduced human dermal fibroblasts are at about 100% transgenic per cell. having an integrated transgene copy number of 0.1 to about 1.

95. 85. The method of claim 84, wherein the transduced human dermal fibroblasts are at about 100% transgenic per cell. having an integrated transgene copy number of 0.4 to about 1.

96. 85. The method of claim 84, wherein the transduced human dermal fibroblasts are at about 100% transgenic per cell. having an integrated transgene copy number of from 0.4 to about 0.

75.

100. 85. The method of claim 84, wherein the transducing lentiviral vector is a lentiviral vector. The method is characterized in that the vector is in the form of a viral vector particle.

101. 101. The method of claim 100, wherein the transducing lentiviral vector particle comprises: A method characterized in that the compound is INXN-2002.

102. 101. The method of claim 100, wherein the transducing lentiviral vector particle comprises: A method characterized in that the compound is INXN-2004.

103. 87. The method of claim 86, wherein the transduced human dermal fibroblasts or the keratinocytes The transduced human skin, wherein the site has not been subjected to spinoculation or a second transduction. characterized by increased C7 expression compared to skin fibroblasts or keratinocytes. How to do it.

104. 87. The method of claim 86, wherein the transduced human dermal fibroblasts or the keratinocytes The transduced human skin, wherein the site has not been subjected to spinoculation or a second transduction. At least 100-fold increased expression of C7 compared to skin fibroblasts or keratinocytes. A method comprising:

105. 87. The method of claim 86, wherein the transduced human dermal fibroblasts or the keratinocytes The transduced human skin, wherein the site has not been subjected to spinoculation or a second transduction. At least 200-fold increased expression compared to skin fibroblasts or keratinocytes. A method characterized by:

106. Integrated transcriptome per cell in genetically modified human skin fibroblasts or keratinocytes A method for increasing gene copy number, comprising: (a) a nucleotide sequence encoding the COL7A1 gene or a functional variant thereof; The transducing lentiviral vector was transduced into human skin fibroblasts obtained from C7-deficient patients or contacting keratinocytes to form a first transduction composition; (b) subjecting said first transduction composition to spinoculation to form transduced human skin. forming fibroblasts or keratinocytes; (c) subjecting the transduced human skin fibroblasts or keratinocytes of step (b) to a second and contacting the transducing lentiviral vector to form a second transducing composition. Top and (d) subjecting said first transduction composition to spinoculation to form transduced human skin. forming fibroblasts or keratinocytes; In a method comprising: Integrated transgene copy number of the transduced human skin fibroblasts or keratinocytes transduced human skin that has not been subjected to a spinoculation or second transduction step At least 5, 10, 15, 20, 25, 27 , 28, 29, 30, 35, 40, 45, or 50 times more.

107. 107. The method of claim 106, wherein the first and second transducing lentiviral vectors The method is characterized in that the agent is INXN-2004.

108. 107. The method of claim 106, wherein the cells are human dermal fibroblasts. How to signify.

109. 107. The method of claim 106, wherein the transduced human skin fibroblasts or keratinosa The integrated transgene copy number of the site is determined by spinoculation or a second transduction step. At least 100% of the transduced human skin fibroblasts or keratinocytes were transduced with 100% keratinocytes. The method according to claim 1, wherein the amount of the ion exchange is five times greater than the amount of the ion exchange.

110. 107. The method of claim 106, wherein the transduced human skin fibroblasts or keratinosa The integrated transgene copy number of the site is determined by spinoculation or a second transduction step. At least 100% of the transduced human skin fibroblasts or keratinocytes were transduced with 100% keratinocytes. The method according to claim 1, wherein the amount of the compound is 10 times greater than the amount of the compound.

111. 107. The method of claim 106, wherein the transduced human skin fibroblasts or keratinosa The integrated transgene copy number of the site is determined by spinoculation or a second transduction step. At least 100% of the transduced human skin fibroblasts or keratinocytes were transduced with 100% keratinocytes. 27 times more.

112. 107. The method of claim 106, wherein the transduced human skin fibroblasts or keratinosa The integrated transgene copy number of the site is determined by spinoculation or a second transduction step. Approximately five times more transduced human skin fibroblasts or keratinocytes than untreated transduced human skin fibroblasts or keratinocytes. A method characterized by:

113. 107. The method of claim 106, wherein the transduced human skin fibroblasts or keratinosa The integrated transgene copy number of the site is determined by spinoculation or a second transduction step. Approximately 10 times more transduced human skin fibroblasts or keratinocytes than untreated transduced human skin fibroblasts or keratinocytes. A method characterized by:

114. 107. The method of claim 106, wherein the transduced human skin fibroblasts or keratinosa The integrated transgene copy number of the site is determined by spinoculation or a second transduction step. Approximately 27 times more transduced human skin fibroblasts or keratinocytes than untreated transduced human skin fibroblasts or keratinocytes. A method characterized by:

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