WO2022018884A1 - 栄養障害型表皮水疱症の治療薬 - Google Patents

栄養障害型表皮水疱症の治療薬 Download PDF

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WO2022018884A1
WO2022018884A1 PCT/JP2020/044096 JP2020044096W WO2022018884A1 WO 2022018884 A1 WO2022018884 A1 WO 2022018884A1 JP 2020044096 W JP2020044096 W JP 2020044096W WO 2022018884 A1 WO2022018884 A1 WO 2022018884A1
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cells
ability
acid sequence
blister
derived
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French (fr)
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克人 玉井
康 菊池
智樹 玉越
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University of Osaka NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to a therapeutic agent for nutritionally impaired epidermolysis bullosa.
  • Epidermolysis bullosa is a disease in which the epidermis peels off from the dermis and causes blisters (blisters) and skin ulcers when force is applied to the skin due to the loss or disappearance of the adhesive structural molecules responsible for the adhesion of skin tissue. be.
  • the type in which the epidermis is torn and blisters are formed is simple epidermolysis bullosa
  • the type in which blisters are formed by peeling between the epidermis and the dermis is junctional epidermolysis bullosa, and the part between the epidermis and the dermis is peeled off.
  • the type of disease is called epidermolysis bullosa.
  • Nutritional disorder type epidermolysis bullosa is the most common type of epidermolysis bullosa, accounting for about 50% of all epidermolysis bullosa, and is a hereditary disease caused by a mutation in the COL7A1 gene that encodes type VII collagen.
  • the epidermal basal cells at the bottom of the epidermis are bound to a sheet-like structure called the basement membrane.
  • Type VII collagen forms a fiber called anchoring fibril in the dermis and connects the basement membrane and the dermis.
  • Skin sheets are difficult to manufacture because they require advanced process control and culture technology, and are expensive, so there is a demand for therapeutic agents that are easier to manufacture.
  • the present disclosure is a composition for use in the treatment of dystrophic epidermolysis bullosa, comprising blistering-derived cells of a dystrophic epidermolysis bullosa patient genetically modified to produce type VII collagen. Regarding the composition.
  • the present disclosure provides a therapeutic agent for nutritionally impaired epidermolysis bullosa.
  • FIG. 1 is a photograph showing the appearance of blister-derived cells up to 20 days after the start of culture.
  • FIG. 2 shows the results of FACS analysis of blister-derived cells and human bone marrow-derived mesenchymal stem cells (BM-MSC).
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • FIG. 3 blisters-derived cells and human BM-MSC are cultured under conditions for inducing differentiation into osteoblasts, adipocytes, and chondrocytes, and are stained with alkaline phosphatase (ALP), oil red O, and alcian blue, respectively. The result of the above is shown.
  • FIG. 4 shows the cleavage of genomic DNA by the designed sgRNA (sgAAVS1- # 1 to # 3) and its cleavage efficiency.
  • FIG. 1 is a photograph showing the appearance of blister-derived cells up to 20 days after the start of culture.
  • FIG. 2 shows the results of FACS analysis of blister-derived cells and human bone
  • FIG. 5 is an explanatory diagram of genome editing in which the COL7A1 gene is introduced into the AAVS1 region.
  • HA-R and HA-L indicate the homologous sequence part
  • SA indicates the splice acceptor sequence
  • T2A indicates the T2A sequence encoding the T2A peptide
  • Puro indicates the puromycin resistance gene
  • CAG indicates the CAG promoter sequence.
  • the length from F2 to R2 in the wild-type genome (top) is 1952 bp
  • the length from F1 to R1 in the genome into which the COL7A1 gene is introduced (bottom) is 1246 bp
  • the length from F2 to R2 is 14249 bp. be.
  • FIG. 6 is an explanatory diagram of the production of an epidermolysis bullosa model mouse.
  • the photo on the right shows the formed blisters.
  • FIG. 7 shows a skin tomographic image of an epidermolysis bullosa model mouse in which blister-derived cells are injected into the blister.
  • the photo on the left shows the results of immunostaining for type VII collagen
  • the photo on the right shows the results of immunostaining for DAPI staining and type VII collagen.
  • Control shows the results of mice injected with unmodified blister-derived cells
  • CAG-hCOL7 shows the results of mice injected with COL7A1 gene-introduced blister-derived cells.
  • Dystrophic Epidermolysis Bullosa is a hereditary disease caused by a mutation in the COL7A1 gene that encodes type VII collagen, and type VII collagen is not produced at all or its function is impaired by the mutation. It is known that type VII collagen is produced as its characteristic. Type VII collagen forms a fiber called anchoring fibril in the dermis and connects the basement membrane and the dermis. Type VII collagen contains a first non-collagen region, a collagen region, and a second non-collagen region from the N-terminal, and forms a triple chain at the collagen region portion characterized by a repeating sequence of glycine-XY, forming a triple chain and a C-terminal.
  • Mutations include mutations in which glycine in the collagen region is replaced with other amino acids, stop codon mutations that stop protein translation, and splice site mutations.
  • the mutation may be in one of the alleles or in both.
  • the malnourished epidermolysis bullosa includes a dominant malnutrition type and a recessive malnutrition type, and the recessive malnutrition type includes a severe generalized type and other generalized types with relatively mild symptoms.
  • the malnutrition-type epidermolysis bullosa in the present specification may be any type of malnutrition-type epidermolysis bullosa, and may be caused by a mutation in any COL7A1 gene.
  • the blister-derived cells of a dystrophic epidermolysis bullosa patient refer to adherent cells collected from within the blister of a dystrophic epidermolysis bullosa patient, and in the present disclosure, "DEB patient blister-derived cells” or Also called “blister-derived cells”.
  • the cells can be obtained by culturing the blister contents of a nutritionally impaired epidermolysis bullosa patient on a solid phase.
  • the blister contents can be collected from the blisters of a nutritionally impaired epidermolysis bullosa patient by means such as a syringe.
  • the solid phase means a solid support to which cells can adhere, and includes, for example, a culture vessel made of plastic or glass, such as a culture dish, a flask, or a multi-well plate.
  • the solid phase is a plastic culture vessel.
  • the solid phase may be coated, and examples of the coating substance include collagen I, laminin, vitronectin, fibronectin, poly-L-lysine, and poly-L-ornithine.
  • the solid phase is coated with collagen I. Culturing can be carried out in a general incubator under conditions such as "37 ° C, 5% CO 2 ", "37 ° C, 5% O 2 , 5% CO 2".
  • the culture medium may be any medium that can be used for culturing animal cells, for example, MEM, MEM ⁇ , DMEM, GMEM, RPMI 1640, MesenCult TM (STEMCELL Technologies), Messengerle Stem Cell Growth Medium 2 (PromoCell). , MSCGM Mesenchymal Stem Cell Growth Medium (Lonza), Cellartis MSC Xeno-Free Culture Medium (Takara Bio), and a mixed medium thereof.
  • the culture period may be long enough for the cells to adhere to the solid phase, eg, 1 day to several months (eg, 2, 3 or 4 months), 1 day to 1 month, 1 day. It can be from several weeks (eg, two, three or four weeks), one day to one week.
  • blister-derived cells of DEB patients have one or more characteristics selected from the following 1) -6): 1) Adhesive to the 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) It does not have the ability to differentiate into osteoblasts, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells. 5) It does not have the ability to differentiate into adipocytes, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells.
  • chondrocytes It does not have the ability to differentiate into chondrocytes, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells.
  • a cell "does not have the ability to differentiate" into osteoblasts, fat cells or chondrocytes means osteoblasts, fat cells or or using normal differentiation induction conditions and detection methods (staining, etc.). It means that differentiation into cartilage cells cannot be detected.
  • the DEB patient blister-derived cells are CD73-positive, CD105-positive, and CD90-positive cells.
  • DEB patient blister-derived cells are CD45-negative, CD34-negative, CD11b-negative, CD79A-negative, HLA-DR-negative, and CD31-negative cells.
  • DEB patient blisters are cells that are less capable of differentiating into osteoblasts, adipocytes, and chondrocytes than bone marrow-derived mesenchymal stem cells. be.
  • DEB patient blisters-derived cells have a lower ability to differentiate into osteoblasts and adipocytes than bone marrow-derived mesenchymal stem cells, and have no ability to differentiate into chondrocytes. It is a cell.
  • blister-derived cells of a nutritionally impaired epidermolysis bullosa patient genetically modified to produce type VII collagen are used.
  • “cells genetically modified to produce type VII collagen” means cells genetically modified to produce functional (ie, capable of forming anchoring fibrils) type VII collagen.
  • the gene modification of a cell means both the modification of a gene in the genome of the cell and the modification of the cell to express the gene from an extragenome nucleic acid construct (for example, a vector). That is, the expression “gene-modify to produce type VII collagen” refers to modifying cells to express type VII collagen from the COL7A1 gene in the genome, and to VII from the COL7A1 gene of the extragenomic nucleic acid construct. It involves modifying cells to express type collagen.
  • “cells genetically modified to produce type VII collagen” include cells that express type VII collagen from the COL7A1 gene in the genome and cells that express type VII collagen from the COL7A1 gene, which is a nucleic acid construct outside the genome. And are included.
  • Gene modification of cells can be performed by introducing the COL7A1 gene or by modifying the mutation of the COL7A1 gene in the genome.
  • the introduction of the COL7A1 gene can be performed either by introducing the COL7A1 gene into the cell genome or by allowing a nucleic acid construct containing the COL7A1 gene to be present in the cell so that the COL7A1 gene is expressed from an extragenome nucleic acid construct. Can be done.
  • the COL7A1 gene is introduced into the genome of a cell, it may be introduced at a specific position or may be introduced at random.
  • the COL7A1 gene is introduced into the COL7A1 locus of the genome, or a safe harbor such as the AAVS1 region.
  • DEB patient blister-derived cells are cells of the dystrophic epidermolysis bullosa patient (ie, autologous cells) to which the cells are administered, but are different from those of the patient receiving the cells. It may be a patient's cell (ie, an allogeneic cell).
  • the cells of the epidermolysis bullosa patient with malnutrition include cells that do not produce type VII collagen and cells that produce type VII collagen but whose function is reduced due to mutation.
  • the "cells of a patient with epidermolysis bullosa" may be any of them.
  • the DEB patient blister-derived cells may be cells that can produce type VII collagen in the vicinity of the epidermal basement membrane when administered to the patient.
  • cells are used in the sense of including those grown as needed.
  • Cell proliferation can be performed by culturing the cells.
  • blister-derived cells in patients with dystrophy-type epidermolysis bullosa
  • gene-modified cells are cells obtained by genetic modification. Includes those that have been propagated.
  • genetically modified cells may be grown until the amount required for the genetic modification is obtained. Also, after genetic modification, cells may be grown until the amount required for treatment is obtained.
  • the term "cell” can mean one cell or a plurality of cells depending on the context. Further, the cell may be a cell population consisting of one type of cell, or may be a cell population including a plurality of types of cells.
  • the COL7A1 gene means a nucleic acid sequence encoding type VII collagen, and is used to include cDNA as well as a sequence containing one or more introns (eg, a genomic sequence or a minigene).
  • 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 encode functional type VII collagen (ie, capable of forming anchoring fibrils), and its sequence is not limited.
  • CDNA sequence of human COL7A1 gene (8835 bp) (SEQ ID NO: 1) Amino acid sequence of human type VII collagen (2944 AA) (SEQ ID NO: 2) *
  • the COL7A1 gene is a nucleic acid having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the nucleic acid sequence of SEQ ID NO: 1. Contains or consists of the nucleic acid sequence. In another embodiment, the COL7A1 gene is 1-30, 1-20, 1-10, 1-5, 1-3, 1-2 or 1 in the nucleic acid sequence of SEQ ID NO: 1. The base contains or consists of the nucleic acid sequence inserted, deleted, substituted, or added. In a further embodiment, the COL7A1 gene comprises or consists of the nucleic acid sequence of SEQ ID NO: 1.
  • type VII collagen has 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2. Contains or consists of the amino acid sequence. In another embodiment, type VII collagen is 1-30, 1-20, 1-10, 1-5, 1-3, 1-2 or 1 in the amino acid sequence of SEQ ID NO: 2. Contains or consists of the amino acid sequence of which the amino acid residue of is inserted, deleted, substituted, or added. In a further embodiment, type VII collagen comprises or consists of the amino acid sequence of SEQ ID NO: 2.
  • the COL7A1 gene is an amino acid having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2. Contains or consists of the nucleic acid sequence encoding the sequence. In another embodiment, the COL7A1 gene is 1-30, 1-20, 1-10, 1-5, 1-3, 1-2 or 1 in the amino acid sequence of SEQ ID NO: 2. The amino acid residue comprises or consists of a nucleic acid sequence encoding an inserted, deleted, substituted, or added amino acid sequence.
  • sequence identity with respect to a nucleic acid sequence or amino acid sequence coincides between two sequences that are optimally aligned (maximum match) over the entire region of the sequence to be compared. Means the proportion of base or amino acid residues.
  • sequence to be compared may have insertions, additions or deletions (eg, gaps, etc.) in the optimal alignment of the two sequences.
  • Sequence identity can be calculated using programs such as FASTA, BLAST, and CLUSTAL W provided in public databases (eg, DDBJ (http://www.ddbj.nig.ac.jp)). Alternatively, it can be obtained by using commercially available sequence analysis software (for example, Vector NTI (registered trademark) software, GENETYX (registered trademark) ver. 12).
  • cells are genetically modified by genome editing such as CRISPR systems (eg, CRISPR / Cas9, CRISPR / Cpf1), TALENs, ZFNs.
  • CRISPR systems eg, CRISPR / Cas9, CRISPR / Cpf1
  • TALENs TALENs
  • ZFNs ZFNs.
  • the cell is genetically modified by a viral vector such as a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector.
  • the cells are genetically modified by CRISPR / Cas9.
  • the cells are genetically modified with a retroviral vector or a lentiviral vector.
  • the sequence can be inserted into the cleavage site of the genome by causing cleavage in the genome and introducing a donor vector containing the target sequence into the cell.
  • the sequence to be inserted into the genome can be the COL7A1 gene, or a sequence for replacement with a site containing a mutation in the COL7A1 gene (eg, a partial sequence of the COL7A1 gene).
  • the donor vector may contain regulatory sequences such as promoters and enhancers that control the expression of the sequence of interest, as well as other elements such as drug resistance genes for cell selection, at both ends of the genome. It may contain homologous sequences at both ends of the insertion site.
  • the donor vector can be introduced at the desired site by non-homologous end binding or homologous recombination.
  • a viral vector such as a plasmid, an adeno-associated virus vector, or an integrase-deficient lentivirus vector can be used.
  • endonucleases such as Cas9 or Cas12 (eg, Cas12a (also known as Cpf1), Cas12b, Cas12e) recognize the PAM sequence, which is a specific base sequence, and the action of the endonuclease double-strands the target DNA. To disconnect. If the endonuclease is Cas9, it cleaves about 3-4 bases upstream of the PAM sequence.
  • endonucleases include S. pyogenes, S. aureus, N. meningitidis, S. thermophilus, or T. denticola Cas9, L. bacterium ND2006 or Acidaminococcus sp. BV3L6 Cpfl.
  • the PAM sequence is endonuclease dependent, for example, the PAM sequence of Cas9 in S. pyogenes is NGG.
  • the gRNA contains a sequence (target sequence) of about 20 bases upstream of the PAM sequence or a sequence complementary thereto on the 5'end side, and plays a role of recruiting endonucleases to the target sequence.
  • the sequence of the portion of the gRNA other than the target sequence (or a sequence complementary thereto) can be appropriately determined by those skilled in the art depending on the endonuclease used.
  • gRNA is a crRNA (CRISPRRNA) that contains a target sequence or a sequence complementary to it and is responsible for the sequence specificity of gRNA, and a tracrRNA (Trans-activating crRNA) that forms a double strand and contributes to the formation of a complex with Cas9. And can be included.
  • crRNA and tracrRNA may exist as different molecules.
  • the endonuclease is Cpf1
  • crRNA alone functions as a gRNA.
  • a gRNA containing an element necessary for the function of the gRNA on a single strand may be particularly referred to as sgRNA.
  • the gRNA sequence can be determined by tools available for target sequence selection and gRNA design, such as CRISPRdirect (https://crispr.dbcls.jp/).
  • a vector containing a nucleic acid sequence encoding a gRNA and a nucleic acid sequence encoding an endonuclease may be introduced and expressed in the cell, or an extracellularly prepared gRNA and an endonuclease protein may be introduced into the cell. ..
  • the endonuclease may include 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.
  • Vectors, gRNAs, and endonucleases can be introduced into cells by lipofection, electroporation, microinjection, calcium phosphate method, DEAE-dextran method, etc., but are not limited to these methods.
  • the gRNA that can be used to introduce the COL7A1 gene into the genome comprises any of the sequences of SEQ ID NOs: 3-5 or a sequence complementary thereto.
  • the COL7A1 gene can be introduced into the cell genome by using a retroviral vector or a lentiviral vector having integrase activity.
  • the retroviral vector and the lentiviral vector may be integrase-deficient. Integrase-deficient vectors lack integrase activity, for example, due to mutations in the integrase gene.
  • an integrase-deficient vector, an adenovirus vector, or an adeno-associated virus vector is used, the sequence integrated into the vector is usually not introduced into the cell genome.
  • type VII collagen is expressed from the COL7A1 gene of the vector existing in the cell (intranuclear).
  • the viral vector contains a sequence encoding the COL7A1 gene, and may contain regulatory sequences such as promoters and enhancers that control the expression of the COL7A1 gene, and other elements such as drug resistance genes for cell selection.
  • the viral vector may be prepared by any method known in the art.
  • retroviral and lentiviral vectors are viral vector plasmids containing LTR sequences (5'LTR and 3'LTR) at both ends, packaging signals, and sequences of interest, viral structures such as Gag, Pol, Env. It can be made by introducing into packaging cells with one or more plasmid vectors expressing the proteins, or by introducing into packaging cells expressing these structural proteins.
  • the packaging cells include, but are not limited to, 293T cells, 293 cells, HeLa cells, COS1 cells, COS7 cells, and the like.
  • the viral vector may be pseudotyped and may express an enveloped protein such as, for example, the vesicular stomatitis virus G protein (VSV-G).
  • VSV-G vesicular stomatitis virus G protein
  • the viral vector is a lentiviral vector.
  • Lentivirus vectors include HIV (human immunodeficiency virus) (for example, 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), but are not limited to these.
  • the lentiviral vector is HIV.
  • the lentiviral vector can be produced as follows. First, a viral vector plasmid encoding the viral genome and one or more plasmid vectors expressing Gag, Pol, and Rev (and optionally Tat) and one or more plasmid vectors expressing enveloped proteins such as VSV-G. , Introduced into packaging cells. Viral vector plasmids are LTR sequences at both ends (5'LTR and 3'LTR), packaging signals, and promoters that control the COL7A1 gene and its expression (eg, CMV promoter, CAG promoter, EF1 ⁇ promoter, PGK promoter, or hCEF). Promoter) is included.
  • LTR sequences at both ends 5'LTR and 3'LTR
  • promoters that control the COL7A1 gene and its expression (eg, CMV promoter, CAG promoter, EF1 ⁇ promoter, PGK promoter, or hCEF). Promoter) is included.
  • the 5'LTR functions as a promoter that induces transcription of the viral RNA genome, it may be replaced with another promoter such as the CMV promoter in order to enhance the expression of the RNA genome.
  • the viral RNA genome is transcribed from the vector plasmid and packaged to form the viral core.
  • the virus core is transported to the cell membrane of the packaging cell, encapsulated in the cell membrane, and released as virus particles from the packaging cell.
  • the released virus particles can be recovered from the culture supernatant of the packaging cells.
  • virus particles can be recovered by conventional purification methods such as centrifugation, filter filtration, column purification and the like.
  • lentiviral vectors can be manufactured using kits such as Lentiviral High Titer Packaging Mix, Lenti-X TM Packaging Single Shots (Takara Bio Inc.), and ViraSafe TM Lentivirus Complete Expression System (Cell Biolabs Inc.). can.
  • Adeno-associated virus vectors can be prepared using a kit such as AAVpro (R) Helper Free System (Takara Bio Inc. ).
  • the cells into which the target sequence has been introduced can be confirmed by Southern blotting or PCR.
  • the sequence of interest may be introduced into at least one of the alleles.
  • DEB patient blister-derived cells are the most abundant cells contained in the composition.
  • DEB patient blister-derived cells make up 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98 or 99% or more of the cells contained in the composition.
  • the compositions of the present disclosure are substantially free of cells other than DEB patient blister-derived cells. "Substantially free of cells other than DEB patient blister-derived cells” means that only cells obtained by substantially the same method as the method for obtaining DEB patient blister-derived cells described herein are included. means.
  • the number of cells contained in the composition is an amount required to exert the desired effect (also referred to herein as an effective amount), and those skilled in the art will appreciate the age, weight, and medical condition of the patient, as well as the type of cells. It can be appropriately determined in consideration of factors such as the gene modification method.
  • the number of cells is not limited, but is, for example, 1 cell to 1 ⁇ 10 7 cells, 1 ⁇ 10 cells to 1 ⁇ 10 7 cells, 1 ⁇ 10 2 cells to 1 ⁇ 10 7 cells, 1 ⁇ 10 3 cells to 1 ⁇ .
  • composition may include pharmaceutically acceptable vehicles and / or additives in addition to the cells.
  • Pharmaceutically acceptable vehicles include water, medium, saline, glucose, infusions containing D-sorbitol, D-mannitol and the like, phosphate buffered saline (PBS) and the like.
  • PBS phosphate buffered saline
  • the additive include a solubilizing agent, a stabilizer, a preservative and the like.
  • the dosage form of the composition is not particularly limited, but is a parenteral administration preparation, for example, an injection.
  • Injections include solution injections, suspension injections, emulsion injections, and time-prepared injections.
  • the composition may be frozen or may contain cryoprotectants such as DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, dextran, sucrose and the like.
  • compositions of the present disclosure may be administered systemically or topically.
  • the composition is administered to the affected area of a nutritionally impaired epidermolysis bullosa patient.
  • the affected area means a blister or an area in the vicinity thereof.
  • the composition is administered intracutaneously or intrablister of the blister portion.
  • the composition is administered intrablister.
  • administration in the blister means administration in the space under the epidermis of the blister portion. Intrablister administration can reduce patient distress as compared to intradermal or subcutaneous administration, and type VII collagen can be well expressed near the basement membrane.
  • the number of cells administered per site is an amount (effective amount) necessary for exerting the desired effect, and those skilled in the art will be able to determine the patient's age, weight, and medical condition, as well as the cell type and gene modification method. It can be determined as appropriate in consideration of the factors of.
  • the number of cells is not limited, but for example, 1 cell to 1 ⁇ 10 7 cells, 1 ⁇ 10 cells to 1 ⁇ 10 7 cells, 1 ⁇ 10 2 cells to 1 ⁇ 10 7 cells, 1 ⁇ 10 3 cells to 1 ⁇ 10 7 cells, 1 ⁇ 10 4 cells to 1 ⁇ 10 7 cells, 1 ⁇ 10 5 cells to 1 ⁇ 10 7 cells, 1 ⁇ 10 5 cells to 5 ⁇ 10 6 cells, 5 ⁇ 10 5 cells to 1 ⁇ 10 6 cells, or 1 x 10 5 cells to 1 x 10 6 cells.
  • the dose per blister may be a standard blister having a diameter of 7 to 8 mm when the blister is approximately circularly approximated, and the above dose may be adjusted according to the size thereof.
  • a composition for use in the treatment of dystrophic epidermolysis bullosa comprising blister-derived cells of a dystrophic epidermolysis bullosa patient genetically modified to produce type VII collagen.
  • the composition according to 1 above, wherein the blister-derived cells have been genetically modified by introducing the COL7A1 gene.
  • the COL7A1 gene contains a nucleic acid sequence having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the nucleic acid sequence of SEQ ID NO: 1 Containing a nucleic acid sequence encoding an amino acid sequence having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2.
  • composition according to any one of 1 to 4 above, wherein the blister-derived cells have one or more characteristics selected from the following 1) to 6): 1) Adhesive to the 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) It does not have the ability to differentiate into osteoblasts, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells. 5) It does not have the ability to differentiate into adipocytes, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells.
  • the composition according to any one of 1 to 10 above, wherein the blister-derived cells have been genetically modified by genome editing.
  • composition according to 11 above wherein the genome editing is performed by CRISPR / Cas9.
  • composition according to any one of 1 to 10 above, wherein the blister-derived cells are genetically modified by a viral vector.
  • the viral vector is a retrovirus vector or a lentiviral vector.
  • the viral vector is a lentiviral vector.
  • a method for producing a composition for use in the treatment of nutritionally impaired epidermolysis bullosa comprising genetically modifying a blister-derived cell of a dystrophic epidermolysis bullosa patient to produce type VII collagen, and preparing a composition comprising the genetically modified blister-derived cell.
  • a method for treating dystrophic epidermolysis bullosa wherein a composition containing blister-derived cells of a dystrophic epidermolysis bullosa patient genetically modified to produce type VII collagen is administered to the patient. How to include.
  • [18] 17 The method according to 17 above, wherein the blister-derived cells have been genetically modified by introducing the COL7A1 gene. [19] 18.
  • the method according to 18 above wherein the COL7A1 gene has been introduced into the genome of a blister-derived cell.
  • the COL7A1 gene contains a nucleic acid sequence having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the nucleic acid sequence of SEQ ID NO: 1 Containing a nucleic acid sequence encoding an amino acid sequence having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2.
  • the method of any of 16 and 26-29 above, wherein the blister-derived cells are genetically modified by genome editing.
  • the viral vector is a lentiviral vector.
  • composition comprising blister-derived cells of a dystrophy-type epidermolysis bullosa patient genetically modified to produce type VII collagen for the manufacture of a pharmaceutical for the treatment of dystrophic epidermolysis bullosa.
  • 41 The use according to 41, wherein the composition is administered intrablister.
  • a gRNA comprising any of the sequences of SEQ ID NOs: 3 to 5 or a sequence complementary thereto.
  • a method for producing cells which comprises a step of culturing the contents of blisters of a patient with epidermolysis bullosa with malnutrition on a solid phase.
  • the cell according to 50 which has one or more characteristics selected from 1) to 6) below: 1) Adhesive to the solid phase, 2) One or more surface markers selected from the group consisting of CD73, CD105 and CD90 are positive.
  • One or more surface markers selected from the group consisting of CD45, CD34, CD11b, CD79A, HLA-DR and CD31 are negative, 4) It does not have the ability to differentiate into osteoblasts, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells. 5) It does not have the ability to differentiate into adipocytes, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells. 6) It does not have the ability to differentiate into chondrocytes, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells.
  • [52] Cell production method including the following steps: 1) The process of culturing the contents of blisters in patients with epidermolysis bullosa with malnutrition on a solid phase, 2) Culture step A step of genetically modifying the cells obtained in 1) to produce type VII collagen.
  • the cell according to 53 or 54 above which has been genetically modified by introducing the COL7A1 gene.
  • the COL7A1 gene contains a nucleic acid sequence having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the nucleic acid sequence of SEQ ID NO: 1 Containing a nucleic acid sequence encoding an amino acid sequence having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2.
  • the cell according to 56 [58] The cell according to any one of 53 to 57 above, which has been genetically modified by genome editing. [59] The cell according to 58 above, wherein the genome editing was performed by CRISPR / Cas9.
  • One or more surface markers selected from the group consisting of CD45, CD34, CD11b, CD79A, HLA-DR and CD31 are negative, 4) It does not have the ability to differentiate into osteoblasts, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells. 5) It does not have the ability to differentiate into adipocytes, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells. 6) It does not have the ability to differentiate into chondrocytes, or its differentiation ability is lower than that of bone marrow-derived mesenchymal stem cells.
  • the cells obtained by such a method are also referred to as "blister-derived cells” below.
  • the following surface marker analysis and differentiation induction experiments used cells of the 3rd passage, and cells of the 3rd to 4th passages were used for gene transfer.
  • an equal amount 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 It was confirmed that equivalent blisters-derived cells could be obtained even when Medium (Takara Bio Inc., Y50200) was used.
  • Blister-derived cell characterization a) Surface marker analysis (FACS) Regarding the blisters-derived cells obtained in 1. above and human bone marrow-derived mesenchymal stem cells (hereinafter, also referred to as BM-MSC) [purchased from PromoCell (Heidelberg, Germany) or Lonza (Basel, Switzerland)], the following. Surface marker analysis was performed according to the procedure of: Cells were peeled from the plate using Accutase-Solution (PromoCell, C-41310), washed with medium, and then placed in two tubes based on the cell count measurement results. 100,000 cells were separated.
  • FACS Surface marker analysis
  • the cells were washed once with 2 ml of Flow Cytometry Staining Buffer (1X), resuspended in 300 ⁇ l of Flow Cytometry Staining Buffer (1X), and then analyzed with BD FACSAria (BD). FACS analysis was also performed on CD31 according to the following procedure to confirm the presence or absence of expression in blister-derived cells and human BM-MSC: cells were peeled from the plate using Accutase-Solution (PromoCell, C-41310), and the medium was used. After washing with, 100,000 cells were separated into two tubes based on the measurement result of the cell number.
  • BD FACSAria BD FACSAria
  • both blister-derived cells and BM-MSC were positive for CD73, CD105 and CD90, and negative for CD45, CD34, CD11b, CD79A, HLA-DR and CD31 (Fig. 2).
  • Induction of differentiation (osteoblasts, adipocytes and chondrocytes)
  • the blisters-derived cells and BM-MSC obtained in 1. above were induced to differentiate into osteoblasts, adipocytes and chondrocytes under the following conditions.
  • Induction of differentiation into osteoblasts Cells are cultured in medium containing 0.1 ⁇ M Dexamethasone, 0.2 mM Ascorbic acid 2-phosphate, 10 mM Glycerol 2-phosphate (all numerical values are final concentrations) at 37 ° C. and 5% CO 2 for 3 weeks (2 weeks). (Culture exchange) was performed to induce differentiation into osteoblasts.
  • Alkaline phosphatase (ALP) staining was performed using the TRACP & ALP Assay Kit (Takara Bio Inc., MK301) according to the product manual.
  • Induction of differentiation into adipocytes Cells were cultivated in medium containing 1 ⁇ M Dexamethasone, 0.5 mM 3-Isobutyl-1-methylanxthine (IBMX), 10 ⁇ g / mL Insulin, 100 ⁇ M Indomethacin (all numbers are final concentrations) under 37 ° C., 5% CO 2 conditions. Insulin differentiation into adipocytes was induced by culturing for 3 weeks (medium exchange twice a week).
  • the cells were stained with Oil Red O using the Lipit assay kit (Cosmo Bio Co., Ltd., AK09F) according to the product manual.
  • Induction of differentiation into chondrocytes The components of Human Mesenchymal Stem Cell (hMSC) Chondrogenic Differentiation Medium Bullet Kit (tm) (Lonza, PT-3003) were mixed as instructed to prepare a cartilage differentiation-inducing medium (incomplete medium).
  • Recombinant Human TGF-beta 3 Protein R & D Systems, 243-B3 was added to this to a final concentration of 10 ng / ml, and a cartilage differentiation-inducing medium (complete medium) was prepared for each use.
  • the third passage cells were peeled off with Accutase-Solution PromoCell (C-41310), washed with a medium, and then 250,000 cells were separated into 15 ml polypropylene conical tubes based on the cell number measurement results.
  • the cells were washed twice with cartilage differentiation-inducing medium (incomplete medium), the supernatant was removed, and then suspended in 500 ⁇ l of cartilage differentiation-inducing medium (complete medium).
  • Cell pellets were formed by centrifugation at 150 g for 5 minutes, the lid was loosened and placed in a CO 2 incubator (37 ° C, 5% CO 2 ), and then the medium (complete medium) was changed every 2 to 3 days.
  • the pellet was taken out and fixed with 4% paraformaldehyde, frozen sections were prepared, and chondrocyte-derived proteoglycan was stained by Alcian blue staining.
  • chondrocyte-derived proteoglycan was stained by Alcian blue staining.
  • the same differentiation-inducing operation was performed on human bone marrow-derived mesenchymal stem cells.
  • BM-MSC was positive in all of ALP staining, Oil Red O staining, and Alcian blue staining.
  • blistering-derived cells were positive for ALP staining (however, the staining intensity was lower than BM-MSC), positive for Oil Red O staining (however, the staining intensity was lower than that for BM-MSC), and negative for alcyan blue staining. There was (Fig. 3).
  • sgRNAs Three types were prepared in order to select a position in the human genome that has good cleavage efficiency by the CRISPR-Cas9 system in the AAVS1 (Adeno-associated virus integration site 1) region.
  • the AAVS1 region is a safe region (safe harbor) that is not easily affected by gene transfer. Since the CRISPR-Cas9 system recognizes the base sequence of "NGG” and cleaves 3 bases upstream of it, it selects the region where "GG" is located at the end and selects the target sequence of 20 bases upstream of "NGG". Included sgRNAs (sgAAVS1- # 1 to # 3) were designed (Fig. 4, top; Table 1).
  • a plasmid expressing the Cas9 protein and sgRNA was prepared by cloning the oligonucleotide consisting of any of SEQ ID NOs: 3 to 5 and its complementary strand into the Bbs1 site of eSpCas9 (1.1) (Addgene plasmid # 71814). Created (eSpCas9 (1.1)-sgAAVS1- # 1, eSpCas9 (1.1)-sgAAVS1- # 2, eSpCas9 (1.1)-sgAAVS1- # 3, respectively).
  • This plasmid (2.5 ⁇ g) was introduced into HEK293 cells (human fetal kidney cell line) seeded in 6 well dishes by Lipofectamin 3000 (Thermo Fisher Scientific). Forty-eight hours after transfection, genomic DNA was extracted from the cells and the region containing the target site was amplified by PCR. The PCR amplified fragments were single-stranded by heat treatment, annealed by slow cooling, and then treated with a mismatch site-specific endonuclease. This was fractionated by electrophoresis, the degree of insertion or deletion mutation introduced by genome cleavage was measured by the band density, and the genome editing efficiency was calculated by the following formula (in the formula, a is digested). Band concentration that was not present, b and c indicate the band concentration that was cleaved).
  • COL7A1 gene into blister-derived cells
  • a plasmid expressing the COL7A1 gene was designed under the control of the CAG promoter (Fig. 5).
  • the COL7A1 cDNA was obtained from the Flexi ORF sequence-verified clone (Promega, Madison, WI, USA).
  • the COL7A1 cDNA was subcloned into the pENTR1A plasmid (Thermo Fisher Scientific, A10462) to give pENTR1A-COL7A1.
  • COL7A1 cDNA was introduced from pENTR1A-COL7A1 to pAAVS1-P-CAG-DEST (Addgene plasmid # 80490) by Gateway reaction using LR recombinase (Thermo Fisher Scientific) to obtain donor plasmid pAAVS1-P-CAG-COL7A1.
  • the blister-derived cells obtained in 1. above were suspended in a dedicated buffer of the Neon transfection system (Thermo Fisher Scientific), and the Cas9-sgRNA expression plasmid (eSpCas9 (1.1) -sgAAVS1- # 3) and donor plasmid (pAAVS1-P) were suspended.
  • eSpCas9 1.1
  • pAAVS1-P donor plasmid
  • the above plasmid was introduced into blister-derived cells by electroporation under the conditions of 1,200 V, 20 ms, and 2 pulses, and then seeded and cultured on a 6-well plate.
  • As the medium an equal amount mixed medium of Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009) and MSCGM Mesenchymal Stem Cell Growth Medium (Lonza, PT-3001) was used. Puromycin was added to a final concentration of 0.5 ⁇ g / mL 48 hours after transfection, cultured for about 2 weeks, and the selected cells were used in the transplantation experiment into the following mice.
  • the blister-derived cells have a higher expression level and secretion amount of type VII collagen than BM-MSC and fibroblasts. Therefore, blister-derived cells into which the COL7A1 gene has been introduced are expected to exert a higher therapeutic effect than BM-MSC and fibroblasts in gene therapy for dystrophic epidermolysis bullosa.

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