Cell sheet

Abstract

To provide a cell sheet implanted on skin of a patient of dystrophic epidermolysis bullosa, and a method for producing the sheet.SOLUTION: A cell sheet is composed of cells collected from body fluid inside a blister of a patient of dystrophic epidermolysis bullosa. Type VII collagen gene is introduced into the cell.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to cell sheets.

Background Art

[0002] Epidermolysis bullosa is a disease in which, when force is applied to the skin, the epidermis peels off from the dermis, resulting in blisters or skin ulcers due to the deficiency or disappearance of adhesion structural molecules responsible for skin tissue adhesion. Among these, the disease type in which the epidermis is torn and blisters form is called simplex epidermolysis bullosa, the disease type in which blisters form due to the separation between the epidermis and the basement membrane is called junctional epidermolysis bullosa, and the disease type in which the separation occurs between the basement membrane and the dermis is called dystrophic epidermolysis bullosa.

[0003] Dystrophic epidermolysis bullosa is the most common disease type, accounting for about 50% of all epidermolysis bullosa cases, and is a hereditary disease caused by mutations in the COL7A1 gene that encodes type VII collagen. In the structure of the skin, the epidermal basal cells at the bottom of the epidermis are connected to a sheet-like structure called the basement membrane. Type VII collagen forms fibers called anchoring fibrils in the dermis and connects the basement membrane and the dermis. Therefore, when there is an abnormality in the type VII collagen gene, the adhesion function between the basement membrane and the dermis is impaired, resulting in dystrophic epidermolysis bullosa in which blisters form between the basement membrane and the dermis. Among dystrophic epidermolysis bullosa cases, severe recessive dystrophic epidermolysis bullosa is an extremely severe hereditary blistering skin disease in which skin symptoms similar to burns persist throughout the body from immediately after birth, and squamous cell carcinoma of the skin (scar cancer) frequently develops at a high rate from around 30 years of age, leading to death.

[0004] There is currently no effective treatment for epidermolysis bullosa, and the development of gene therapy to radically suppress blister formation is required. As such gene therapy, a treatment technique has been disclosed in which skin cells of a patient are collected, genetically engineered to produce type VII collagen, cultured to form a skin sheet, and transplanted into the patient (see Patent Document 1). In addition, it has been proposed that genome editing be performed on mesenchymal stem cells in which the activity of type VII collagen has been inactivated, and the mesenchymal stem cells capable of producing type VII collagen be differentiated into keratinocytes or fibroblasts and cultured to form a skin sheet for use in treating patients (see Patent Document 2).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] This application provides a cell sheet effective for treating dystrophic epidermolysis bullosa.

Means for Solving the Problems

[0007] One embodiment of the cell sheet of the present invention is composed of cells obtained from the body fluid in the blisters of a patient with dystrophic epidermolysis bullosa and into which the type VII collagen gene has been introduced. This cell sheet is applied to the skin of a patient with dystrophic epidermolysis bullosa.

[0008] In one aspect of the method for producing a cell sheet, first, the body fluid in the blister of a patient with dystrophic epidermolysis bullosa is seeded in a culture medium. Next, the cells attached to the bottom of the culture vessel are obtained. Next, the type VII collagen gene is introduced into these cells. Next, the cells into which the type VII collagen gene has been introduced are cultured. Finally, the cultured cells are taken out of the culture vessel as a cell sheet.

Advantages of the Invention

[0009] The present invention is useful for the treatment of dystrophic epidermolysis bullosa.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Mode for Carrying Out the Invention

[0011] Unless otherwise specifically defined, the terms used in this disclosure have the meanings generally understood by those skilled in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, etc. Definitions of some of the terms used in this disclosure are described below, but these definitions take precedence over general understanding in this disclosure.

[0012] One embodiment of this disclosure relates to a cell sheet. This cell sheet contains cells derived from the body fluid within the blisters of a patient with dystrophic epidermolysis bullosa. The VII type collagen gene is introduced into these cells. This cell sheet is transplanted onto the skin of a patient with dystrophic epidermolysis bullosa.

[0013] Preferably, a VII type collagen gene expression cassette is introduced into the cells. The VII type collagen gene expression cassette preferably contains an EF1α promoter and a COL7A1 gene arranged downstream of the EF1α promoter.

[0014] Within the cell sheet, it is preferable that the cells overlap in the thickness direction of the sheet, that is, the normal direction. Also, in the cell sheet, it is preferable that the cells form a plurality of layers. Thereby, the mechanical strength of the cell sheet is improved, and a larger number of cells per unit area can be supplied to the affected area. The thickness of the cell sheet is preferably about 5 - 50 μm, and more preferably about 10 - 25 μm. With this thickness, the mechanical strength of the cell sheet can be ensured, and sufficient oxygen can be supplied to the cells inside the sheet.

[0015] When the cell sheet is viewed in a longitudinal section, it is preferable that the cell density near the surface of the sheet is higher than the cell density at the center of the sheet. More preferably, on both surfaces of the cell sheet, the cell density near the surface is higher than the cell density at the center of the sheet. When the cells are dense near the surface of the sheet, the strength of the cell sheet increases, and it is less likely to tear when peeled off or transplanted. As the center part of the sheet, a part located exactly in the middle between one surface and the other surface of the sheet is exemplified.

[0016] Dystrophic Epidermolysis Bullosa (DEB) is a genetic disease caused by mutations in the COL7A1 gene encoding type VII collagen. It is known that either no type VII collagen is produced or type VII collagen with reduced function due to mutations is produced. Type VII collagen forms fibers called anchoring fibrils in the dermis and connects the basement membrane and the dermis. Type VII collagen contains, from the N-terminus, a first non-collagenous region, a collagenous region, and a second non-collagenous region, forms a triple helix in the collagenous region part characterized by a glycine-X-Y repeat sequence, binds two molecules at the C-terminus, and binds the N-terminus to the basement membrane. Mutations include mutations in which glycine in the collagenous region is replaced by other amino acids, stop codon mutations that stop protein translation, splice site mutations, and the like. Mutations can be present in one of the alleles or in both. Dystrophic Epidermolysis Bullosa includes dominant dystrophic and recessive dystrophic types, and the recessive dystrophic type includes severe generalized type and other generalized types with relatively mild symptoms. Dystrophic Epidermolysis Bullosa in this specification may be any type of dystrophic epidermolysis bullosa and may be caused by any mutation in the COL7A1 gene.

[0017] A blister refers to a collection of liquids such as body fluids and tissue fluids under the epidermis. Preferably, the blister is in a form in which liquid accumulates in a space formed between the epidermis and the dermis due to the separation of the epidermis from the dermis. More preferably, the blister is in a form in which liquid accumulates in a space formed between the basement membrane of the epidermis and the dermis due to the separation of the basement membrane from the dermis.

[0018] In the present disclosure, the blister-derived cells of a patient with dystrophic epidermolysis bullosa refer to adherent cells collected from the blisters of a patient with dystrophic epidermolysis bullosa, and are also referred to as "DEB patient blister-derived cells" or "blister-derived cells" in the present disclosure. Such cells can be obtained by culturing the blister contents of a patient with dystrophic epidermolysis bullosa on a solid phase. In one embodiment, the blister contents are the liquid accumulated in the blister, and this liquid is referred to as the "intra-blister solution" in this specification. The blister contents can be collected from the blisters of a patient with dystrophic epidermolysis bullosa by means such as a syringe. For example, in the case of the intra-blister solution, a syringe needle is punctured into the blister, and with the tip of the syringe needle positioned in the space formed between the epidermis and the dermis, the plunger of the syringe is pulled to aspirate the intra-blister solution into the syringe barrel. In one embodiment, the blister-derived cells can be obtained by seeding the intra-blister solution in a medium without treatment with enzymes such as collagenase and dispase and culturing on a solid phase. Specifically, the intra-blister solution collected from the blister is directly seeded in the medium, and after incubating the medium on a solid phase for a predetermined time, the cells attached to the solid phase can be used as blister-derived cells. At this time, it is preferable to obtain the cells that have formed colonies on the solid phase. The intra-blister solution is preferably seeded in the medium within 3 hours after collection, more preferably within 2 hours, and even more preferably within 1 hour. In the present disclosure, the solid phase means a solid support to which cells can adhere, and includes, for example, culture containers such as plastic or glass culture dishes, flasks, and multi-well plates. In a certain embodiment, the solid phase is a plastic culture container. The solid phase may be coated, and examples of the coating substance include collagen I, laminin, vitronectin, fibronectin, poly-L-lysine, poly-L-ornithine, and the like. In a certain embodiment, the solid phase is coated with collagen I. The culture can be carried out under conditions such as "37°C, 5% CO2" or "37°C, 5% O2, 5% CO2" in a general incubator. The culture medium may be any medium that can be used for culturing animal cells, such as MEM, MEMα, DMEM, GMEM, RPMI 1640, MesenCult TM(STEMCELL Technologies), Mesenchymal Stem Cell Growth Medium 2 (PromoCell), MSCGM Mesenchymal Stem Cell Growth Medium (Lonza), Cellartis MSC Xeno-Free Culture Medium (Takara Bio), and mixed media thereof, etc. Among them, MesenCult TM , media for mesenchymal stem cells such as Mesenchymal Stem Cell Growth Medium 2, MSCGM Mesenchymal Stem Cell Growth Medium, and Cellartis MSC Xeno-Free Culture Medium are preferably used. Also, the medium is preferably a serum-free medium. The culture period may be a period sufficient for the cells to adhere to the solid phase, for example, 1 day to several months (e.g., 2, 3, or 4 months), 1 day to 1 month, 1 day to several weeks (e.g., 2, 3, or 4 weeks), or 1 day to 1 week.

[0019] In certain embodiments, the blister-derived cells of DEB patients have one or more characteristics selected from the following 1) to 6): 1) Having adhesiveness to a solid phase, 2) One or more surface markers selected from the group consisting of CD73, CD105, and CD90 are positive, 3) One or more surface markers selected from the group consisting of CD45, CD34, CD11b, CD79A, HLA-DR, and CD31 are negative, 4) Having no ability to differentiate into osteoblasts or having a lower differentiation ability compared to bone marrow-derived mesenchymal stem cells, 5) Having no ability to differentiate into adipocytes or having a lower differentiation ability compared to bone marrow-derived mesenchymal stem cells, 6) Having no ability to differentiate into chondrocytes or having a lower differentiation ability compared to bone marrow-derived mesenchymal stem cells. In the present disclosure, the fact that a certain cell "does not have the ability to differentiate" into osteoblasts, adipocytes or chondrocytes means that differentiation into osteoblasts, adipocytes or chondrocytes cannot be detected using normal differentiation induction conditions and detection methods (such as staining).

[0020] In certain embodiments, cells derived from blisters of DEB patients are cells that are positive for CD73, positive for CD105, and positive for CD90. In another embodiment, cells derived from blisters of DEB patients are cells that are negative for CD45, negative for CD34, negative for CD11b, negative for CD79A, negative for HLA-DR, and negative for CD31. In a further embodiment, cells derived from blisters of DEB patients are cells that have lower abilities to differentiate into osteoblasts, adipocytes, and chondrocytes compared to bone marrow-derived mesenchymal stem cells in all cases.

[0021] In the present disclosure, cells derived from blisters of dystrophic epidermolysis bullosa patients that have been genetically modified to produce type VII collagen are used. The "cells genetically modified to produce type VII collagen" in the present disclosure means cells that have been genetically modified to produce functional (i.e., capable of forming anchoring fibrils) type VII collagen.

[0022] In the present disclosure, genetic modification of cells means either modifying the genes of the cell's genome or modifying the cell to express genes from nucleic acid constructs outside the genome (for example, vectors). That is, the expression "genetically modify to produce type VII collagen" includes modifying the cell to express type VII collagen from the COL7A1 gene in the genome and modifying the cell to express type VII collagen from the COL7A1 gene of a nucleic acid construct outside the genome. Also, the "cells genetically modified to produce type VII collagen" includes cells that express type VII collagen from the COL7A1 gene in the genome and cells that express type VII collagen from the COL7A1 gene of a nucleic acid construct outside the genome.

[0023] The genetic modification of cells can be carried out by introducing the COL7A1 gene or by correcting mutations in the COL7A1 gene of the genome. The introduction of the COL7A1 gene can be achieved by introducing the COL7A1 gene into the genome of the cells or by introducing a nucleic acid construct containing the COL7A1 gene into the cells so that the COL7A1 gene is expressed from an extra-genomic nucleic acid construct. When introducing the COL7A1 gene into the genome of the cells, it may be introduced at a specific position or randomly. In certain embodiments, the COL7A1 gene is introduced into the COL7A1 locus of the genome or into a safe harbor such as the AAVS1 region.

[0024] The cells derived from the blisters of DEB patients may be the cells of a dystrophic epidermolysis bullosa patient (i.e., autologous cells) into which the cells are administered, or the cells of a dystrophic epidermolysis bullosa patient different from the patient receiving the administration of the cells (i.e., allogeneic cells). The cells of a dystrophic epidermolysis bullosa patient include cells that do not produce type VII collagen and cells that produce type VII collagen but have a reduced function due to mutations. However, the "cells of a dystrophic epidermolysis bullosa patient" in the present disclosure may be any of them.

[0025] The cells derived from the blisters of DEB patients may be any cells that can produce type VII collagen near the epidermal basement membrane when administered to a patient.

[0026] In the present disclosure, the cells are used in the sense of including those that have been proliferated as necessary. The proliferation of the cells can be carried out by culturing the cells. For example, "cells derived from blisters (of a dystrophic epidermolysis bullosa patient)" include those that have been proliferated after being obtained from a patient, and "genetically modified cells" include those that have been proliferated from the cells obtained by a genetic modification operation. When performing a genetic modification, the cells may be proliferated until the amount required for the genetic modification is obtained. Also, after the genetic modification, the cells may be proliferated until the amount required for treatment is obtained.

[0027] As used herein, the term "cell" can mean one cell or a plurality of cells, depending on the context. The cell may be a cell population consisting of one type of cell, or may be a cell population containing a plurality of types of cells.

[0028] As used herein, the COL7A1 gene means a nucleic acid sequence encoding type VII collagen, and is used in the sense that it also includes cDNA and sequences containing one or more introns (e.g., genomic sequences or minigenes). The representative nucleic acid sequence of the human COL7A1 gene (cDNA) is shown in SEQ ID NO: 1, and the representative amino acid sequence of human type VII collagen is shown in SEQ ID NO: 2. The cDNA sequence of the COL7A1 gene is disclosed in GenBank: NM_000094.3, and the genomic sequence is disclosed in GenBank: AC121252.4. The COL7A1 gene may be any gene encoding a functional (i.e., capable of forming anchoring fibrils) type VII collagen, and its sequence is not limited. cDNA sequence (8835 bp) of the human COL7A1 gene (SEQ ID NO: 1) Amino acid sequence of human type VII collagen (2944 AA) (SEQ ID NO: 2)

[0029] In certain embodiments, the COL7A1 gene comprises, or consists of, a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 1. In another embodiment, the COL7A1 gene comprises, or consists of, a nucleic acid sequence in which 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 bases are inserted, deleted, substituted or added in the nucleic acid sequence of SEQ ID NO: 1. In a further embodiment, the COL7A1 gene comprises, or consists of, the nucleic acid sequence of SEQ ID NO: 1.

[0030] In certain embodiments, type VII collagen comprises, or consists of, an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 2. In another embodiment, type VII collagen comprises, or consists of, an amino acid sequence in which 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acid residues are inserted, deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2. In a further embodiment, type VII collagen comprises, or consists of, the amino acid sequence of SEQ ID NO: 2.

[0031] In certain embodiments, the COL7A1 gene comprises, or consists of, a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 2. In another embodiment, the COL7A1 gene comprises, or consists of, a nucleic acid sequence encoding an amino acid sequence in which 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acid residues are inserted, deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2.

[0032] As used herein, "sequence identity" with respect to a nucleic acid sequence or an amino acid sequence means the percentage of nucleotide or amino acid residues that match between two sequences that are optimally aligned (in a state where the match is maximized) over the entire region of the sequences to be compared. Here, the sequences to be compared may have insertions, additions or deletions (such as gaps, etc.) in the optimal alignment of the two sequences. Sequence identity can be calculated using programs such as FASTA, BLAST, CLUSTAL W, etc. provided in public databases (for example, DDBJ (http: / / www.ddbj.nig.ac.jp)). Alternatively, it can also be determined using commercially available sequence analysis software (for example, Vector NTI (registered trademark) software, GENETYX (registered trademark) ver. 12).

[0033] The method for genetically modifying cells is not particularly limited. In one embodiment, the cells are genetically modified by genome editing such as the CRISPR system (for example, CRISPR / Cas9, CRISPR / Cpf1), TALEN, ZFN, etc. In another embodiment, the cells are genetically modified by viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, etc. In a further embodiment, the cells are genetically modified by CRISPR / Cas9. In a further embodiment, the cells are genetically modified by a retroviral vector or a lentiviral vector.

[0034] In the case of genome editing, by causing a cleavage in the genome and introducing a donor vector containing the target sequence into the cell, the sequence can be inserted into the cleavage site of the genome. The sequence to be inserted into the genome can be the COL7A1 gene or a sequence for substituting a site containing a mutation in the COL7A1 gene (for example, a partial sequence of the COL7A1 gene). In addition to the target sequence, the donor vector may contain regulatory sequences such as a promoter and an enhancer that control the expression of the target sequence, and other elements such as a drug resistance gene for cell selection, and may contain sequences homologous to both ends of the insertion site in the genome at both ends thereof. The donor vector can be introduced into the desired site by non-homologous end joining or homologous recombination. As the donor vector, a plasmid or a viral vector such as an adeno-associated virus vector or an integrase-deficient lentiviral vector can be used.

[0035] In the CRISPR system, endonucleases such as Cas9 or Cas12 (e.g., Cas12a (also called Cpf1), Cas12b, Cas12e) recognize a PAM sequence, which is a specific base sequence, and cleave the double-stranded target DNA by the action of the endonuclease. If the endonuclease is Cas9, it cleaves about 3 - 4 bases upstream of the PAM sequence. Examples of endonucleases include Cas9 from S. pyogenes, S. aureus, N. meningitidis, S. thermophilus, or T. denticola, and Cpfl from L. bacterium ND2006 or Acidaminococcus sp. BV3L6. The PAM sequence depends on the endonuclease. For example, the PAM sequence of Cas9 from S. pyogenes is NGG. The gRNA contains, at the 5'-end side, a sequence (target sequence) of about 20 bases upstream of the PAM sequence or a sequence complementary thereto, and plays a role in recruiting the endonuclease to the target sequence. Among the gRNAs, the sequence of the part other than the target sequence (or a sequence complementary thereto) can be appropriately determined by those skilled in the art according to the endonuclease to be used. The gRNA may contain a crRNA (CRISPR RNA) that contains the target sequence or a sequence complementary thereto and is responsible for the sequence specificity of the gRNA, and a tracrRNA (Trans-activating crRNA) that forms a double strand and contributes to the formation of a complex with Cas9. The crRNA and tracrRNA may exist as separate molecules. When the endonuclease is Cpf1, only the crRNA functions as the gRNA. In this specification, a gRNA that contains the elements necessary for the function of the gRNA on a single strand may be particularly described as sgRNA. The sequence of the gRNA can be determined by tools available for the selection of the target sequence and the design of the gRNA, such as CRISPRdirect (https: / / crispr.dbcls.jp / ).

[0036] A vector containing a nucleic acid sequence encoding a gRNA and a nucleic acid sequence encoding an endonuclease may be introduced into cells for expression, or a gRNA prepared extracellularly and the protein of the endonuclease may be introduced into cells. Note that the endonuclease may have a nuclear localization signal. The nucleic acid sequence encoding the gRNA and the nucleic acid sequence encoding the endonuclease may be present on different vectors. The vector, gRNA, and endonuclease can be introduced into cells by lipofection, electroporation, microinjection, calcium phosphate method, DEAE-dextran method, etc., but are not limited to these methods.

[0037] In certain embodiments, the gRNA that can be used for introduction into the genome of the COL7A1 gene contains any one of the sequences of SEQ ID NOs: 3 to 5 or a sequence complementary thereto.

[0038] In the case of using a viral vector, when a retroviral vector or a lentiviral vector having integrase activity is used, the COL7A1 gene can be introduced into the genome of cells. The retroviral vector and the lentiviral vector may be integrase-deficient. The integrase-deficient vector lacks integrase activity due to, for example, a mutation in the integrase gene. When an integrase-deficient vector, an adenoviral vector, or an adeno-associated viral vector is used, usually, the sequence incorporated into the vector is not introduced into the genome of cells. For example, when an integrase-deficient lentiviral vector or an adenoviral vector incorporating the COL7A1 gene is used, type VII collagen is expressed from the COL7A1 gene of the vector present intracellularly (in the nucleus).

[0039] The viral vector contains a sequence encoding the COL7A1 gene and may include regulatory sequences such as promoters and enhancers that control the expression of the COL7A1 gene, as well as other elements such as drug resistance genes for cell selection. The viral vector may be produced by any method known in the art. For example, retroviral vectors and lentiviral vectors can be produced by introducing a viral vector plasmid containing the LTR sequences (5'LTR and 3'LTR) at both ends, a packaging signal, and the sequence of interest, together with one or more plasmid vectors expressing viral structural proteins such as Gag, Pol, and Env, into packaging cells, or by introducing them into packaging cells expressing these structural proteins. Examples of packaging cells include, but are not limited to, 293T cells, 293 cells, HeLa cells, COS1 cells, and COS7 cells. The viral vector may be pseudotyped and may express an envelope protein such as the vesicular stomatitis virus G protein (VSV-G). By infecting the target cells with the produced viral vector, the sequence of interest can be introduced into the target cells.

[0040] In certain embodiments, the viral vector is a lentiviral vector. Examples of lentiviral vectors include, but are not limited to, HIV (human immunodeficiency virus) (e.g., HIV-1 and HIV-2), SIV (simian immunodeficiency virus), FIV (feline immunodeficiency virus), MVV (Maedi-Visna virus), EV1 (Maedi-Visna-like virus), EIAV (equine infectious anemia virus), and CAEV (caprine arthritis encephalitis virus). In certain embodiments, the lentiviral vector is HIV.

[0041] As an example, a lentiviral vector can be produced as follows. First, a viral vector plasmid encoding a viral genome, one or more plasmid vectors expressing Gag, Pol, and Rev (and optionally Tat), and one or more plasmid vectors expressing an envelope protein such as VSV-G are introduced into packaging cells. The viral vector plasmid contains LTR sequences (5'LTR and 3'LTR) at both ends, a packaging signal, and the COL7A1 gene and a promoter controlling its expression (e.g., CMV promoter, CAG promoter, EF1α promoter, PGK promoter, or hCEF promoter). The 5'LTR functions as a promoter that induces transcription of the viral RNA genome, but may be replaced with another promoter such as the CMV promoter to enhance the expression of the RNA genome. Inside the cell, the viral RNA genome is transcribed from the vector plasmid, packaged, and a viral core is formed. The viral core is transported to the cell membrane of the packaging cell, encapsulated in the cell membrane, and released as viral particles from the packaging cell. The released viral particles can be recovered from the culture supernatant of the packaging cells. For example, the viral particles can be recovered by ordinary purification methods such as centrifugation, filter filtration, and column purification. Also, lentiviral vectors can be produced using kits such as Lentiviral High Titer Packaging Mix, Lenti-X TM Packaging Single Shots (Takara Bio Inc.), ViraSafe TM Lentiviral Complete Expression System (Cell Biolabs Inc.). An adeno-associated virus vector can be produced using kits such as AAVpro (登録商標) Helper Free System (Takara Bio Inc.).

[0042] In one aspect, the present disclosure provides a plasmid used in the production of a lentiviral vector, the plasmid having an EF1α promoter and a COL7A1 gene disposed downstream of the EF1α promoter. In a further aspect, the present disclosure provides a lentiviral vector having an EF1α promoter and a COL7A1 gene disposed downstream of the EF1α promoter.

[0043] A representative sequence of the EF1α promoter is shown in SEQ ID NO: 6. EF1α promoter (SEQ ID NO: 6) In one embodiment, the EF1α promoter comprises, or consists of, a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 6. In another embodiment, the EF1α promoter comprises, or consists of, a nucleic acid sequence in which 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 bases are inserted, deleted, substituted or added in the nucleic acid sequence of SEQ ID NO: 6. In a further embodiment, the EF1α promoter comprises, or consists of, the nucleic acid sequence of SEQ ID NO: 6.

[0044] Cells into which the target sequence has been introduced can be confirmed by Southern blotting or PCR. The target sequence only needs to be introduced into at least one of the alleles.

[0045] In one embodiment, the cell sheet is transplanted to the affected area of a patient with dystrophic epidermolysis bullosa. In a preferred embodiment, the cell sheet is transplanted to a part where the epidermis of the skin is defective. Specific examples of the part where the epidermis of the skin is defective include skin ulcer surfaces. Also, the cell sheet may be transplanted to a part where the epidermis has peeled off from the dermis. In one embodiment, the cell sheet is transplanted by attaching it to the affected area of the skin. At that time, it is preferable to attach the cell sheet to the affected area so that the cell sheet adheres to the dermis.

[0046] Hereinafter, embodiments of the method for producing a cell sheet will be described. FIG. 1 shows an overview of the method for producing a cell sheet. The outline of each step is as follows. 1. Collect the body fluid in the blisters of a patient with dystrophic epidermolysis bullosa. 2. Seed at least a part of the collected body fluid in a medium and culture it. 3. Obtain the cells attached to the bottom of the culture vessel. And, if necessary, passage these cells. 4. Introduce the type VII collagen gene into the obtained cells. 5.Culture the cells into which the type VII collagen gene has been introduced, and passage them as necessary. 6.Seed the cells into which the type VII collagen gene has been introduced in a culture vessel. 7.Remove the cultured cells from the culture vessel as a cell sheet.

[0047] 1.Collection of the blister fluid The method for collecting the body fluid / tissue fluid in the blister has been described above, so it will be omitted.

[0048] 2.Sowing and culturing of the blister fluid Seed at least a part of the collected body fluid / tissue fluid in a medium and culture it. At least a part of the body fluid / tissue fluid refers to a constituent component of the body fluid / tissue fluid. Examples of a constituent component include the precipitate after centrifuging the body fluid / tissue fluid. Also, fractions obtained using a device capable of separating specific cells, such as magnetic beads or a flow cytometer, are also included as a constituent component. As described above, it is desirable to seed the body fluid / tissue fluid in the blister, a constituent component of the body fluid / tissue fluid, or cells obtained from the body fluid / tissue fluid in the medium without enzyme treatment. Note that the seeding and culturing methods have been described above, so they will be omitted.

[0049] 3.Passage of adherent cells After seeding and culturing, obtain the cells attached to the bottom of the culture vessel. Then, passage the cells as appropriate and repeat as necessary. Thereby, the number of cells required for gene introduction can be obtained. During passage, the cells can be frozen and stored as necessary. Cryopreservation of blister-derived cells can be performed by a general operation process using a general cell cryopreservation solution. The cryopreservation solution may be any solution that can be used for cryopreservation of animal cells. For example, CELLBANKER (登録商標) 1 (Xenogen Pharma), STEM-CELLBANKER GMP grade (Xenogen Pharma), STEM-CELLBANKER DMSO Free GMP grade (Xenogen Pharma), HSC-BANKER (登録商標)GMP grade (manufactured by Xenogen Pharma), or a medium supplemented with 5 - 10% DMSO, etc. Among these, STEM-CELLBANKER GMP grade, HSC-BANKER (登録商標) Cryopreservation solutions for stem cells such as GMP grade are preferably used. Also, the cell cryopreservation solution is preferably serum-free.

[0050] 4. Gene Transfer The type VII collagen gene is introduced into the subcultured cells. The gene transfer method has been described above and will be omitted here. In this step, it is preferable to introduce the type VII collagen gene into the blister-derived cells using a lentiviral vector.

[0051] 5. Subculture of Gene-Transfected Cells The cells into which the type VII collagen gene has been introduced are cultured, and if necessary, cell subculture is appropriately repeated as needed. This allows for obtaining the number of cells required for creating a cell sheet. Also, during subculture, if necessary, these cells can be frozen and stored. For cell cryopreservation, the cryopreservation solution described above can be used.

[0052] 6. Seeding of Gene-Transfected Cells The blister-derived cells into which the type VII collagen gene has been introduced are seeded into a culture vessel. At this time, it is preferable to seed the cells into the culture vessel so that they become overconfluent. Specifically, it is preferable to seed the cells at a confluency of 150% or more, more preferably at 300% or more, and even more preferably at 600% or more. By seeding the cells at such a confluency, a cell sheet with a layer structure suitable for sheet transplantation can be obtained.

[0053] Also, the blister-derived cells into which the type VII collagen gene has been introduced are preferably seeded into a culture vessel at a cell density of 1x10 4 cells / cm 2 or higher, and more preferably at 5x10 4 cells / cm 2It is more preferable to seed at the above cell density, 1x10 5 cells / cm 2 It is even more preferable to seed at the above cell density. When cells are seeded at such a density, the number of cell layers in the cell sheet becomes optimal, the mechanical strength is excellent, and oxygen can reach the cells inside the cell sheet.

[0054] As the culture vessel, a normal culture vessel whose surface is made of polystyrene can be used. That is, it is not necessary to use a temperature-responsive culture vessel. The present inventor has discovered that blister-derived cells can be detached from the bottom of the culture vessel without using a temperature-responsive culture vessel. Of course, blister-derived cells may be cultured in a temperature-responsive culture vessel. Examples of the temperature-responsive culture vessel include a culture vessel whose surface is processed with a temperature-responsive polymer. The temperature-responsive polymer changes from hydrophobic to hydrophilic at a specific temperature, for example, 32°C. Therefore, for example, at 37°C, cells adhere to the temperature-responsive polymer, and when the temperature is lowered to, for example, 20°C, the cells can no longer adhere to the temperature-responsive polymer and detach from the bottom of the culture vessel.

[0055] 7. Cell sheet extraction The cultured blister-derived cells are taken out as a cell sheet from the culture vessel. The present inventor has discovered that blister-derived cells can be detached as a cell sheet from the culture vessel without being treated with enzymes such as trypsin and dispase. The cell sheet composed of blister-derived cells can be detached and taken out only with tweezers even from the bottom of a normal culture vessel that is not a temperature-responsive culture vessel. However, in order to maintain the cell morphology, it is desirable to use a support. Of course, a cell sheet may be prepared using a temperature-responsive culture vessel, and the cell sheet may be detached and taken out by lowering the temperature of the vessel. Also in this case, it is desirable to use a support in order to maintain the cell morphology. The support means a membrane material or sheet material that can adhere to the cell sheet and reinforce the strength of the cell sheet, and examples thereof include non-woven fabric, hydrophilic PVDF membrane, nitrocellulose membrane, gauze, and a membrane made of a bioabsorbable material.

[0056] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the embodiments described below.

Example

[0057] 1. Obtaining cells derived from blisters The blister fluid of a patient with dystrophic epidermolysis bullosa was collected and centrifuged at 300 g for 5 minutes. The obtained precipitate was suspended in a medium (Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009) supplemented with penicillin and streptomycin at final concentrations of 100 unit / mL and 100 μg / mL, respectively), seeded in a collagen I-coated 6-well plate, and cultured at 37°C and 5% CO2 to obtain adherent cells. The time required from the collection of the blister fluid to seeding in the medium varied depending on the patient, but was within the range of 18 minutes to 1 hour. Thereafter, the medium was appropriately changed and subcultured to grow to the desired number of cells (the culture progress up to 20 days after the start of culture is shown in Fig. 2). The cells obtained by such a method are also referred to as "cells derived from blisters" hereinafter. Cells at the 3rd passage were used for the following surface marker analysis and differentiation induction experiments, and cells at the 3rd to 4th passages were used for gene introduction. In addition, it was confirmed that equivalent cells derived from blisters can be obtained when using an equal-volume mixed medium of Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009) and MSCGM Mesenchymal Stem Cell Growth Medium (Lonza, PT-3001), or Cellartis MSC Xeno-Free Culture Medium (Takara Bio, Y50200) instead of the above medium. As a preliminary study, the correlation between the time from the collection of the blister fluid from the patient's blister to seeding in the medium and the number of colonies formed on the plate after culture was examined. As a result, a large number of colonies were obtained when seeded within 1 hour after the collection of the blister fluid, and extremely few colonies, or almost no colonies, were obtained when seeded more than 3 hours after the collection.

[0058] 2. Characterization of Blister-Derived Cells a) Surface Marker Analysis (FACS) For the blister-derived cells obtained in 1. above and human bone marrow-derived mesenchymal stem cells (hereinafter also referred to as BM-MSCs) [purchased from PromoCell (Heidelberg, Germany) or Lonza (Basel, Switzerland)], surface marker analysis was performed according to the following procedure: The cells were detached from the plate using Accutase-Solution (PromoCell, C-41310), washed with medium, and then, based on the cell count measurement results, 100,000 cells were aliquoted into two tubes each. The cells were washed once with Flow Cytometry Staining Buffer (1X) (R&D Systems, FC001) and resuspended in 100 μl of Flow Cytometry Staining Buffer (1X). To block Fc receptors, 5 μl of Human TruStain FcX TM (BioLegend, 422301) was added and reacted on ice for 10 minutes. To one tube, 10 μl each of CD73-CFS Mouse IgG2B, CD90-APC Mouse IgG2A, and Negative Marker Cocktail (CD45-PE Mouse IgG1, CD34-PE Mouse IgG1, CD11b-PE Mouse IgG2B, CD79A-PE Mouse IgG1, HLA-DR-PE Mouse IgG1) included in the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems, FMC020) was added, and further Brilliant Violet 421 TM5 μl of anti-human CD105 antibody (BioLegend, 323219) was added and reacted at room temperature in the dark for 30 minutes. For the other tube used as a negative control, the same amount of each isotype control antibody was added and reacted at room temperature in the dark for 30 minutes. The cells were washed once with 2 ml of Flow Cytometry Staining Buffer (1X), resuspended in 300 μl of Flow Cytometry Staining Buffer (1X), and then analyzed with a BD FACSAria (BD). Also, FACS analysis of CD31 was performed according to the following procedure to confirm its expression in blister-derived cells and human BM-MSCs: The cells were detached from the plate using Accutase-Solution (PromoCell, C-41310), washed with the medium, and then 100,000 cells were aliquoted into two tubes based on the cell count measurement results. The cells were washed with PBS containing 2% FBS and resuspended in 100 μl of PBS containing 2% FBS. To block Fc receptors, 5 μl of Human TruStain FcX TM (BioLegend, 422301) was added and reacted on ice for 10 minutes. To one tube, 5 μl (protein amount 0.4 μg) of APC anti-human CD31 antibody (BioLegend, 303116) was added and reacted on ice in the dark for 60 minutes. For the other tube used as a negative control, 2 μl (protein amount 0.4 μg) of APC Mouse IgG1, κ Isotype Ctrl Antibody (BioLegend, 400120) was added and reacted on ice in the dark for 60 minutes. The cells were washed once with 2 ml of PBS containing 2% FBS, resuspended in 300 μl of PBS containing 2% FBS, and analyzed with a BD FACSAria (BD).

[0059] As a result of FACS analysis, both blister-derived cells and BM-MSCs were positive for CD73, CD105, and CD90, and negative for CD45, CD34, CD11b, CD79A, HLA-DR, and CD31 (Figure 3).

[0060] b) Differentiation induction (osteoblasts, adipocytes, and chondrocytes) For the blister-derived cells and BM-MSC obtained in 1. above, differentiation induction into osteoblasts, adipocytes, and chondrocytes was performed under the following conditions. Differentiation induction into osteoblasts: The cells were cultured in a medium containing 0.1 μM Dexamethasone, 0.2 mM Ascorbic acid 2-phosphate, and 10 mM Glycerol 2-phosphate (all numerical values are final concentrations) at 37°C and 5% CO2 for 3 weeks (medium change twice a week) to induce differentiation into osteoblasts. Alkaline phosphatase (ALP) staining was performed using a TRACP & ALP Assay Kit (Takara Bio Inc., MK301) according to the product manual. Differentiation induction into adipocytes: The cells were cultured in a medium containing 1 μM Dexamethasone, 0.5 mM 3-Isobutyl-1-methylanxthine (IBMX), 10 μg / mL Insulin, and 100 μM Indomethacin (all numerical values are final concentrations) at 37°C and 5% CO2 for 3 weeks (medium change twice a week) to induce differentiation into adipocytes. Oil Red O staining of the cells was performed using a LipidTox Assay Kit (Cosmo Bio Co., Ltd., AK09F) according to the product manual. Differentiation induction into chondrocytes: The components of the Human Mesenchymal Stem Cell (hMSC) Chondrogenic Differentiation Medium Bullet Kit(tm) (Lonza, PT-3003) were mixed as instructed to prepare a chondrogenic differentiation induction medium (incomplete medium). Recombinant Human TGF-beta 3 Protein (R&D Systems, 243-B3) was added to this at a final concentration of 10 ng / ml to prepare a chondrogenic differentiation induction medium (complete medium) each time it was used. The third passage cells were detached with Accutase-Solution (PromoCell, C-41310), washed with the medium, and then, based on the cell count results, 250,000 cells were aliquoted into a 15 ml polypropylene conical tube. The cells were washed twice with the chondrogenic differentiation induction medium (incomplete medium), the supernatant was removed, and then they were suspended in 500 μl of the chondrogenic differentiation induction medium (complete medium). They were centrifuged at 150 g for 5 minutes to form a cell pellet, the lid was loosened, and they were left standing in a CO2 incubator (37 °C, 5% CO2), and then the medium (complete medium) was changed every 2 - 3 days. After 3 weeks, the pellet was taken out, fixed with 4% paraformaldehyde, frozen sections were prepared, and proteoglycans derived from chondrocytes were stained by Alcian blue staining. For the human bone marrow-derived mesenchymal stem cells, the same differentiation induction procedure was also performed as a positive control.

[0061] The results of the above differentiation induction experiment are shown in Figure 4. BM-MSC was positive in all of the ALP staining, Oil Red O staining, and Alcian blue staining. The cells derived from the blisters were positive in ALP staining (however, the staining intensity was lower than that of BM-MSC), positive in Oil Red O staining (however, the staining intensity was lower than that of BM-MSC), and positive in Alcian blue staining (however, the staining intensity was lower than that of BM-MSC).

[0062] c) Evaluation of the expression ability and secretion ability of type VII collagen The degree to which blister-derived cells express and secrete type VII collagen was examined by Western blotting. In this experiment, blister-derived cells obtained from patients in whom type VII collagen is expressed but the expressed type VII collagen is considered to be almost non-functional due to amino acid mutations were used. First, human epidermal keratinocytes (hereinafter referred to as KC), human skin fibroblasts (hereinafter referred to as FB), human bone marrow-derived mesenchymal stem cells (hereinafter referred to as MSC), and blister-derived cells from patients with epidermolysis bullosa (hereinafter referred to as BFC; also called blister fluid cell) were each cultured in the medium shown in Table 1 below.

Table 1

[0063] When the cells reached 90 - 95% confluence, they were washed with D-PBS(-), and each medium (without supplements) supplemented with ascorbic acid (Nacalai Tesque, 13048-42, final concentration 50 μg / ml) and protease inhibitor cocktail (SIGMA, P1860-1ML, diluted 1 / 400-fold) was added, followed by culturing in a CO2 incubator for 24 hours. After culturing, the medium was concentrated using the methanol-chloroform precipitation method. Also, cell lysates were prepared from the cells using RIPA buffer (Nacalai Tesque, 08714-04). Each lysate was corrected based on the protein concentration, and electrophoresis samples were prepared using LDS sample buffer and sample reducing agent (Invitrogen, NP0007 and NP0009, respectively). After electrophoresis using a 3-8% NuPAGE gel (Invitrogen, EA0375BOX), the samples were transferred to a PVDF membrane (Millipore, IPVH07850), and antibody reactions were performed using Anti-Col7 (Atlas, HPA042420) as the primary antibody and Anti-Rabbit IgG-HRP (GE helthcare, NA9340-1ML) as the secondary antibody. Then, bands were detected using Chemi-lumi-one ultra (Nacalai Tesque, 11644-40) and Chemi DOC (BioRad, 17001402JA), and analyzed and quantified using Image Lab software (BioRad, 1709690). Western blot was also performed on the concentrated medium in the same manner to quantify the concentration of type VII collagen.

[0064] The results are shown in Fig. 5. As shown in Fig. 5 (upper), as a result of performing Western blotting on the cell lysate, the expression level of COL7A in the cells derived from blisters was higher than that in dermal fibroblasts and bone marrow-derived mesenchymal stem cells, and was comparable to that in epidermal keratinocytes. Also, as shown in Fig. 5 (lower), as a result of performing Western blotting on the concentrate of the medium in which the cells were cultured, the secretion amount of COL7A from the cells derived from blisters was the largest among all the cells. From the above results, it is inferred that the cells derived from blisters are optimal as cells for expressing type VII collagen by gene introduction.

[0065] 3. Design of Genome Editing To select a position with good cleavage efficiency by the CRISPR-Cas9 system in the AAVS1 (Adeno-associated virus integration site 1) region in the human genome, three types of sgRNAs were prepared. The AAVS1 region is a safe harbor that is less affected by gene introduction. Since the CRISPR-Cas9 system recognizes the base sequence of "NGG" and cleaves 3 bases upstream thereof, a region with "GG" arranged at the end was selected, and sgRNAs (sgAAVS1-#1 to #3) containing a target sequence of 20 bases upstream of "NGG" were designed (Fig. 6, upper; Table 2). [Table 2]

[0066] After annealing an oligonucleotide consisting of any of the sequences of SEQ ID NOs: 3 to 5 and its complementary strand, and cloning it into the BbsI site of eSpCas9(1.1) (Addgene plasmid # 71814), plasmids expressing Cas9 protein and sgRNA were created (eSpCas9(1.1)-sgAAVS1-#1, eSpCas9(1.1)-sgAAVS1-#2, eSpCas9(1.1)-sgAAVS1-#3, respectively). This plasmid (2.5 μg) was introduced into HEK293 cells (human fetal kidney cell line) seeded in a 6-well dish using Lipofectamin 3000 (Thermo Fisher Scientific). 48 hours after transfection, genomic DNA was extracted from the cells, and the region containing the target site was amplified by PCR. The PCR amplification fragment was made single-stranded by heat treatment, annealed by slow cooling, and then treated with a mismatch site-specific endonuclease. This was fractionated by electrophoresis, and the degree of insertion or deletion mutation introduced by genomic cleavage was measured by the intensity of the band, and the genomic editing efficiency was calculated by the following formula (where a is the band concentration not digested, and b and c are the band concentrations digested). TIFF2025094280000003.tif15150

[0067] It was confirmed that a short DNA fragment different from the control was generated by any of the sgRNAs of sgAAVS1-#1 to #3, and double-strand breakage occurred (Figure 6, bottom). In the following experiments, sgAAVS1-#3 with the highest cleavage efficiency was used.

[0068] 4. Introduction of COL7A1 Gene into Blister-derived Cells For the introduction of the COL7A1 gene into the AAVS1 region, a plasmid expressing the COL7A1 gene under the control of the CAG promoter was designed (Figure 7). The COL7A1 cDNA was obtained from a Flexi ORF sequence-verified clone (Promega, Madison, WI, USA). The COL7A1 cDNA was subcloned into the pENTR1A plasmid (Thermo Fisher Scientific, A10462) to obtain pENTR1A-COL7A1. The COL7A1 cDNA was introduced from pENTR1A-COL7A1 into pAAVS1-P-CAG-DEST (Addgene plasmid # 80490) by a Gateway reaction using LR recombinase (Thermo Fisher Scientific) to obtain the donor plasmid pAAVS1-P-CAG-COL7A1.

[0069] The blister-derived cells obtained in 1. above were suspended in the dedicated buffer of the Neon transfection system (Thermo Fisher Scientific) and mixed with the Cas9-sgRNA expression plasmid (eSpCas9(1.1)-sgAAVS1-#3) and the donor plasmid (pAAVS1-P-CAG-COL7A1) as follows.

Table 3

[0070] Using the Neon transfection system, the plasmid was introduced into the blister-derived cells by electroporation under the conditions of 1,200 V, 20 ms, and 2 pulses, and then seeded in a 6-well plate and cultured. As the culture medium, an equal-volume mixed medium of Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009) and MSCGM Mesenchymal Stem Cell Growth Medium (Lonza, PT-3001) was used. 48 hours after transfection, puromycin was added to a final concentration of 0.5 μg / mL, and the cells selected after culturing for about 2 weeks were used for the transplantation experiment into mice in the following "5. Transplantation of Genetically Modified Blister-Derived Cells into Mice".

[0071] In addition, a donor plasmid expressing the COL7A1 gene under the control of the PGK promoter was prepared, and this plasmid was introduced into various cells including blister-derived cells. Then, the expression level and secretion amount of COL7A1 in the modified cells were evaluated by Western blotting in the same manner as in "c) Evaluation of the Expression Ability and Secretion Ability of Type VII Collagen" in the above "2. Characterization of Blister-Derived Cells". In this experiment, blister-derived cells obtained from patients not expressing type VII collagen were used. The results are shown in Figure 8. As shown in Figure 8 (upper), more type VII collagen was detected from the lysate of blister-derived cells than from the lysate of fibroblasts. Similarly, as shown in Figure 8 (lower), more type VII collagen was also detected from the medium in which blister-derived cells were cultured than from the medium in which fibroblasts were cultured. From the above results, it was found that when the COL7A gene was introduced into cells by CRISPR-Cas9, blister-derived cells expressed and secreted more type VII collagen than fibroblasts. (It has been confirmed by immunostaining and Western blotting of the culture supernatant that cells expressing and secreting type VII collagen can also be obtained even when blister-derived cells are genetically modified with a COL7A1 gene donor plasmid using the EF1α promoter.)

[0072] 5. Infection Efficiency of Lentivirus on Blister-Derived Cells The infection efficiency of lentivirus on vesicle-derived cells was analyzed. The cells used in this experiment and the medium used for cell culture are shown in Table 4 below.

Table 4

[0073] Preparation of RetroNectin-coated plate: RetroNectin [Takara Bio Inc. (Shiga, Japan), T100B] was diluted with PBS (Dulbecco's Phosphate Buffered Saline (without Ca, Mg)) [Nacalai Tesque Inc. (Kyoto, Japan), 14249-95] to a concentration of 40 μg / mL, and 100 μL / well was added to a 96-well plate without surface treatment [Corning Inc. (Tokyo, Japan), 3370]. Then, the plate was left standing at 4°C overnight. Before using the plate, the RetroNectin solution was removed, and the plate was washed twice with PBS, and the following operations were performed. Seeding of cells and lentivirus infection: Vesicle-derived cells were detached from the plate using Accutase-Solution [PromoCell GmbH (Heidelberg, Germany), C-41310], human bone marrow-derived mesenchymal stem cells were detached using Trypsin / EDTA for Mesenchymal Stem Cells [Lonza Group Ltd. (Basel, Switzerland), CC-3232], and normal adult dermal fibroblasts were detached using Trypsin / EDTA Solution [Lonza Group Ltd. (Basel, Switzerland), CC-5012], and the cells were collected using their respective culture media. The number of cells in the collected cells was measured, and the cells were seeded into the above-mentioned RetroNectin-coated 96-well plate at 2500 cells / well. Then, lentivirus carrying the GFP gene: pLenti-C-mGFP [ORIGENE Technologies Inc. (Rockville, USA), PS100071] was added to each well at an MOI of 1 or MOI of 5, and the cells were cultured in a CO2 incubator for 72 hours. Detection of GFP-positive cells: The detection of the GFP-positive cell rate was performed using a fluorescence microscope [Keyence Corporation (Tokyo, Japan), BZ-X710]. The results are shown in Fig. 9.

[0074] In addition, GFP-positive cells of each cell type infected at an MOI of 1 were quantified by the following method. First, the ratio of the number of GFP-positive cells to the total number of cells in the field of view was defined as the GFP-positive cell rate. This measurement was repeated three times, and statistical analysis was performed (*: P < 0.05, Dunnett's test). The results are shown in Fig. 10.

[0075] As shown in Fig. 9, it was revealed that the GFP-positive cell rate and the fluorescence intensity of GFP were higher in blister-derived cells compared to mesenchymal stem cells and fibroblasts. Furthermore, as shown in Fig. 10, it was clarified that the GFP-positive cell rate of blister-derived cells was significantly higher compared to mesenchymal stem cells and fibroblasts. These results suggest that the gene delivery efficiency by lentivirus is higher in blister-derived cells compared to mesenchymal stem cells and fibroblasts.

[0076] 6. Preparation of a lentiviral vector plasmid carrying the type VII collagen gene A lentiviral vector plasmid equipped with an EF1α promoter and a COL7A1 gene expression cassette as shown in Fig. 11 (left) was prepared. First, COL7A1 cDNA was excised from a Flexi ORF sequence-verified clone (Promega) containing COL7A1 cDNA by SpeI and XbaI treatment that generates paired sequences, and ligated to pLVSIN-EF1α Puro (Takara Bio Inc.) treated with XbaI to produce pLVSIN-EF1α-C7 Puro. This was further treated with NotI and MluI to remove the PGK-Puro cassette, and pLVSIN-EF1α-COL7A1 was produced.

[0077] Furthermore, a lentiviral vector plasmid equipped with a PGK promoter and a COL7A1 gene in an expression cassette as shown in Fig. 11 (right) was prepared. pLVSIN-EF1α-Col7A1 was treated with ClaI and SwaI to excise the EF1α promoter region, and the PGK promoter region (501 bp) amplified by PCR using the AAVS1 hPGK-PuroR-pA donor plasmid (addgene #22072) as a template was incorporated by Gibson assembly to produce pLVSIN-PGK-COL7A1. KOD One (Toyobo Co., Ltd.) was used for the amplification of the PGK promoter. The NEBuilder HiFi DNA Assembly kit (New England Biolabs) was used for Gibson assembly.

[0078] 7. Production of a lentiviral vector carrying the type VII collagen gene A lentiviral vector carrying the COL7A1 gene as shown in Fig. 12 was produced. Transfection using a lentiviral plasmid: The plasmid shown in Fig. 11 was used as a lentiviral vector plasmid. Also, for the packaging plasmid, Lentiviral High Titer Packaging Mix [Takara Bio Inc. (Shiga, Japan), 6194] was used. First, Lenti-X 293T cells [Takara Bio Inc. (Shiga, Japan), 632180], a cell line for lentiviral packaging, were seeded in a 100 mm dish [Corning Inc. (New York, USA), 353003] at 5,000,000 cells / dish and cultured overnight in a CO2 incubator. The medium for Lenti-X 293T cells used was DMEM [Nacalai Tesque Inc. (Kyoto, Japan), 08457-55] containing 10% FBS and penicillin and streptomycin (added to final concentrations of 100 units / mL and 100 μg / mL, respectively). Next, the vector plasmid and the packaging plasmid were transfected using polyethyleneimine. After culturing the transfected cells overnight in a CO2 incubator, the medium was changed. For the transfection reagent, OPTI-MEM [Thermo Fisher Science (Tokyo, Japan), 31985062] and PEI-MAX [Polysciences Inc. (Warrington, USA), 24765-100] were used. The transfection protocol followed the recommended protocol for PEI-MAX. Also, for the mixing ratio of the vector plasmid and the packaging plasmid, the recommended protocol for Lentiviral High Titer Packaging Mix was followed. Recovery and purification of lentiviral vectors: The culture supernatant of Lenti-X 293T cells 72 hours after transfection was collected and centrifuged at 300 g for 5 min to remove cell debris. The supernatant was filtered using a 0.45 μm filter [Merck (Tokyo, Japan), SLHVR33RS] to further remove cell debris. Next, the filtered supernatant was centrifuged (6000 g, 4 °C, 20 hr) to precipitate the lentiviral vector, and the pellet was resuspended in 1.5 mL of PBS. Next, a layer of 55% sucrose / PBS solution (1 mL), 20% sucrose / PBS solution (2.5 mL), and lentiviral vector solution (1.5 mL) was formed in an ultracentrifugation tube [Beckman Coulter (Tokyo, Japan), 344058] and ultracentrifuged (41000 rpm, 4 °C, 2 hr). The ultracentrifuge used was [Beckman Coulter (Tokyo, Japan), L-90K], and the rotor used was [Beckman Coulter (Tokyo, Japan), SW55Ti]. After ultracentrifugation, the layer of lentiviral vector that appeared between the 55% sucrose / PBS solution and the 20% sucrose / PBS solution was collected and diluted to 1 mL using PBS. Next, a layer of 20% sucrose / PBS solution (4 mL) and lentiviral vector solution (1 mL) was formed in the ultracentrifugation tube and ultracentrifuged again (41000 rpm, 4 °C, 2 hr). The pellet of the precipitated lentiviral vector was thoroughly suspended in 400 μL of DMEM to obtain a lentiviral vector solution.

[0079] The titer of the lentiviral vector was measured as follows. Preparation of RetroNectin-coated plate: RetroNectin [Takara Bio Inc. (Shiga, Japan), T100B] was diluted with PBS to 100 μg / mL and added to a flat-bottom 48-well plate without surface treatment [IWAKI (Tokyo, Japan), 1830-048] at 100 μL / well. Then, the plate was left standing at 4 °C overnight. Before using the plate, the RetroNectin solution was removed and blocked with PBS containing 2% FBS at room temperature for 30 min, and the following operations were performed. Cell seeding and lentivirus infection: In a 48-well plate coated with RetroNectin, the LVSIN-EF1α-COL7A1 lentiviral vector or the LVSIN-PGK-COL7A1 lentiviral vector (see Fig. 12) was added to each well in an arbitrary volume of viral solution and centrifuged at 2000 g at 32 °C for 2 hours. Then, the viral solution was removed and the plate was washed once with PBS. Next, the blister-derived cells were detached from the plate using Accutase-Solution and collected using the medium. The number of cells in the collected cells was measured and seeded in the above-mentioned RetroNectin-coated 48-well plate so as to be 12,500 cells / well. The cells on the 14th day after infection were detached from the plate using Accutase-Solution and collected using the medium. Next, Maxwell (登録商標) RSC Instrument [Promega Corporation (Tokyo, Japan), AS4500] and Maxwell (登録商標) Genomic DNA was extracted from the collected cells using the RSC Blood DNA Kit [Promega Corporation (Tokyo, Japan), AS1400]. Lenti-X TM The VCN (Vector Copy Number) was calculated from the recovered genomic DNA using the Provirus Quantitation Kit [Takara Bio Inc. (Shiga, Japan), 631239]. The virus titer (TU / mL, TU: Transduction Unit) was calculated from the VCN and the volume of the viral solution at the time of virus infection. The calculation formula is as follows. TIFF2025094280000006.tif18150 Based on the virus titer thus obtained, the volume of the viral solution required for setting the multiplicity of infection (MOI) in the lentivirus infection experiment was calculated.

[0080] 8. Analysis of the efficiency of type VII collagen gene transfer by immunostaining The efficiency of type VII collagen gene transfer by the lentiviral vector into blister-derived cells was analyzed. Preparation of RetroNectin-coated plate: The plate was coated with RetroNectin in the same manner as described in "11. Production of lentiviral vector carrying type VII collagen gene" above. Seeding of cells and lentiviral infection: The LVSIN-EF1α-COL7A1 lentiviral vector or LVSIN-PGK-COL7A1 lentiviral vector (see Fig. 12) was added to each well of a 48-well plate coated with RetroNectin so that the multiplicity of infection (MOI) was 0.5, 1, or 2, and centrifuged at 2000 g at 32 °C for 2 hours. Then, the viral solution was removed and the plate was washed once with PBS. Next, the cells derived from blisters were detached from the plate using Accutase-Solution and collected using a medium. The number of the collected cells was measured and seeded into the above-described RetroNectin-coated 48-well plate so that the number of cells per well was 12,500. Immunostaining: The cells derived from blisters 14 days after lentiviral infection were detached from the plate using Accutase-Solution and collected using a medium. The number of the collected cells was measured and seeded into a CC2-coated chamber slide [Thermo Fisher Science (Tokyo, Japan), 154852] so that the number of cells per well was 50,000, and cultured in a CO2 incubator. After 24 hours, immunostaining was performed using an anti-type VII collagen antibody (clone LH7.2) [Sigma Aldrich (Tokyo, Japan), C6805] and Alexa488 [Thermo Fisher Science (Tokyo, Japan), A-11001]. Then, the expression of type VII collagen was analyzed using a confocal fluorescence microscope [Nikon (Tokyo, Japan), Nikon A1R HD25].

[0081] The results are shown in Figs. 13 and 14. Expression of type VII collagen increased in an MOI-dependent manner in cells infected with either the EF1α promoter- or PGK promoter-containing lentiviral vectors. The highest percentage of type VII collagen-positive cells was observed in cells infected at an MOI of 2, which was approximately 30% for the LVSIN-EF1α-COL7A1 lentiviral vector and approximately 16% for the LVSIN-PGK-COL7A1 lentiviral vector. Generally, cells derived from blisters infected with the LVSIN-EF1α-COL7A1 lentiviral vector had a higher percentage of type VII collagen-positive cells and stronger staining intensity compared to cells derived from blisters infected with the LVSIN-PGK-COL7A1 lentiviral vector.

[0082] 9. Analysis of the efficiency of type VII collagen gene transfer by flow cytometry (FACS) The efficiency of type VII collagen gene transfer by lentiviral vectors into cells derived from blisters was analyzed. Generation of lentivirus-infected cells: The procedure was the same as that in "8. Analysis of the efficiency of type VII collagen gene transfer by immunostaining" above. FACS analysis: Cells derived from blisters 14 days after lentiviral infection were detached from the plate using Accutase-Solution and collected using medium. The number of collected cells was measured, and aliquots were taken to obtain 300,000 cells / sample. Permeabilization was performed using eBioscience TM Permeabilization Buffer [Thermo Fisher Science (Tokyo, Japan), 00-8333-56]. Subsequently, immunostaining was performed using an anti-type VII collagen antibody (clone LH7.2) [Sigma Aldrich (Tokyo, Japan), C6805] and Alexa488 [Thermo Fisher Science (Tokyo, Japan), A-11001], and the expression of type VII collagen was analyzed using a flow cytometer (BD FACSCanto TM II) [Becton Dickinson Japan (Tokyo, Japan)].

[0083] The results are shown in Fig. 15. Also, the percentage of cells contained in the framed part in each FACS data is shown as the type VII collagen-positive cell rate in Fig. 16 (left). Furthermore, the mean fluorescence intensity (MFI) within the framed region is shown as the mean fluorescence intensity of type VII collagen-positive cells in Fig. 16 (right). As shown in Fig. 15 and Fig. 16 (left), the type VII collagen-positive cell rate increased in an MOI-dependent manner. The highest type VII collagen-positive cell rates were all those infected with an MOI of 2, approximately 18% for the EF1α-COL7A1 lentivirus and approximately 12% for the PGK-COL7A1 lentivirus. Also, as shown in Fig. 16 (right), the MFI of EF1α-COL7A1 lentivirus-infected cells was approximately 2.2 times higher than that of PGK-COL7A1 lentivirus-infected cells. From this, it is considered that in blister-derived cells transfected with a gene using a lentiviral vector, the EF1α promoter expresses a larger amount of COL7A1 than the PGK promoter.

[0084] Also, as a preliminary study, blister-derived cells, human bone marrow-derived mesenchymal stem cells, and normal adult dermal fibroblasts were infected with an EF1α-COL7A1 lentiviral vector or a PGK-COL7A1 lentiviral vector, and immunostaining and flow cytometry (FACS) were performed in the same manner as above to measure the type VII collagen-positive cell rate. As a result, the type VII collagen-positive cell rate was higher in blister-derived cells than in human bone marrow-derived mesenchymal stem cells and normal adult dermal fibroblasts.

[0085] 10. VCN (Vector Copy Number) of Lentivirus-Infected Blister-Derived Cells The VCN of blister-derived cells infected with a lentiviral vector carrying type VII collagen was analyzed. First, blister-derived cells were infected with a lentiviral vector in the same procedure as described in "8. Analysis of the efficiency of type VII collagen gene transfer by immunostaining". Cells on days 7, 14, 21, and 28 after infection were detached from the plate using Accutase-Solution and collected using the medium. Next, Maxwell (登録商標) RSC Instrument [Promega Corporation (Tokyo, Japan), AS4500] and Maxwell (登録商標) RSC Blood DNA Kit [Promega Corporation (Tokyo, Japan), AS1400] were used to extract genomic DNA from the collected cells. Lenti-X TM Provirus Quantitation Kit [Takara Bio Inc. (Shiga, Japan), 631239] was used to calculate VNC from the recovered genomic DNA.

[0086] The results are shown in Figure 17. As shown in the figure, VCN increased in an MOI-dependent manner. Also, VCN decreased with the passage of the number of days after infection, but VCN became stable after 21 days of infection. This result suggests that the type VII collagen gene inserted into the genome of blister-derived cells by the lentiviral vector can persist in the genome for a long period.

[0087] 11. Preparation of cell sheets The blister fluid of a patient with dystrophic epidermolysis bullosa was collected. The obtained blister fluid was seeded onto a collagen I-coated 6-well plate. As the medium, a medium prepared by adding penicillin and streptomycin to an equal-volume mixed medium of Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009) and MSCGM Mesenchymal Stem Cell Growth Medium (Lonza, PT-3001) at final concentrations of 50 units / mL and 50 μg / mL, respectively, was used. By culturing the plate at 37°C and 5% CO2, adherent cells were obtained on the bottom of the plate. Thereafter, the obtained blister-derived cells were appropriately subcultured with medium changes to grow to the desired number of cells.

[0088] Next, the COL7A1 gene was introduced into the blister-derived cells by the same procedure as in the above "4. Introduction of the COL7A1 gene into blister-derived cells". The CAG promoter was used as the promoter. Thereafter, the blister-derived cells into which the gene had been introduced were appropriately subcultured with medium changes to grow to the desired number of cells.

[0089] Thereafter, 0.4×10 6 cells / cm 2 (corresponding to 700% confluence), the gene-introduced blister-derived cells were seeded onto a 12-well plate. As the medium, an equal-volume mixed medium of Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009) and MSCGM Mesenchymal Stem Cell Growth Medium (Lonza, PT-3001) was used. The plate was cultured at 37°C and 5% CO2 for 2 days. As a result, a cell sheet was formed on the bottom of the 12-well plate. A 1×1 cm square cut was made in the cell sheet with a blade, and the cell sheet was peeled off the bottom of the plate with forceps. The cell sheet during peeling is shown in Fig. 18 (upper left). The cell sheet taken out from the plate is shown in Fig. 18 (upper middle).

[0090] Figure 18 (upper right) shows a photograph of hematoxylin-eosin staining (HE staining) of the obtained cell sheet. As shown in the figure, it was confirmed that in the obtained cell sheet, cells overlapped in the thickness direction of the sheet. That is, in the obtained cell sheet, cells formed multiple layers. When observing the HE staining in detail, it was observed that the cell density was high near the surface of the sheet, and in the interior, especially in the central part, the cell density was not as high as that near the surface. The thickness of the obtained cell sheet was 15 to 20 μm.

[0091] As a preliminary study, 0.1×10 6 cells / cm 2 (corresponding to a confluence of 175%) and 0.2×10 6 cells / cm 2 (corresponding to a confluence of 350%), the gene-introduced blister-derived cells were seeded in a 12-well plate. When seeded at 0.2×10 6 cells / cm 2 , a cell sheet was formed, but the mechanical strength was inferior to that when seeded at 0.4×10 6 cells / cm 2 , and the result was that it was slightly more likely to tear. When seeded at 0.1×10 6 cells / cm 2 , a cell sheet was also formed, but the mechanical strength was even more inferior to that when seeded at 0.2×10 6 cells / cm 2 , and the result was that it was easily torn and difficult to handle. Also, after seeding the gene-introduced blister-derived cells in a 12-well plate at 0.4×10 6 cells / cm 2 and culturing for 1 day and 3 days. When cultured for 1 day, a cell sheet was formed, but the mechanical strength was inferior to that when cultured for 2 days, and it was likely to tear. When cultured for 3 days, the strength of the cell sheet was slightly improved compared to when cultured for 2 days.

[0092] 12. Application of the cell sheet to the skin lesion As shown in Fig. 18 (lower left), the cell sheet obtained in "11. Preparation of cell sheet" was transplanted onto the skin lesion of an epidermolysis bullosa model mouse. First, the full-thickness skin of a neonatal Col7Aa1 gene knockout mouse (Col7a1- / -) showing blister formation was excised and grafted onto the back of an immunodeficient mouse (NOD-SCID). Nine to ten days after skin grafting, as shown in Fig. 18 (second from the left in the lower row), an epidermolysis bullosa model mouse with engrafted skin grafts derived from knockout mice was obtained. Next, as shown in Fig. 18 (second from the right in the lower row), incisions were made with a scalpel on three sides of a 1×1 cm square on the epidermis of the lesion, the epidermis was peeled off to expose the dermis. Then, as shown in Fig. 18 (lower right), the cell sheet was attached to the exposed dermis. Finally, the incised epidermis was returned to its original position and fixed with medical tape. Also, as a comparative control, a suspension of blister-derived cells into which the COL7A1 gene was introduced by the same procedure as in "4. Introduction of COL7A1 gene into blister-derived cells" was administered intradermally.

[0093] Four weeks after transplantation of the cell sheet, the skin at the transplantation site was collected and immunostained using an anti-type VII collagen antibody (clone LH7.2; Sigma Aldrich, C6805) to analyze the deposition of type VII collagen on the basement membrane by confocal fluorescence microscopy [Nikon Corporation (Tokyo, Japan), Nikon A1R HD25]. The results are shown in Figs. 19 to 21. Furthermore, seven days after transplantation of the cell sheet, in situ hybridization (ISH) using a probe that does not bind to mouse Col7a1 mRNA but specifically binds to human COL7A1 mRNA, and multiplex staining of immunostaining for human type VII collagen protein were performed according to the TECHNICAL NOTE of RNAscope 登録商標 RED Assay and Immunofluorescence (Advanced Cell Diagnostics). The probe for ISH targets 1510 - 4172 of NM_000094.4 and was designed and manufactured by request to Advanced Cell Diagnostics (Newark, CA). The ISH part of the multiplex staining was RNAscope 登録商標Performed using 2.5 HD Detection Reagents-RED (Advanced Cell Diagnostics, 322360). The immunostaining part was performed using an anti-type VII collagen antibody (Atlas Antibodies, HPA042420). The results are shown in Figs. 22 and 23. Furthermore, 32 days after the transplantation of the cell sheet, the skin at the transplantation site was observed with an electron microscope. Sample preparation for electron microscopy observation by chemical fixation and resin embedding ultra-thin section method, and observation and photography by transmission electron microscope were requested from Tokai Electron Microscopy Analysis Co., Ltd. (Nagoya City, Japan). The results are shown in Fig. 24.

[0094] As shown in Figs. 19 and 20, in the skin transplanted with the cell sheet, deposition of type VII collagen was observed near the basement membrane over a very long distance. On the other hand, in the skin administered intradermally, deposition of type VII collagen was seen in the deep dermis, and almost no deposition of type VII collagen was seen near the basement membrane. When the fluorescence intensity of immunostaining of type VII collagen in the basement membrane of the skin transplanted with the cell sheet was quantified, it was higher than that of normal human skin as shown in Fig. 21.

[0095] As shown in Fig. 22, in the skin transplanted with the cell sheet, human COL7A1 mRNA was detected directly under the epidermis. This suggests that the cells derived from the cell sheet remain near the dermal surface. As shown in Fig. 23, the cells derived from the cell sheet form a thick layer here and there near the dermal surface. In the epidermis, old cells are lost and gradually replaced by new cells. On the other hand, inside the dermis, the cells are known to remain for a long time and maintain the function of the skin. The fact that the transplanted cells remain in the dermis with thickness suggests that these cells may remain in the dermis for a long time and provide drug efficacy over a long period.

[0096] As shown in Fig. 24, in the skin transplanted with the cell sheet, anchoring fibrils were formed near the basement membrane. On the other hand, in the skin without transplantation of the cell sheet, no anchoring fibrils were formed.

[0097] 13. Preparation of cell sheets into which type VII collagen gene has been introduced using a lentiviral vector Using the lentiviral vector produced in "7. Production of lentiviral vector carrying type VII collagen gene", the type VII collagen gene was introduced into the blister-derived cells obtained in "11. Preparation of cell sheets". Specifically, in the same manner as in "8. Analysis of type VII collagen gene transduction efficiency by immunostaining", the LVSIN-EF1α-COL7A1 lentiviral vector or the LVSIN-PGK-COL7A1 lentiviral vector was infected into the blister-derived cells at an MOI of 2. Using the obtained lentivirus-infected blister-derived cells, a cell sheet was prepared in the same manner as in "11. Preparation of cell sheets". Then, in the same manner as in "12. Application of cell sheet to skin lesion", the prepared cell sheet was transplanted onto the skin lesion of the epidermolysis bullosa model mouse.

[0098] Four weeks after transplantation, the skin at the transplantation site was collected and immunostained using an anti-type VII collagen antibody (clone LH7.2; Sigma Aldrich, C6805) to examine the deposition of type VII collagen on the basement membrane. The results are shown in Figures 25 and 26. As shown in Figure 25, deposition of type VII collagen was observed along the basement membrane even in the blister-derived cell sheet transfected with the LVSIN-PGK-COL7A1 lentiviral vector. As shown in the left panels of Figures 25 and 26, in the blister-derived cell sheet transfected with the LVSIN-EF1α-COL7A1 lentiviral vector, deposition of type VII collagen was observed along the basement membrane over a very long distance. In addition, as shown in the right panels of Figures 25 and 26, the fluorescence intensity of the immunostaining for type VII collagen was also high in the blister-derived cell sheet transfected with the LVSIN-EF1α-COL7A1 lentiviral vector.

Claims

**Claim 1** A cell sheet, which is derived from the body fluid in the blisters of a patient with dystrophic epidermolysis bullosa, contains cells into which a type VII collagen gene has been introduced, and is transplanted onto the skin of a patient with dystrophic epidermolysis bullosa. **Claim 2** A type VII collagen gene expression cassette has been introduced into the cells, The cell sheet according to claim 1, wherein the type VII collagen gene expression cassette contains an EF1α promoter and a COL7A1 gene arranged downstream of the EF1α promoter. **Claim 3** The cell sheet according to claim 1, wherein the cells overlap in the thickness direction of the sheet. **Claim 4** The cell sheet according to claim 3, wherein in the longitudinal section of the cell sheet, the cell density near the surface of the sheet is higher than the cell density at the center of the sheet. **Claim 5** The cell sheet according to claim 1, which is transplanted onto a portion where the epidermis is damaged. **Claim 6** A method for producing a cell sheet to be transplanted onto the skin of a patient with dystrophic epidermolysis bullosa, comprising: seeding at least a part of the body fluid in the blisters of a patient with dystrophic epidermolysis bullosa into a culture medium; obtaining cells adhered to the bottom of a culture vessel; introducing a type VII collagen gene into the cells; culturing the cells into which the type VII collagen gene has been introduced; taking out the cultured cells from the culture vessel as a cell sheet; The method for producing a cell sheet, characterized by comprising the above steps. **Claim 7** The method for producing a cell sheet according to claim 6, wherein the cells obtained from the body fluid in the blisters are seeded into the culture medium without enzyme treatment. **Claim 8** The method for producing a cell sheet according to claim 6, wherein a type VII collagen gene is introduced into the cells using a lentiviral vector. **Claim 9** The method for producing a cell sheet according to claim 6, further comprising a step of seeding the cells into which the type VII collagen gene has been introduced so as to be over-confluent. **Claim 10** 1x10 4 cells / cm 2 The method for producing a cell sheet according to claim 6, further comprising the step of seeding a culture vessel with cells into which a type VII collagen gene has been introduced at the above cell density. **Claim 11** The method for producing a cell sheet according to claim 6, wherein the cultured cells are taken out from the culture vessel as a cell sheet without enzyme treatment.

Images