The present application claims priority with respect to Japanese Patent Application No. 2020-125620, which is incorporated herein by reference in its entirety.
The present disclosure relates to compositions for use in treating dystrophic epidermolysis bullosa.
Epidermolysis bullosa is a disease in which adhesive structural molecules responsible for adhesion of the skin tissue are lost or disappeared, and then the epidermis peels off from the dermis and blisters or skin ulcers occur when force is applied to the skin. The disease includes simple epidermolysis bullosa, in which the epidermis is torn to form blisters, junctional epidermolysis bullosa, in which the epidermis is peeled from the basement membrane to form blisters, and dystrophic epidermolysis bullosa, in which the basement membrane is peeled from the dermis.
Dystrophic epidermolysis bullosa is the most common type of epidermolysis bullosa, accounting for about 50% of all epidermolysis bullosa. It is a hereditary disease caused by a mutation in the COL7A1 gene, which encodes type VII collagen. In the structure of the skin, 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 fibers called anchoring fibrils in the dermis and connects the basement membrane and the dermis. Therefore, if there is an abnormality in the type VII collagen gene, the adhesive 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, severe recessive dystrophic epidermolysis bullosa is a very serious hereditary bullous skin disease that has continued burn-like skin symptoms throughout the body immediately after birth, and cutaneous spinous cell carcinoma (scar cancer) occurs frequently from around 30 years old and leads to death.
There is currently no effective treatment for epidermolysis bullosa, and the development of gene therapy that radically suppresses blistering is required. As such gene therapy, a therapeutic technique is 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 to the patient (Patent Document 1). Also, it has been proposed to subject mesenchymal stem cells lacking the type VII collagen activity to genome editing, differentiate the mesenchymal stem cells capable of producing type VII collagen thus obtained into keratinocytes or fibroblasts, culture the cells to form a skin sheet, and use the skin sheet for treating a patient (see Patent Document 2).
Manufacturing the skin sheet requires advanced process control and culture technology, and then involves high-difficulty and high-cost. Therapeutic agents that are easier to manufacture are required.
In one aspect, the present disclosure relates to a composition for use in the treatment of dystrophic epidermolysis bullosa, comprising a blister-derived cell of a patient with dystrophic epidermolysis bullosa that is genetically modified to produce type VII collagen.
The present disclosure provides compositions for use in treating dystrophic epidermolysis bullosa.
Unless otherwise specified, the terms used in the present disclosure have meanings generally understood by those skilled in the art in the fields such as organic chemistry, medical science, pharmaceutical science, molecular biology, and microbiology. Definitions of some terms used in the present disclosure are provided below, and these definitions supersede the general understandings in the present disclosure.
Dystrophic epidermolysis bullosa (DEB) is a hereditary disease caused by a mutation in the COL7A1 gene, which encodes type VII collagen, and is known to be characterized in that no type VII collagen is produced or type VII collagen with reduced function due to the mutation is produced. The type VII collagen forms fibers called anchoring fibrils in the dermis and connects the basement membrane and the dermis. The type VII collagen contains a first non-collagen region, a collagen region, and a second non-collagen region from the N-terminus, and forms a triple chain at the collagen region, which is characterized by a repeating sequence of glycine-X-Y. Two molecules bind to each other at the C-terminus and the N-terminus binds to the basement membrane. Examples of mutations include a mutation in which glycine in the collagen region is replaced by a different amino acid, a stop codon mutation that stops protein translation, and a splice site mutation. The mutation may be in one of the alleles or in both. Dystrophic epidermolysis bullosa includes dominant dystrophic epidermolysis bullosa and recessive dystrophic epidermolysis bullosa, and the recessive dystrophic epidermolysis bullosa include severe generalized recessive dystrophic epidermolysis bullosa and other generalized types with relatively mild symptoms. The dystrophic epidermolysis bullosa herein may be any type of dystrophic epidermolysis bullosa, and the causal mutation in the COL7A1 gene may be any mutation.
A blister is an accumulation of fluid such as body fluid or tissue fluid under the epidermis. A preferable blister is a blister in which fluid is accumulated in the space formed between the epidermis and the dermis due to detachment of the epidermis from the dermis. More preferable blister is a blister in which fluid is accumulated in the space formed between the basement membrane of the epidermis and the dermis due to detachment of the basement membrane from the dermis.
In the present disclosure, a blister-derived cell of a patient with dystrophic epidermolysis bullosa refers to an adherent cell collected from a blister of a patient with dystrophic epidermolysis bullosa, and herein also referred to as “a DEB patient blister-derived cell” or “a blister-derived cell”. The cell can be obtained by culturing a blister content of a patient with dystrophic epidermolysis bullosa on a solid phase. In one embodiment, the blister content is a fluid that has accumulated within the blister, which is herein referred to as “a blister fluid”. The blister content can be collected from a blister of a patient with dystrophic epidermolysis bullosa with a tool such as a syringe. For example, when a injection needle of a syringe is pierced into a blister so that the tip of the injection needle is positioned in the space formed between the epidermis and the dermis and the plunger of the syringe is pulled, a blister fluid can be aspirated into the syringe. In one embodiment, a blister-derived cell can be obtained by seeding a blister content in a medium without any enzymatic treatment such as collagenase or dispase treatment and culturing on a solid phase. Specifically, when a blister fluid collected from a blister is directly seeded in a medium and the medium is incubated on a solid phase for a certain period of time, a cell adhering to the solid phase can be used as a blister-derived cell. In this case, it is preferable to obtain cells that have formed a colony on the solid phase. The blister fluid is preferably seeded in the medium within 3 hours, more preferably within 2 hours, and even more preferably within 1 hour after collection. In the present disclosure, a solid phase means a solid support to which a cell can adhere, and includes, for example, plastic or glass culture vessels such as culture dishes, flasks and multiwell plates. In one embodiment, the solid phase is a plastic culture vessel. The solid phase may be coated, and examples of substances for coating include collagen I, laminin, vitronectin, fibronectin, poly-L-lysine, and poly-L-ornithine. In one embodiment, the solid phase is coated with collagen I. The culturing can be performed in a general incubator under the condition such as “37° C., 5% CO2” or “37° C., 5% O2, 5% CO2”. The culture medium may be any medium that can be used for culturing animal cells, and can be, for example, MEM, MEMα, DMEM, GMEM, RPMI 1640, MesenCult™ (STEMCELL Technologies), Mesenchymal Stem Cell Growth Medium 2 (PromoCell), MSCGM Mesenchymal Stem Cell Growth Medium (Lonza), Cellartis MSC Xeno-Free Culture Medium (Takara Bio), or a mixture thereof. Among them, a medium for culturing mesenchymal stem cells such as MesenCult™, Mesenchymal Stem Cell Growth Medium 2, MSCGM Mesenchymal Stem Cell Growth Medium, Cellartis MSC Xeno-Free Culture Medium is preferably used. The medium is preferably a serum-free medium. The culture period may be any period sufficient for the cell to adhere to the solid phase, and can be e.g., 1 day to several months (for example, for 2, 3 or 4 months), 1 day to 1 month, 1 day to several weeks (for example, 2, 3 or 4 weeks), 1 days to a week.
In an embodiment, the DEB patient blister-derived cell has one or more characteristics selected from:
In the present disclosure, the expression that a cell is “incapable of differentiating” into an osteoblast, an adipocyte or a chondrocyte means that differentiation of the cell into an osteoblast, an adipocyte or a chondrocyte cannot be detected by a standard detection method (such as staining) after induction of differentiation with a standard condition.
In an embodiment, the DEB patient blister-derived cell is a CD73-positive, CD105-positive, and CD90-positive cell. In another embodiment, the DEB patient blister-derived cell is a CD45-negative, CD34-negative, CD11b-negative, CD79A-negative, HLA-DR-negative, and CD31-negative cell. In a further embodiment, the DEB patient blister-derived cell is a cell that is less capable of differentiating into an osteoblast, an adipocyte and a chondrocyte than a bone marrow-derived mesenchymal stem cell. In a further embodiment, the DEB patient blister-derived cell is a cell that is less capable of differentiating into an osteoblast and an adipocyte than a bone marrow-derived mesenchymal stem cell, and incapable of differentiating into a chondrocyte.
In the present disclosure, a blister-derived cell of a patient with dystrophic epidermolysis bullosa that is genetically modified to produce type VII collagen is used. As used herein, the term “cell that is genetically modified to produce type VII collagen” means a cell that is genetically modified to produce a functional type VII collagen (ie, a type VII collagen capable of forming anchoring fibrils).
In the present disclosure, genetic modification of a cell means both modification of a gene in the genome of the cell and modification of the cell to express a gene from a nucleic acid construct outside the genome (such as a vector). That is, the expression “genetically modifying a cell to produce type VII collagen” includes modifying a cell to express type VII collagen from a COL7A1 gene in the genome, and modifying a cell to express type VII collagen from a COL7A1 gene in a nucleic acid construct outside the genome. Also, “a cell that is genetically modified to produce type VII collagen” includes a cell that expresses type VII collagen from a COL7A1 gene in the genome and a cell that expresses type VII collagen from a COL7A1 gene in a nucleic acid construct outside the genome.
Genetic modification of a cell can be carried out by introducing a COL7A1 gene or by correcting a mutation in the COL7A1 gene in the genome. The introduction of a COL7A1 gene can be carried out either by introducing a COL7A1 gene into the genome of the cell or by placing a nucleic acid construct comprising a COL7A1 gene in the cell so that the COL7A1 gene is expressed from the nucleic acid construct outside the genome. When a COL7A1 gene is introduced into the genome of a cell, the COL7A1 gene may be introduced at a specific site or may be introduced at random. In an embodiment, the COL7A1 gene is introduced into the COL7A1 locus of the genome, or a safe harbor such as the AAVS1 region.
The DEB patient blister-derived cell may be a cell of a patient with dystrophic epidermolysis bullosa to which the cell is to be administered (ie, an autologous cell), or a cell obtained from a subject other than the patient (ie, an allogeneic cell). The cell of a patient with dystrophic epidermolysis bullosa includes a cell that does not produce type VII collagen and a cell that produces type VII collagen with reduced function due to a mutation, and the “cell of a patient with dystrophic epidermolysis bullosa” as used herein may be any of them.
The DEB patient blister-derived cell may be any cell as long as it produces type VII collagen in the vicinity of the epidermal basement membrane when administered to a patient.
In the present disclosure, the term “cell” is used in the sense of including a cell after proliferation as needed. Proliferation of a cell can be carried out by culturing the cell. For example, “a blister-derived cell (of a patient with dystrophic epidermolysis bullosa)” includes a cell that is proliferated from a cell collected from a patient, and “a genetically modified cell” includes a cell that is proliferated from a cell obtained by genetic modification. When genetic modification is carried out, a cell may be prolifelated until the amount required for the genetic modification is obtained. Also, after genetic modification, the cell may be prolifelated until the amount required for treatment is obtained.
As used herein, the term “cell” can mean a single cell or multiple cells, depending on the context. Further, the cell may be a cell population composed of one type of cell or a cell population including a plurality of types of cells.
As used herein, the term “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 (for example, 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 genome sequence is disclosed in GenBank: AC121252.4. The sequence of a COL7A1 gene is not limited to any specific sequence as long as it encodes a functional type VII collagen (ie, a type VII collagen capable of forming anchoring fibrils).
In an embodiment, the COL7A1 gene comprises or consists of 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. In a different embodiment, the COL7A1 gene comprises or consists of a nucleic acid sequence wherein 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 base(s) is inserted, deleted, substituted, or added with respect to 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.
In an embodiment, the type VII collagen comprises or consists of 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. In a different embodiment, the type VII collagen comprises or consists of an amino acid sequence wherein 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acid residue(s) is inserted, deleted, substituted, or added with respect to the amino acid sequence of SEQ ID NO: 2. In a further embodiment, the type VII collagen comprises or consists of the amino acid sequence of SEQ ID NO: 2.
In an embodiment, the COL7A1 gene comprises or consists of a nucleic acid sequence that encodes 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. In a different embodiment, the COL7A1 gene comprises or consists of a nucleic acid sequence that encodes an amino acid sequence wherein 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acid residue(s) is inserted, deleted, substituted, or added with respect to the amino acid sequence of SEQ ID NO: 2.
As used herein, the term “sequence identity” with respect to a nucleic acid sequence or an amino acid sequence means the proportion of bases or amino acid residues that match between two sequences that are optimally aligned (aligned to be maximally matched) over the entire region of the sequence to be compared. The sequence to be compared may have an insertion, an addition or a deletion (eg, a gap) in the optimal alignment of the two sequences. The sequence identity can be calculated using a program such as FASTA, BLAST, or CLUSTAL W provided in a public database (for example, DDBJ (http://www.ddbj.nig.ac.jp)). Alternatively, the sequence identity can be obtained using a commercially available sequence analysis software (for example, Vector NTI® software, GENETYX® ver. 12).
The cell may be genetically modified by any method. In an embodiment, the cell is genetically modified by genome editing such as the CRISPR system (eg, CRISPR/Cas9, CRISPR/Cpf1), TALEN, or ZFN. In a different embodiment, the cell is genetically modified with a viral vector such as a retroviral vector, lentiviral vector, adenoviral vector, or adeno-associated viral vector. In a further embodiment, the cell is genetically modified with CRISPR/Cas9. In a further embodiment, the cell is genetically modified with a retroviral vector or a lentiviral vector.
In genome editing, causing cleavage in the genome and introducing a donor vector comprising a sequence of interest into the cell can insert the sequence of interest into the cleavage site of the genome. The sequence to be inserted into the genome can be a COL7A1 gene or a sequence to be replaced with a portion containing a mutation in the COL7A1 gene (for example, a partial sequence of a COL7A1 gene). In addition to the sequence of interest, the donor vector may comprise a regulatory sequence such as a promoter or enhancer that controls the expression of the sequence of interest, or other elements such as a drug resistance gene for cell selection, and also may comprise, at both ends, sequences homogeneous to both ends of the insertion site of the genome. The donor vector can be introduced into a desired site as a result of non-homologous end binding or homologous recombination. As the donor vector, a plasmid, an adeno-associated viral vector, an integrase-deficient lentiviral vector, or any of other viral vectors can be used.
In the CRISPR system, an endonuclease such as Cas9 or Cas12 (eg, Cas12a (also called Cpf1), Cas12b, Cas12e) recognizes a specific base sequence, called PAM sequence, and the double strand of the target DNA is cleaved by the action of the endonuclease. When the endonuclease is Cas9, it cleaves about 3-4 bases upstream of the PAM sequence. Examples of endonucleases include Cas9 of S. pyogenes, S. aureus, N. meningitidis, S. thermophilus, or T. denticola, and Cpfl of L. bacterium ND2006 or Acidaminococcus sp. BV3L6. The PAM sequence varies depending on the endonuclease, and the PAM sequence of Cas9 in S. pyogenes is NGG, for example. A gRNA comprises a sequence of about 20 bases upstream of the PAM sequence (target sequence) or a sequence complementary thereto on the 5′ end side, and plays a role of recruiting an endonuclease to the target sequence. The sequences other than the target sequence (or a sequence complementary thereto) of a gRNA can be appropriately determined by those skilled in the art depending on the endonuclease to be used. A gRNA may comprises a crRNA (CRISPR RNA), which comprises the target sequence or a sequence complementary thereto and is responsible for the sequence specificity of the gRNA, and a tracrRNA (Trans-activating crRNA), which contributes to the formation of a complex with Cas9 by forming a double strand. The crRNA and tracrRNA may exist as separate molecules. When the endonuclease is Cpf1, the crRNA alone functions as a gRNA. In the present specification, a gRNA comprising elements necessary for the function as a gRNA on a single strand may be particularly referred to as a sgRNA. The gRNA sequence can be determined by a tool available for target sequence selection and gRNA design, such as CRISPRdirect (https://crispr.dbcls.jp/).
A vector comprising a nucleic acid sequence encoding a gRNA and a nucleic acid sequence encoding an endonuclease may be introduced into and expressed in a cell, or a gRNA and an endonuclease protein that have been prepared extracellularly may be introduced into a cell. The endonuclease may include a nuclear localization signal. The nucleic acid sequence encoding a gRNA and the nucleic acid sequence encoding an endonuclease may be present on different vectors. Methods for introducing the vector, gRNA, and endonuclease into a cell include, but are not limited to, lipofection, electroporation, microinjection, calcium phosphate method, and DEAE-dextran method.
In an embodiment, a gRNA that can be used for the introduction of a COL7A1 gene into the genome comprises any of the sequences of SEQ ID NOs: 3 to 5 or a sequence complementary thereto.
In the case of viral vectors, a COL7A1 gene can be introduced into the genome of a cell when a retroviral vector or a lentiviral vector having integrase activity is used. Alternatively, an integrase-deficient retroviral or lentiviral vector may be used. Integrase-deficient vectors lack integrase activity, for example, due to a mutation in the integrase gene. When an integrase-deficient vector, or an adenoviral vector or an adeno-associated viral vector is used, the sequence incorporated into the vector is not usually introduced into the genome of a cell. For example, when a COL7A1 gene is incorporated into an integrase-deficient lentiviral vector or an adenoviral vector, type VII collagen is expressed from the COL7A1 gene of the vector existing in the cell (in the nucleus).
A viral vector comprises a sequence encoding a COL7A1 gene and may contain a regulatory sequence such as a promoter or enhancer that controls the expression of the COL7A1 gene and other elements such as a drug resistance gene for cell selection. A viral vector may be prepared by any method known in the art. For example, a retroviral or lentiviral vector can be prepared by introducing a viral vector plasmid comprising LTR sequences at both ends (5′ LTR and 3′ LTR), a packaging signal, and a sequence of interest into a packaging cell with one or more plasmid vectors expressing structural proteins of the virus, such as Gag, Pol, and Env, or into a packaging cell that expresses such 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). The sequence of interest can be introduced into a target cell by infecting the target cell with a viral vector thus prepared.
In an embodiment, the viral vector is a lentiviral vector. Examples of lentiviral vectors include, but are not limited to, 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). In an embodiment, the lentiviral vector is HIV.
As an example, a lentiviral vector can be prepared as follows. First, a viral vector plasmid encoding the viral genome, one or more plasmid vectors expressing Gag, Pol, and Rev (and optionally Tat), and one or more plasmid vectors expressing envelope proteins such as VSV-G are introduced into a packaging cell. The viral vector plasmid comprises LTR sequences at both ends (5′ LTR and 3′ LTR), a packaging signal, and a COL7A1 gene and a promoter that controls its expression (eg, 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 a different promoter, such as CMV promoter, to enhance the expression of the RNA genome. Within the cell, the viral RNA genome is transcribed from the vector plasmid and packaged to form a viral core. The viral core is transported to the cell membrane of the packaging cell, encapsulated in the cell membrane, and released as a viral particle from the packaging cell. The released virus particle can be recovered from the culture supernatant of the packaging cell. For example, the virus particle can be recovered by any of conventional purification methods such as centrifugation, filter filtration, and column purification. A lentiviral vector can also be prepared by using a kit such as Lentiviral High Titer Packaging Mix, Lenti-X™ Packaging Single Shots (Takara Bio Inc.), and ViraSafe™ Lentivirus Complete Expression System (Cell Biolabs Inc.). An adeno-associated viral vector can be prepared by using a kit such as AAVpro® Helper Free System (Takara Bio Inc.).
In one aspect, the present disclosure provides a plasmid comprising an EF1α promoter and a COL7A1 gene located downstream of the EF1α promoter that is used for producing a lentiviral vector. In a further aspect, the disclosure provides a lentiviral vector comprising an EF1α promoter and a COL7A1 gene located downstream of the EF1α promoter.
A representative sequence of the EF1α promoter is shown in SEQ ID NO:6.
In an embodiment, the EF1α promoter comprises or consists of 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: 6. In a different embodiment, the EF1α promoter comprises or consists of a nucleic acid sequence wherein 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 base(s) is inserted, deleted, substituted, or added with respect to 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.
A cell into which a sequence of interest has been introduced can be detected by Southern blotting or PCR. The sequence of interest need only be introduced into at least one of the alleles.
In an embodiment of the composition of the present disclosure, the DEB patient blister-derived cell is the most abundant cell in the composition. In a further embodiment, the DEB patient blister-derived cell accounts for 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more of cells comprised in the composition. In a further embodiment, the composition of the present disclosure is substantially free of cells other than the DEB patient blister-derived cell. The expression “substantially free of cells other than the DEB patient blister-derived cell” means that the composition only comprises a cell obtained by a method that is substantially identical to the method for obtaining the DEB patient blister-derived cell as described herein.
The number of cells comprised in a composition is an amount required to exert a desired effect (also referred to herein as an effective amount), and it is appropriately determined by those skilled in the art in consideration of factors such as the age, body weight, and medical condition of the patient, the type of cells and method for genetic modification. The number of cells is not limited to, but can be, for example, 1 cell to 1×107 cells, 1×10 cells to 1×107 cells, 1×102 cells to 1×107 cells, 1×103 cells to 1×107 cells, 1×104 cells to 1×107 cells, 1×105 cells to 1×107 cells, 1×105 cells to 5×106 cells, 5×105 cells to 1×106 cells, or 1×105 cells to 1×106 cells. The composition may comprise a pharmaceutically acceptable carrier and/or an additive in addition to the cell. Examples of pharmaceutically acceptable carriers include water, medium, saline, infusion containing glucose, D-sorbitol, D-mannitol or others, and phosphate buffered saline (PBS). Examples of additives include solubilizers, stabilizers, and preservatives. The dosage form of the composition is not particularly limited to, but can be a parenteral preparation such as an injection. Examples of injections include solution injections, suspension injections, emulsion injections, and injections to be prepared before use. The composition may be frozen and may contain a cryoprotectant such as DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, dextran, or sucrose.
The composition of the present disclosure can be administered systemically or topically. In an embodiment, the composition is administered to an affected area of a patient with dystrophic epidermolysis bullosa. As used herein, the affected area means a blister or an area in the vicinity of a blister. In a further embodiment, the composition is administered intradermally at the site of a blister or administered into a blister. In a further embodiment, the composition is administered into a blister. In the present specification, administration into a blister means administration to the space under the epidermis of a blister. The composition is preferably administered into the space formed between the epidermis and the dermis due to detachment of the epidermis from the dermis. The composition is more preferably administered into the space formed between the basement membrane of the epidermis and the dermis due to detachment of the basement membrane from the dermis. For example, the composition can be administered into a blister by piercing the blister with an injection needle of a syringe containing the composition in the syringe and ejecting the composition from the tip of the injection needle positioned in the space formed between the epidermis and the dermis. The blister may be a blister naturally formed as a pathological condition of epidermolysis bullosa, or may be a blister artificially formed. In patients with epidermolysis bullosa, a blister can be artificially formed, for example, by pinching or rubbing the patient's skin. The intrablister administration can reduce the patient's pain compared to intradermal or subcutaneous administration, and type VII collagen can be well expressed around the basement membrane. The number of cells administered per site is an amount required to exert a desired effect (effective amount), and it is appropriately determined by those skilled in the art in consideration of factors such as the age, body weight, and medical condition of the patient, the type of cells, and method for genetic modification. The number of cells is not limited to, but can be for example, 1 cell to 1×107 cells, 1×10 cells to 1×107 cells, 1×102 cells to 1×107 cells, 1×103 cells to 1×107 cells, 1×104 cells to 1×107 cells, 1×105 cells to 1×107 cells, 1×105 cells to 5×106 cells, 5×105 cells to 1×106 cells, or 1×105 cells to 1×106 cells. In an embodiment, the number of cells to be administered per blister is 1 cell to 1×107 cells, 1×10 cells to 1×107 cells, 1×102 cells to 1×107 cells, 1×103 cells to 1×107 cells, 1×104 cells to 1×107 cells, 1×105 cells to 1×107 cells, 1×105 cells to 5×106 cells, 5×105 cells to 1×106 cells, or 1×105 cells to 1×106 cells. The amount to be administered per blister may be adjusted according to the size of the blister, and the above amount may be considered to be an amount for a standard blister having a diameter of 7 to 8 mm when circularly approximated. When the cells are administered into a blister, a preferred dosage is 1×105 to 1×107 cells per cm2 of blister area, and a more preferred dosage is 5×105 to 5×106 cells per cm2 of blister area.
Exemplary embodiments of the present invention are described below.
derived cell is derived from a blister fluid of the patient with dystrophic epidermolysis bullosa.
A cell produced by the method according to any one of items 54 to 56.
The present invention is described in more detail with reference to the examples hereinafter, but not limited to the embodiments described below.
Blister fluid from a patient with dystrophic epidermolysis bullosa was collected and centrifuged at 300 g for 5 minutes. The resulting precipitate was suspended in a medium (Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009) supplemented with penicillin and streptomycin to a final concentration of 100 unit/mL and 100 μg/mL, respectively), and seeded in a 6-well plate coated with collagen I and cultured at 37° C. and 5% CO2 to obtain adherent cells. The duration time from collection of the blister fluid to seeding in the medium varied from patient to patient, but was within the range of 18 minutes to 1 hour. Thereafter, medium exchange and passaging were carried out appropriately until the cells proliferated to the desired cell number (culture progress up to 20 days after initiation of culture is shown in
The blister-derived cells obtained in the section 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)] were subjected to surface marker analysis according to the following procedure. The cells were removed from the plate with Accutase-Solution (PromoCell, C-41310), and after washed with medium, 100,000 cells each were put into two tubes based on cell count results. The cells were washed once with Flow Cytometry Staining Buffer (1×) (R&D Systems, FC001), and resuspended with 100 μl of Flow Cytometry Staining Buffer (1×). To block Fc receptors, 5 μl of Human TruStain FcX™ (BioLegend, 422301) was added and allowed to react on ice for 10 minutes. To one of the tubes, 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), which were contained in Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems, FMC020), and further 5 μl of Brilliant Violet 421™ anti-human CD105 Antibody (BioLegend, 323219) were added, and allowed to react for 30 minutes at room temperature in the dark. Since the other tube was used as a negative control, the same amount of each isotype control antibody was added and allowed to react for 30 minutes at room temperature in the dark. The cells were washed once with 2 ml of Flow Cytometry Staining Buffer (1×), resuspended in 300 μl of Flow Cytometry Staining Buffer (1×), and analyzed by BD FACSAria (BD). In addition, whether CD31 was expressed in blister-derived cells and human BM-MSCs was analyzed by FACS analysis according to the following procedure. The cells were removed from the plate with Accutase-Solution (PromoCell, C-41310), and after washed with medium, 100,000 cells each were put into two tubes based on cell count results. The cells were washed once with 2% FBS-containing PBS and resuspended in 100 μl of 2% FBS-containing PBS. To block Fc receptors, 5 μl of Human TruStain FcX™ (BioLegend, 422301) was added and allowed to react on ice for 10 minutes. To one of the tubes, 5 μl of APC anti-human CD31 Antibody (BioLegend, 303116) (0.4 μg of protein) was added and allowed to react for 60 minutes on ice in the dark. Since the other tube was used as a negative control, 2 μl of APC Mouse IgG1, κ Isotype Ctrl Antibody (BioLegend, 400120) (0.4 μg of protein) was added and allowed to react for 60 minutes on ice in the dark. The cells were washed once with 2 ml of 2% FBS-containing PBS, resuspended in 300 μl of 2% FBS-containing PBS, and analyzed by BD FACSAria (BD).
FACS analysis showed that 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 (
The blister-derived cells obtained in the section 1 above and BM-MSCs were induced to differentiate into osteoblasts, adipocytes and chondrocytes under the following conditions.
Induction of Differentiation into Osteoblasts:
Cells were cultured in a medium containing 0.1 μM Dexamethasone, 0.2 mM Ascorbic acid 2-phosphate, 10 mM Glycerol 2-phosphate (all values were final concentrations) at 37° C. and 5% CO2 for 3 weeks (the medium was changed twice a week) to induce differentiation into osteoblasts. The cells were stained by Alkaline phosphatase (ALP) staining using TRACP & ALP Assay Kit (Takara Bio Inc., MK301) according to the product manual.
Induction of Differentiation into Adipocytes:
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 values were final concentrations) at 37° C. and 5% CO2 for 3 weeks (the medium was changed twice a week) to induce differentiation into adipocytes. The cells were stained by Oil Red O staining using a LipiD Assay kit (Cosmo Bio, AK09F) according to the product manual.
Induction of Differentiation into Chondrocytes:
A chondrogenic differentiation medium (incomplete medium) was prepared by mixing the components of Human Mesenchymal Stem Cell (hMSC) Chondrogenic Differentiation Medium Bullet Kit (tm) (Lonza, PT-3003) according to the instructions. Recombinant Human TGF-beta 3 Protein (R&D Systems, 243-B3) was added to the medium to a final concentration of 10 ng/ml to prepare a chondrogenic differentiation medium (complete medium) each time it was used. After the third passage cells were detached with Accutase-Solution PromoCell, C-41310) and washed with medium, 250,000 cells each were put into 15 ml polypropylene conical tubes based on cell count results. The cells were washed twice with the chondrogenic differentiation medium (incomplete medium), the supernatant was removed, and the cells were suspended in 500 μl of the chondrogenic differentiation medium (complete medium). The cells were centrifuged at 150 g for 5 minutes to form a cell pellet, and after the lid of the tube was unscrewed, the tube was placed in a CO2 incubator (37° C., 5% CO2), and then the medium (complete medium) was changed every 2 to 3 days. After 3 weeks, the pellet was removed and fixed with 4% paraformaldehyde, frozen sections were prepared, and chondrocyte-derived proteoglycans were stained by Alcian Blue staining. As a positive control, human bone marrow-derived mesenchymal stem cells were also subjected to the same differentiation-inducing operation.
In the above differentiation induction experiments, BM-MSCs were positive in all of ALP staining, Oil Red O staining, and Alcian Blue staining. On the other hand, the blister-derived cells were positive in ALP staining (but staining intensity was lower than that of BM-MSCs), positive in Oil Red O staining (but staining intensity was lower than that of BM-MSCs), and negative in Alcian blue staining (
How much blister-derived cells expressed and secreted type VII collagen was examined by Western blotting. In this experiment, blister-derived cells were obtained from a patient who expressed type VII collagen, but whose expressed type VII collagen was thought to have little function due to amino acid mutation. 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 of an epidermolysis bullosa patient (hereinafter BFC; also referred to as blister fluid cells) were cultured in the media shown in Table 1 below.
When the cells reached 90-95% confluence, the cells were washed with D-PBS(−). Each medium (no supplement added) containing ascorbic acid (Nacalai Tesque, 13048-42, final concentration of 50 ug/ml) and a protease inhibitor cocktail (SIGMA, P1860-1ML, 1/400 dilution) was added to the cells, and the cells were cultured in a CO2 incubator for 24 hours. After the culture, the medium was concentrated using a methanol-chloroform precipitation method. A cell lysate was also prepared from the cells using RIPA buffer (Nacalai Tesque, 08714-04). Each lysate was corrected based on protein concentration, and a sample for electrophoresis was prepared using LDS sample buffer and sample reducing agent (Invitrogen, NP0007 and NP0009, respectively). After electrophoresis with 3-8% NuPAGE gel (Invitrogen, EA0375BOX), the gel was transferred to a PVDF membrane (Millipore, IPVH07850), and the membrane was reacted with Anti-Col7 (Atlas, HPA042420) as a primary antibody and Anti-Rabbit IgG-HRP (GE healthcare, NA9340-1ML) as a secondary antibody. Then, the bands were detected using Chemi-lumi-one ultra (Nacalai Tesque, 11644-40) and Chemi DOC (BioRad, 17001402JA), and analyzed and quantified with Image Lab software (BioRad, 1709690). The western blotting was also performed on the concentrated medium in the same manner to quantify the concentration of type VII collagen.
The results are shown in
Three types of sgRNAs were prepared in order to select a site with good cleavage efficiency by CRISPR-Cas9 system in AAVS1 (Adeno-associated virus integration site 1) region in the human genome. The AAVS1 region is a safe region that is not easily affected by gene transfer (safe harbor). Since the CRISPR-Cas9 system recognized the base sequence of “NGG” and cleaved 3 bases upstream of the sequence, regions each having “GG” at the end were selected and sgRNAs each containing a target sequence of 20 bases upstream of “NGG” were designed (sgAAVS1-#1 to #3) (
An oligonucleotide consisting of a sequence of any one of SEQ ID NOs: 3 to 5 was annealed with its complementary strand and cloned into the Bbs1 site of eSpCas9 (1.1) (Addgene plasmid #71814) to prepare a plasmid that expressed Cas9 protein and sgRNA (eSpCas9(1.1)-sgAAVS1-#1, eSpCas9(1.1)-sgAAVS1-#2, or eSpCas9(1.1)-sgAAVS1-#3). This plasmid (2.5 μg) 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 subjected to heat treatment to be single chains, and they were annealed by slow cooling and then treated with a mismatch site-specific endonuclease. The resulting product was fractionated by electrophoresis, the degree of insertion or deletion mutation introduced by the genome cleavage was measured from the density of the band, and the genome editing efficiency was calculated by the following formula (In the formula, “a” indicates the concentration of the band that was not digested, and “b” and “c” indicate the concentrations of the cleaved bands.).
Indel(%)=10033 (1−√{square root over ((1−fcut))}), fcut=(b+c)/(a+b+c)
All sgRNAs of sgAAVS1-#1 to #3 produced a short DNA fragment different from the control, confirming that double-strand break occurred (
For introduction of a COL7A1 gene into the AAVS1 region, a plasmid expressing a COL7A1 gene under the control of a CAG promoter was designed (
The blister-derived cells obtained in the section 1 above were suspended in a special buffer for 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.
Using the Neon transfection system, the plasmid was introduced into blister-derived cells by electroporation under the conditions of 1200 V, 20 ms, and 2 pulses, and the cells were seeded on a 6-well plate and cultured. The medium was a mixed medium of equal volume of Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009) and MSCGM Mesenchymal Stem Cell Growth Medium (Lonza, PT-3001). Forty-eight hours after transfection, puromycin was added to a final concentration of 0.5 μg/mL, and the cells were cultured for about 2 weeks. The selected cells were treated as described in “5. Transplantation of genetically modified blister-derived cells into mice”.
We also constructed a donor plasmid that expressed a COL7A1 gene under the control of a PGK promoter, and introduced this plasmid into various cells, including blister-derived cells. Then, the expression level and secretion level of COL7A1 in the modified cells were evaluated by Western blotting in the same manner as in “c) Evaluation of ability to express and secrete type VII collagen” in “2. Characterization of blister-derived cells” above. In this experiment, blister-derived cells were obtained from a patient who did not express type VII collagen. The results are shown in
The full-thickness skin of a neonatal Col7A1 gene knockout mouse (Col7a1-/-) showing blistering was excised and transplanted to the back of an immunodeficient mouse (NOD-SCID). Immediately after the transplantation, the skin surface was pinched and rubbed to form a blister, and 1.0×106 genetically modified blister-derived cells prepared in the section 4 above were immediately injected into the space under the epidermis (into the blister) (
We compared type VII collagen deposition between blister-derived cells and mesenchymal stem cells in epidermolysis bullosa model mice. First, the full-thickness skin of a neonatal Col7A1 gene knockout mouse was transplanted to the back of an immunodeficient mouse (NOD-SCID). Then, 1.0×106 of the following cells were injected into the dermis (intradermally) or into the space under the epidermis (into the blister).
After 4 weeks, the skin was collected and immunostained with an anti-type VII collagen antibody (clone LH7.2; Sigma Aldrich, C6805), and photographs of stained images were taken. Then, using an image analysis software, a plurality of photographs were superimposed so that the stained portion of type VII collagen was correctly matched to provide a merged image.
The results are shown in
It was investigated how many blister-derived cells should be transplanted into a blister to obtain the necessary effect. As shown in
The results are shown in
It was investigated how long blister-derived cells administered intradermally to a mouse survived at the administration site. First, as shown on
The results are shown in
The infection efficiency of lentivirus to blister-derived cells was analyzed. The cells used in this experiment and the medium used to culture the cells are shown in Table 4 below.
Preparation of RetroNectin-coated plate: RetroNectin [Takara Bio Inc. (Shiga, Japan), T100B] was diluted to 40 μg/mL with PBS (Dulbecco's phosphate-buffered saline (Ca, Mg-free)) [Nacalai Tesque (Kyoto, Japan), 14249-95] and added to a 96-well plate with no surface treatment [Corning (Tokyo, Japan), 3370] at 100 μL/well. The plate was then left overnight at 4° C. Before the plate was used, the RetroNectin solution was removed, the plate was washed twice with PBS, and the following operations were performed.
Cell seeding and lentivirus infection: The cells were detached from the plate with Accutase-Solution [PromoCell (Heidelberg, Germany), C-41310] for blister-derived cells, Trypsin/EDTA for Mesenchymal Stem Cells [Lonza (Basel, Switzerland), CC-3232] for human bone marrow-derived mesenchymal stem cells, and Trypsin/EDTA Solution [Lonza (Basel, Switzerland), CC-5012] for normal human adult skin fibroblasts, and collected by using the cell culture medium for respective cells. The number of collected cells was counted and the cells were seeded on the RetroNectin-coated 96-well plate at 2500 cells/well. After that, a lentivirus having a GFP gene: pLenti-C-mGFP [ORIGENE (Rockville, USA), PS100071] was added to each well at MOT 1 or MOT 5, and the cells were cultured in a CO2 incubator for 72 hours.
Detection of GFP-positive cells: The GFP-positive cell ratio was detected with a fluorescence microscope [Keyence (Tokyo, Japan), BZ-X710]. The results are shown in
Also, GFP-positive cells after infection with MOT 1 were quantified by the following procedure for each cell type. First, the ratio of the number of GFP-positive cells to the total number of cells in a visual field was defined as the GFP-positive cell ratio. This measurement was repeated three times and statistical processing was performed (*: P<0.05, Dunnett's test). The results are shown in
As shown in
A lentiviral vector plasmid containing an EF1α promoter and a COL7A1 gene in an expression cassette as shown in
Also, a lentiviral vector plasmid containing a PGK promoter and a COL7A1 gene in an expression cassette as shown in
A lentiviral vector having a COL7A1 gene as shown in
Transfection using lentiviral plasmids: The plasmids shown in
Collection and purification of lentiviral vector: The culture supernatant of Lenti-X 293T cells 72 hours after transfection was collected and coarsely 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. The filtered supernatant was then centrifuged (6000 g, 4° C., 20 hr) to pellet the lentiviral vector and the pellet was resuspended in 1.5 mL of PBS. Next, in an ultracentrifuge tube [Beckman Coulter (Tokyo, Japan), 344058], 55% sucrose/PBS solution (1 mL), 20% sucrose/PBS solution (2.5 mL), and the lentiviral vector solution (1.5 mL) were layered and ultracentrifuged (41000 rpm, 4° C., 2 hr). The ultracentrifuge and the rotor used were [Beckman Coulter (Tokyo, Japan), L-90K] and [Beckman Coulter (Tokyo, Japan), SW55Ti]. After ultracentrifugation, the lentiviral vector layer appearing between 55% sucrose/PBS solution and 20% sucrose/PBS solution was collected and diluted to 1 mL with PBS. Next, 20% sucrose/PBS solution (4 mL) and the lentiviral vector solution (1 mL) were layered in an ultracentrifugation tube, and ultracentrifuged again (41000 rpm, 4° C., 2 hr). The precipitated lentiviral vector pellet was well suspended in 400 μL of DMEM to obtain a lentiviral vector solution.
The lentiviral vector titer was determined 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 with no surface treatment [IWAKI (Tokyo, Japan), 1830-048] at 100 μL/well. The plate was then left overnight at 4° C. Before the plate was used, the RetroNectin solution was removed, and the plate was blocked with 2% FBS-containing PBS at room temperature for 30 minutes, followed by the following procedures.
Cell seeding and lentiviral infection: To the RetroNectin-coated 48-well plate, the LVSIN-EF1α-COL7A1 lentiviral vector or LVSIN-PGK-COL7A1 lentiviral vector (see
Based on the virus titer thus obtained, the volume of virus solution required for setting the Multiplicity Of Infection (MOI) in the lentivirus infection experiment was calculated.
We analyzed the efficiency of type VII collagen gene transfer by lentiviral vectors to blister-derived cells.
Preparation of RetroNectin-coated plate: A plate was coated with RetroNectin in the same manner as in “11. Production of lentiviral vector having type VII collagen gene”.
Cell seeding and lentiviral infection: To the RetroNectin-coated 48-well plate, the LVSIN-EF1α-COL7A1 lentiviral vector or LVSIN-PGK-COL7A1 lentiviral vector (see
Immunostaining: The blister-derived cells 14 days after lentivirus infection were detached from the plate with Accutase-Solution and collected by using a medium. The collected cells were counted, seeded on a CC2-coated chamber slide [Thermo Fisher Science (Tokyo, Japan), 154852] at 50000 cells/well, and cultured in a CO2 incubator. After 24 hours, immunostaining was performed by 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 with a confocal fluorescence microscope [Nikon (Tokyo, Japan), Nikon AIR HD25].
Results are shown in
We analyzed the efficiency of type VII collagen gene transfer by lentiviral vectors to blister-derived cells.
Preparation of lentivirus-infected cells: The cells were prepared in the same manner as described in “12. Analysis of type VII collagen gene transfer efficiency by immunostaining”.
FACS analysis: Blister-derived cells 14 days after lentivirus infection were detached from the plate with Accutase-Solution and collected by using a medium. The number of collected cells were measured and the cells were divided to 300000 cells/sample and permeabilized by using eBioscience™ Permeabilization Buffer [Thermo Fisher Science (Tokyo, Japan), 00-8333-56]. Then, immunostaining was performed by 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 by a flow cytometer (BD FACSCanto™ II) [Nippon Becton Dickinson (Tokyo, Japan)].
The results are shown in
Also, as a preliminary study, blister-derived cells, human bone marrow-derived mesenchymal stem cells, and normal human adult skin fibroblasts were infected with the EF1α-COL7A1 lentiviral vector or PGK-COL7A1 lentiviral vector, and immunostaining and flow cytometry (FACS) were performed in the same manner as above to measure the percentage of type VII collagen-positive cells. The blister-derived cells showed a higher percentage of type VII collagen-positive cells than the human bone marrow-derived mesenchymal stem cells and normal human adult skin fibroblasts.
VCN of blister-derived cells infected with the lentiviral vector having the type VII collagen gene was analyzed. First, the blister-derived cells were infected with a lentiviral vector in the same manner as in “12. Analysis of type VII collagen gene transfer efficiency by immunostaining”. The cells 7, 14, 21, and 28 days after infection were detached from the plate with Accutase-Solution and collected by using a medium. Next, from the collected cells, genomic DNA was extracted by using Maxwell® RSC Instrument [Promega (Tokyo, Japan), AS4500] and Maxwell® RSC Blood DNA Kit [Promega (Tokyo, Japan), AS1400]. VCN was calculated from the collected genomic DNA by using the Lenti-X™ Provirus Quantitation Kit [Takara Bio Inc. (Shiga, Japan), 631239].
The results are shown in
We examined whether the blister-derived cells transduced with the lentiviral vector having a type VII collagen gene supplied type VII collagen to the basement membrane of epidermolysis bullosa model mice. First, the full-thickness skin of a neonatal Col7A1 gene knockout mouse was transplanted to the back of an immunodeficient mouse. Immediately after the transplantation, the skin surface was pinched and rubbed to forma blister. Then, 1.0×106 blister-derived cells which were prepared in “12. Analysis of type VII collagen gene transfer efficiency by immunostaining” and infected with the LVSIN-EF1α-COL7A1 lentiviral vector at MOT 5 were immediately injected into the space under the epidermis (into the blister). To the mouse in the control group, 1.0×106 blister-derived cells not infected with the lentiviral vector were injected into the blister. One month later, the skin was collected and immunostained with an anti-type VII collagen antibody (clone LH7.2; Sigma Aldrich, C6805) to examine the deposition of type VII collagen at the basement membrane.
The results are shown in
From the above results, COL7A1 gene-delivered blister-derived cells are expected to exhibit higher therapeutic effects than bone marrow-derived mesenchymal stem cells and fibroblasts for gene therapy for dystrophic epidermolysis bullosa.
Number | Date | Country | Kind |
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2020-125620 | Jul 2020 | JP | national |
Filing Document | Filing Date | Country | Kind |
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PCT/JP2021/027279 | 7/21/2021 | WO |