Ex vivo gene therapy cell for producing blood coagulation factor VIII modifier

Abstract

The present invention addresses the problem of providing a novel cell for ex vivo gene therapy that produces an FVIII-modified body. A cell for isolated gene therapy according to the present invention, which is a means for solving the problem, produces a human FVIII modified body comprising the amino acid sequence at positions 20to 2351st in the amino acid sequence represented by SEQ ID NO: 1 in which the B domain (the amino acid sequence at positions 760 to 1667th) is modified, the number of Asn residues modified by an N-type sugar chain is 4 to 6 from the N-terminal side, and the number of Asn residues modified by the N-type sugar chain is 4 to 6 from the N-terminal side, and the number of Asn residues modified by the N-type sugar chain is 4 to 6 from the N-terminal side. And a modified FVIII having a defect in the function of the furin recognition site.

Description

Technical Field

[0001] The present invention relates to cells for ex vivo gene therapy that produce modified blood coagulation factor VIII.

Background Art

[0002] Blood coagulation factor VIII (hereinafter "FVIII") is one of the factors responsible for the hemostasis mechanism. Congenital abnormalities in the FVIII gene result in a deficiency or decrease in the blood FVIII concentration, leading to a hemostasis disorder called hemophilia A. For the treatment of hemophilia A, plasma-derived preparations of FVIII have been used for a long time, and nowadays, genetically recombinant preparations are also used clinically. Human FVIII is a glycoprotein composed of 2332 amino acid residues, and as a precursor composed of 2351 amino acid residues containing a signal peptide composed of 19 amino acid residues (the amino acid sequence is shown in SEQ ID NO: 1), after being expressed in cells of the main liver, it becomes a full-length body composed of 2332 amino acid residues through cleavage of the signal peptide or sugar chain modification, etc. The full-length body has a domain structure of A1 - A2 - B - A3 - C1 - C2, and there are linker regions called a1, a2, and a3 between the A1 domain and the A2 domain, between the A2 domain and the B domain, and between the B domain and the A3 domain, respectively. The full-length body is converted into a mature body by cleavage between Arg1648 at the C-terminus of the B domain and Glu1649 at the N-terminus of the a3 linker region (positions 1667 and 1668 in the amino acid sequence of SEQ ID NO: 1) by furin in the Golgi apparatus to become a double-stranded heavy chain and light chain, and after being secreted from the cell into the circulating blood, it is converted into an active form by cleavage between Arg372 at the C-terminus of the a1 linker region and Ser373 at the N-terminus of the A2 domain, between Arg740 at the C-terminus of the a2 linker region and Ser741 at the N-terminus of the B domain, and between Arg1689 at the C-terminus of the a3 linker region and Ser1690 at the N-terminus of the A3 domain by thrombin to exert its designated function.

[0003] Since hemophilia A is a genetic disease caused by a single gene and is considered a target disease suitable for gene therapy, in recent years, research and development of gene therapy for hemophilia A have been vigorously carried out (for example, Non-Patent Document 1). Gene therapy can be broadly classified into in vivo gene therapy, which directly administers a vector for expressing a target gene in a living body to a patient, and ex vivo gene therapy, which introduces a target gene into cells taken out from a patient and then returns the cells to the patient for expression of the target gene in the living body.

[0004] As a research and development of ex vivo gene therapy for hemophilia A, attempts have been made to, for example, use a lentiviral vector with excellent safety and effective integration of a target gene into the host cell genome, introduce a polynucleotide encoding an FVIII modified body that produces a B-domain-deleted human FVIII in vivo into cells taken from a patient, and then return the cells to the patient (ID: NCT03818763 introduced in Non-Patent Document 1). However, it can be said that the research and development of ex vivo gene therapy for hemophilia A is still in the developmental stage.

[0005]

Prior Art Documents

[0006]

Non-Patent Documents

[0007] Non-Patent Document 1: Hiroshi Kashiura et al., Progress in Gene Therapy for Hemophilia, Thrombosis and Hemostasis 2021; 32(1): 17-25

[0008]

Summary of the Invention

[0009]

Problems to be Solved by the Invention

[0010] The present invention aims to provide a novel ex vivo gene therapy cell that produces an FVIII modified body.

[0011]

Means for Solving the Problems

[0012] The ex vivo gene therapy cell of the present invention produces a modified body of human FVIII composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 (the B-domain (amino acid sequence at positions 760 to 1667) is altered, the number of Asn residues modified with N-type sugar chains is 4 to 6 from the N-terminal side, and the function of the furin protease recognition site is defective).

[0013] In addition, in the ex vivo gene therapy cell of the present invention, the FVIII modified body is any one of the following (a) to (c).

[0014] (a) A protein composed of the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2

[0015] (b) A protein composed of an amino acid sequence containing one or more deletions, substitutions, additions, or insertions in the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2 and having the same biological activity as the protein of (a)

[0016] (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2 and having the same biological activity as the protein of (a).

[0017] In addition, the ex vivo gene therapy cell of the present invention is such that the FVIII modifier is any one of the following (a) to (c).

[0018] (a) A protein consisting of the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3.

[0019] (b) A protein consisting of an amino acid sequence containing one or more amino acid residue deletions, substitutions, additions or insertions in the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3 and having the same biological activity as the protein of (a).

[0020] (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3 and having the same biological activity as the protein of (a).

[0021] In addition, the ex vivo gene therapy cell of the present invention is such that the FVIII modifier is any one of the following (a) to (c).

[0022] (a) A protein consisting of the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4.

[0023] (b) A protein consisting of an amino acid sequence containing one or more amino acid residue deletions, substitutions, additions or insertions in the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4 and having the same biological activity as the protein of (a).

[0024] (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4 and having the same biological activity as the protein of (a).

[0025] In addition, the ex vivo gene therapy cell of the present invention is such that the FVIII modifier is the Pro residue at position 758 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with another amino acid residue.

[0026] In addition, the cells for ex vivo gene therapy of the present invention are such that the other amino acid residues are polar uncharged amino acid residues selected from Thr residues, Ser residues, Asn residues, and Gln residues.

[0027] In addition, the cells for ex vivo gene therapy of the present invention are such that the FVIII modifier is any one of the following (a) to (c).

[0028] (a) A protein consisting of the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5

[0029] (b) A protein consisting of an amino acid sequence containing one or more deletions, substitutions, additions, or insertions in the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5 and having the same biological activity as the protein of (a)

[0030] (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5 and having the same biological activity as the protein of (a)

[0031] In addition, the cells for ex vivo gene therapy of the present invention are such that the FVIII modifier is any one of the following (a) to (c).

[0032] (a) A protein consisting of the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14

[0033] (b) A protein consisting of an amino acid sequence containing one or more deletions, substitutions, additions, or insertions in the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14 and having the same biological activity as the protein of (a)

[0034] (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14 and having the same biological activity as the protein of (a). In addition, the cells for ex vivo gene therapy of the present invention are such that the FVIII modifier is any one of the following (a) to (c).

[0035] (a) A protein consisting of the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15

[0036] (b) A protein composed of an amino acid sequence having one or more amino acid residue deletions, substitutions, additions or insertions in the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown by SEQ ID NO: 15, and having the same biological activity as the protein in (a).

[0037] (c) A protein composed of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown by SEQ ID NO: 15, and having the same biological activity as the protein in (a). Additionally, the ex vivo gene therapy cell of the present invention is such that the FVIII modifier is any one of the following (a) to (c).

[0038] (a) A protein composed of the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown by SEQ ID NO: 16.

[0039] (b) A protein composed of an amino acid sequence having one or more amino acid residue deletions, substitutions, additions or insertions in the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown by SEQ ID NO: 16, and having the same biological activity as the protein in (a).

[0040] (c) A protein composed of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown by SEQ ID NO: 16, and having the same biological activity as the protein in (a).

[0041] Additionally, the ex vivo gene therapy cell of the present invention is such that the cell is an adipocyte.

[0042] Additionally, the transplantation composition of the present invention contains the ex vivo gene therapy cell of the present invention and a pharmaceutically acceptable carrier. Additionally, the transplantation composition of the present invention further contains an extracellular matrix component.

[0043] Additionally, the transplantation composition of the present invention further contains an angiogenesis factor.

[0044] Additionally, the method for manufacturing the ex vivo gene therapy cell of the present invention is by introducing into a cell that can be transplanted into the body of a subject for ex vivo gene therapy an expression vector of a polynucleotide encoding a modifier of human FVIII (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of Asn residues modified with N - type sugar chains is 4 to 6 from the N - terminal side, and the function of the furin protease recognition site is defective) composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown by SEQ ID NO: 1).

[0045] In addition, the expression vector in the method for manufacturing cells for ex vivo gene therapy of the present invention is a viral vector.

[0046] In addition, the viral vector in the method for manufacturing cells for ex vivo gene therapy of the present invention is a lentiviral vector.

[0047] In addition, the expression vector for manufacturing the cells for ex vivo gene therapy of the present invention carries a polynucleotide encoding at least an amino acid sequence of a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of Asn residues modified with N-linked glycans is 4 to 6 from the N-terminal side, and the function of the furin recognition site is defective).

[0048] In addition, the modified FVIII of the present invention is a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of Asn residues modified with N-linked glycans is 4 to 6 from the N-terminal side, the function of the furin recognition site is defective, and furthermore, the Pro residue at position 758 is replaced with another amino acid residue).

[0049] In addition, in the modified FVIII of the present invention, the other amino acid residue is a polar uncharged amino acid residue selected from Thr residue, Ser residue, Asn residue, and Gln residue.

[0050] In addition, the polynucleotide of the present invention encodes at least the amino acid sequence of the modified FVIII of the present invention.

[0051] In addition, the composition for transplantation of the present invention further contains cells that produce human vWF and / or its fragments in a living body.

[0052] In addition, the cells of the present invention produce a fragment of human vWF in a living body.

[0053] In addition, in the cells of the present invention, the fragment of human vWF is a fragment containing at least the D' domain and the D3 domain as the binding sites for FVIII.

[0054] In addition, the cell of the present invention is such that the fragment of human vWF has the domain structure of SP-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK that the precursor has, has each of the domains of SP, D', and D3, does not have each of the domains of A2, A3, D4, B1, B2, B3, C1, and C2, and the A1 domain may have at most 50 amino acid residues on the N-terminal side, and may optionally have or not have fragments of each of the domains of D1, D2, and CK.

[0055] In addition, the cell of the present invention is such that the fragment of human vWF is any one of the following (a) to (c).

[0056] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 29

[0057] (b) A protein consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 29 and having the same biological activity as the protein of (a)

[0058] (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 29 and having the same biological activity as the protein of (a)

[0059] In addition, the cell of the present invention is such that the fragment of human vWF is any one of the following (a) to (c).

[0060] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 30

[0061] (b) A protein consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 30 and having the same biological activity as the protein of (a)

[0062] (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 30 and having the same biological activity as the protein of (a)

[0063] In addition, the cell of the present invention is such that the fragment of human vWF is any one of the following (a) to (c).

[0064] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 31

[0065] (b) A protein consisting of an amino acid sequence with one or more amino acid residue deletions, substitutions, additions, or insertions in the amino acid sequence shown in SEQ ID NO: 31 and having the same biological activity as the protein in (a).

[0066] (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 31 and having the same biological activity as the protein in (a). In addition, the medicament of the present invention is used to increase the secretion amount of the FVIII modifier into the blood generated by transplanting the ex vivo gene therapy cells of the present invention into the body of a subject for ex vivo gene therapy. The cells that produce human vWF and / or its fragments in vivo are used as the active ingredient.

[0067] In addition, the transplantation composition of the present invention contains cells that produce human vWF and / or its fragments in vivo and a pharmaceutically acceptable carrier.

[0068] In addition, the group of the transplantation composition of the present invention includes a transplantation composition containing the ex vivo gene therapy cells of the present invention and a pharmaceutically acceptable carrier, and a transplantation composition containing cells that produce the human vWF and / or its fragments of the present invention in vivo and a pharmaceutically acceptable carrier.

[0069]

Effects of the Invention

[0070] The present invention can provide new ex vivo gene therapy cells that produce FVIII modifiers.

[0071]

Brief Description of the Drawings

[0072]

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Figure 16

DETAILED DESCRIPTION

[0088] The cell for ex vivo gene therapy of the present invention produces a modified form of human FVIII consisting of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of Asn residues modified with N-linked sugar chains is 4 to 6 from the N-terminal side, and the FVIII modified form has a functional defect in the furin recognition site).

[0089] The FVIII modified form in the present invention is a modified form of human FVIII consisting of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 (a full-length form consisting of 2332 amino acid residues), and the B domain (amino acid sequence at positions 760 to 1667) is altered, and it has the following two structural characteristics in total.

[0090] (A) The number of Asn residues modified with N-linked sugar chains is 4 to 6 from the N-terminal side

[0091] (B) Functional defect in the furin recognition site

[0092] FVIII modifiers with the structural feature (A) are known from the literature of Hongzhi Z. Miao et al. (Bioengineering of coagulation factor VIII for improved secretion. Blood. 2004; 103: 3412-3419). In the B domain of human FVIII, there are 19 Asn residues modified with N-linked glycans. In this literature, it was reported that genes of FVIII modifiers with amino acid sequence alterations in the B domain, where one additional Asn residue modified with N-linked glycan was added to the N-terminal side of the B domain, were introduced into COS-1 cells. When produced in vitro, up to the modifier (N8) with an amino acid sequence having 8 Asn residues was obtained, and the secretion amount of the modifier increased according to the increase in the number of Asn residues (however, the secretion amount of the modifier (N7) with an amino acid sequence having 7 Asn residues was not described).

[0093] FVIII modifiers with the structural feature (B) are known from the literature of G.N. Nguyen et al. (Novel factor VIII variants with a Modified furin cleavage site improve the efficacy of gene therapy for hemophilia A. Journal of Thrombosis and Haemostasis. 2017; 15: 110-121). In this literature, it was reported that by mutating the furin recognition site (Arg1645-His1646-Gln1647-Arg1648 in the full-length form) consisting of 4 amino acid residues counted from the C-terminal side of the B domain of the FVIII modifier (human FVIII-BDD) that retains only 3 amino acid residues counted from the N-terminal side and 11 amino acid residues counted from the C-terminal side (both without Asn residues modified with N-linked glycans) of the B domain of human FVIII to render it functionally defective, the gene of the FVIII modifier (human FVIII-BDDΔfurin) in which cleavage between Arg1648 at the C-terminal of the B domain and Glu1649 at the N-terminal of the a3 linker region by furin does not occur was introduced into hemophilia A / CD4 knockout mice using an adeno-associated virus vector, and the secretion amount of human FVIII-BDDΔfurin produced in vivo was higher than that of human FVIII-BDD produced.

[0094] However, in the literature of Hongzhi Z. Miao et al., there is no record of using an FVIII modifier with the structural feature (A) in ex vivo gene therapy. In the literature of G. N. Nguyen et al., there is no record of using an FVIII modifier with the structural feature (B) in ex vivo gene therapy.

[0095] In addition, an FVIII modifier having both the structural feature (A) and the structural feature (B) is described in Japanese Patent Laid-Open No. 2019-527541, and is also mentioned in the use of a polynucleotide encoding its amino acid sequence in gene therapy, but there is no specific description or evidence.

[0096] In the present invention, "the number of N-glycosylated Asn residues with the structural feature (A) is 4 to 6 from the N-terminal side" corresponds to modifier N4, modifier N5, and modifier N6 in the literature of Hongzhi Z. Miao et al.

[0097] In the present invention, for the "functional defect of the furin recognition site" of the structural feature (B), as long as it is an embodiment in which cleavage does not occur between Arg1648 at the C-terminus of the B domain and Glu1649 at the N-terminus of the a3 linker region by furin, there is no particular limitation. Examples include an embodiment in which at least one of the amino acid residues constituting the furin recognition site composed of 4 amino acid residues counted from the C-terminal side of the B domain is deleted, or an embodiment in which it is replaced with one or more other amino acid residues.

[0098] In the present invention, as a specific example of the FVIII modifier having both the structural feature (A) and the structural feature (B), a protein (abbreviation: N4Δfurin) composed of the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2, where the number of N-glycosylated Asn residues with the structural feature (A) is 4 from the N-terminal side, can be cited.

[0099] As another specific example, a protein (abbreviation: N5Δfurin) composed of the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3, where the number of N-glycosylated Asn residues with the structural feature (A) is 5 from the N-terminal side, can be cited.

[0100] As another specific example, a protein (abbreviation: N6Δfurin) composed of the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4, where the number of N-glycosylated Asn residues with the structural feature (A) is 6 from the N-terminal side, can be cited.

[0101] In addition, in the FVIII modifier of the present invention, the Pro residue at position 758 in the amino acid sequence shown in SEQ ID NO: 1 can also be replaced with other amino acid residues. The Pro residue at position 758 (Pro739 in the full-length form) in the amino acid sequence shown in SEQ ID NO: 1 is located at the thrombin recognition site for cleavage by thrombin between Arg740 at the C-terminus of the a2 linker region and Ser741 at the N-terminus of the B domain. By replacing this Pro residue with a polar uncharged amino acid residue such as a Thr residue, Ser residue, Asn residue, Gln residue, etc., the secretion amount into the blood (increase in blood concentration) or specific activity (activity equivalent to the amount of antigen) can be increased.

[0102] In addition to the common structural feature (A) and the structural feature (B), as a specific example of the FVIII modifier in which the Pro residue at position 758 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with a Thr residue, there can be mentioned a protein (abbreviation: N5Δfurin(P739T)) composed of the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5, where the number of Asn residues modified with N-type sugar chains in the structural feature (A) is 5 when counted from the N-terminal side. In addition to the common structural feature (A) and the structural feature (B), as a specific example of the FVIII modifier in which the Pro residue at position 758 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with a Ser residue, there can be mentioned a protein (abbreviation: N5Δfurin(P739S)) composed of the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14, where the number of Asn residues modified with N-type sugar chains in the structural feature (A) is 5 when counted from the N-terminal side. In addition to the common structural feature (A) and the structural feature (B), as a specific example of the FVIII modifier in which the Pro residue at position 758 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with an Asn residue, there can be mentioned a protein (abbreviation: N5Δfurin(P739N)) composed of the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15, where the number of Asn residues modified with N-type sugar chains in the structural feature (A) is 5 when counted from the N-terminal side. In addition to the common structural feature (A) and the structural feature (B), as a specific example of the FVIII modifier in which the Pro residue at position 758 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with a Gln residue, there can be mentioned a protein (abbreviation: N5Δfurin(P739Q)) composed of the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16, where the number of Asn residues modified with N-type sugar chains in the structural feature (A) is 5 when counted from the N-terminal side.

[0103] The FVIII modifier in the present invention is the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16. In each of the amino acid sequences, there is a deletion, substitution, addition or insertion of one or more amino acid residues, and the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16, and a protein having the same biological activity as the protein composed of each amino acid sequence.In addition, it is composed of the amino acid sequences at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequences at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16, and amino acid sequences having at least 90% sequence identity with the respective amino acid sequences. Moreover, the amino acid sequences at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequences at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16, are proteins having the same biological activity as the proteins composed of the respective amino acid sequences. Among them, "a plurality" refers to an integer of 50 or less, preferably an integer of 30 or less, more preferably an integer of 20 or less, still more preferably an integer of 10 or less, such as 2 to 9, 2 to 7, 2 to 5, 2 to 3.The amino acid sequences at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequences at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15, the amino acid sequences at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16, and the sequence identity with their respective amino acid sequences is preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and still more preferably at least 99%. Furthermore, the % of identity refers to the value calculated using software (e.g., FASTA, DANASYS, BLAST, etc.) for calculating the identity between multiple (two) amino acid sequences with default settings. "Equivalent biological activity" means, for example, the expression of the function as a coagulation factor possessed by FVIII, or biological activities such as its activation or inactivation that are the same in nature.

[0104] The cells used for manufacturing the cells for ex vivo gene therapy of the present invention are not particularly limited as long as they can be transplanted into the body of the subject of ex vivo gene therapy and produce the FVIII modifier in the present invention (expressed as a protein bound to the signal peptide composed of the amino acid sequence at positions 1 to 19 in the amino acid sequence shown in SEQ ID NO: 1) in the body. As a specific example, adipocytes can be mentioned. Among adipocytes, in addition to cells that store fat in a spherical shape and mature adipocytes containing oil droplets, preadipocytes having the ability to differentiate into mature adipocytes are included. The adipocytes can be autologous cells or allogeneic cells. For the cells for ex vivo gene therapy using adipocytes, since it is described in detail in Japanese Patent Publication No. 4879867, the matters described in Japanese Patent Publication No. 4879867 can be appropriately referred to in the present invention.

[0105] The cells for ex vivo gene therapy of the present invention can be produced by introducing an expression vector of a polynucleotide encoding an amino acid sequence of a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 into cells taken from, for example, a subject for ex vivo gene therapy or a healthy person, such as adipocytes collected from adipose tissue by a known method. The modified FVIII has the B domain (amino acid sequence at positions 760 to 1667) altered, the number of Asn residues for N-glycosylation modification is 4 to 6 counted from the N-terminal side, and the function of the furin recognition site is defective.

[0106] A method for introducing an expression vector of a polynucleotide encoding an amino acid sequence of a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of Asn residues modified with N-linked sugar chains is 4 to 6 from the N-terminal side, and the Furin protease recognition site is functionally defective) adopts any known method. The expression vector is preferably a viral vector that is excellent in terms of gene transfer efficiency into cells, replication and maintenance in cells, stability, expression efficiency, etc. As the viral vector, known ones such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, etc. can be used. Among them, a lentiviral vector that is preferably excellent in terms of safety and can effectively integrate the target gene into the host cell genome and enable long-term stable gene expression is used. Gene transfer into target cells using a lentiviral vector can be carried out, for example, by co-transfecting a packaging cell such as 293T cells or COS cells with a packaging plasmid and a lentiviral vector plasmid derived from human immunodeficiency virus type-1 (HIV-1) integrating the target gene, and then infecting the target cells with the lentiviral vector carrying the target gene obtained as the cell culture supernatant (protamine sulfate or polybrene, etc. can also be added to improve the infection efficiency) by a known method. Tools for producing lentiviral vectors carrying the target gene are also commercially available (such as Takara Bio Inc., Funakoshi Co., Ltd., or COSMO-BIO Co., Ltd., etc.). A lentiviral vector of a polynucleotide encoding an amino acid sequence of a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of Asn residues modified with N-linked sugar chains is 4 to 6 from the N-terminal side, and the Furin protease recognition site is functionally defective) can be obtained as the cell culture supernatant after co-transfecting a packaging cell such as 293T cells or COS cells with a packaging plasmid and a lentiviral vector plasmid of a polynucleotide encoding an amino acid sequence of a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of Asn residues modified with N-linked sugar chains is 4 to 6 from the N-terminal side, and the Furin protease recognition site is functionally defective) as the target gene.

[0107] A polynucleotide encoding an amino acid sequence of a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of N-glycosylation-modified Asn residues is 4 to 6 counted from the N-terminal side, and the Furin protease recognition site is functionally defective), for example, encoding a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of N-glycosylation-modified Asn residues is 4 to 6 counted from the N-terminal side, and the Furin protease recognition site is functionally defective) is a polynucleotide of the amino acid sequence of a protein that binds to a signal peptide composed of the amino acid sequence at positions 1 to 19 in the amino acid sequence shown in SEQ ID NO: 1. A polynucleotide encoding the amino acid sequence of a protein that binds to a signal peptide composed of the amino acid sequence at positions 1 to 19 in the amino acid sequence shown in SEQ ID NO: 1 and a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of N-glycosylation-modified Asn residues is 4 to 6 counted from the N-terminal side, and the Furin protease recognition site is functionally defective) can be prepared, for example, by chemical synthesis based on the amino acid sequence of a protein that binds to a signal peptide composed of the amino acid sequence at positions 1 to 19 in the amino acid sequence shown in SEQ ID NO: 1 and a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of N-glycosylation-modified Asn residues is 4 to 6 counted from the N-terminal side, and the Furin protease recognition site is functionally defective) (if necessary, the base sequence codons can also be optimized).

[0108] Specific examples of the polynucleotide of the amino acid sequence of the protein in which a modified human FVIII (the B domain (amino acid sequence positions 760 to 1667) is altered, the number of Asn residues modified with N-type sugar chains is 4 to 6 from the N-terminal side, and the function of the furin recognition site is defective) consisting of the amino acid sequence positions 20 to 2351 shown in SEQ ID NO: 1 is bound to the signal peptide consisting of the amino acid sequence positions 1 to 19 shown in SEQ ID NO: 1 include the polynucleotide consisting of the base sequence shown in SEQ ID NO: 6 (polynucleotide encoding the amino acid sequence of the protein in which N4Δfurin consisting of the amino acid sequence positions 1 to 1607 shown in SEQ ID NO: 2 is bound to the signal peptide), the polynucleotide consisting of the base sequence shown in SEQ ID NO: 7 (polynucleotide encoding the amino acid sequence of the protein in which N5Δfurin consisting of the amino acid sequence positions 1 to 1652 shown in SEQ ID NO: 3 is bound to the signal peptide), the polynucleotide consisting of the base sequence shown in SEQ ID NO: 8 (polynucleotide encoding the amino acid sequence of the protein in which N6Δfurin consisting of the amino acid sequence positions 1 to 1670 shown in SEQ ID NO: 4 is bound to the signal peptide), the polynucleotide consisting of the base sequence shown in SEQ ID NO: 9 (polynucleotide encoding the amino acid sequence of the protein in which N5Δfurin(P739T) consisting of the amino acid sequence positions 1 to 1652 shown in SEQ ID NO: 5 is bound to the signal peptide), the polynucleotide consisting of the base sequence shown in SEQ ID NO: 17 (polynucleotide encoding the amino acid sequence of the protein in which N5Δfurin(P739S) consisting of the amino acid sequence positions 1 to 1652 shown in SEQ ID NO: 14 is bound to the signal peptide), the polynucleotide consisting of the base sequence shown in SEQ ID NO: 18 (polynucleotide encoding the amino acid sequence of the protein in which N5Δfurin(P739N) consisting of the amino acid sequence positions 1 to 1652 shown in SEQ ID NO: 15 is bound to the signal peptide), and the polynucleotide consisting of the base sequence shown in SEQ ID NO: 19 (polynucleotide encoding the amino acid sequence of the protein in which N5Δfurin(P739Q) consisting of the amino acid sequence positions 1 to 1652 shown in SEQ ID NO: 16 is bound to the signal peptide).However, the polynucleotide of the amino acid sequence of the protein that binds to the signal peptide consisting of the amino acid sequence at positions 1 to 19 in the amino acid sequence shown in SEQ ID NO: 1, for the modified human FVIII composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of Asn residues modified with N-linked sugar chains is 4 to 6 from the N-terminal side, and the modified FVIII with a functional defect in the furin recognition site) is not limited to these polynucleotides, and is a polynucleotide that hybridizes under stringent conditions as a complementary strand to these polynucleotides, and contains at least the polynucleotide encoding the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16, and a polynucleotide of a protein having the same biological activity as the protein composed of each amino acid sequence. In addition, a polynucleotide having at least 90%, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and still more preferably at least 99% sequence identity with the base sequences shown in SEQ ID NOs: 6 to 9, 17 to 19, and contains at least the polynucleotide encoding the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15, the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16, and a polynucleotide of a protein having the same biological activity as the protein composed of each amino acid sequence.Among them, "hybridization under stringent conditions" includes, for example, hybridization for 1 to 24 hours in 30 to 50 °C, 3 to 4×SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.2), 0.1 to 0.5% SDS, preferably hybridization for 1 to 24 hours in 40 to 45 °C, 3.4×SSC, 0.3% SDS, and subsequent washing. As the washing conditions, for example, conditions such as continuous washing at room temperature with a solution containing 2×SSC and 0.1% SDS, 1×SSC solution, and 0.2×SSC solution can be cited. However, by exemplifying the combination of the above conditions, those skilled in the art can achieve the same stringency as above by appropriately combining the above elements or other elements (for example, the concentration, length, and GC content of the hybridization probe, the reaction time of hybridization, etc.) that determine the stringency of hybridization.

[0109] Since the cells for ex vivo gene therapy of the present invention are transplanted into the body of a subject for ex vivo gene therapy of hemophilia A, a modified form of human FVIII (the B domain (amino acid sequence at positions 760 to 1667) is altered, the number of Asn residues modified with N-linked sugar chains is 4 to 6 from the N-terminal side, and the FVIII modified form with a functional defect in the furin recognition site) is produced in vivo, and is useful for ex vivo gene therapy of hemophilia A. The number of transplanted cells of the cells for ex vivo gene therapy of the present invention is determined in consideration of the age, body weight, sex, disease state or severity, etc. of the subject for ex vivo gene therapy.

[0110] When the cells for ex vivo gene therapy of the present invention can be produced using adipocytes, they are transplanted by injecting them into the subcutaneous tissue or adipose tissue by a known method. At this time, it is preferable to transplant the cells for ex vivo gene therapy of the present invention together with a pharmaceutically acceptable carrier such as physiological saline, Ringer's solution, phosphate buffer solution, culture solution, serum, body fluid, etc. as a transplantation composition. The transplantation composition may also contain various extracellular matrix components (for example, collagen, laminin, heparan sulfate, gelatin) as scaffold raw materials. In addition, the transplantation composition may contain angiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) (which may be of natural origin or produced by genetic engineering methods) so as to form blood vessels around after transplantation and secrete the produced FVIII modified form into the blood with high efficiency.

[0111] In addition, the cells for ex vivo gene therapy of the present invention can also be transplanted into the body of a subject for ex vivo gene therapy together with cells that produce human von Willebrand factor (hereinafter "vWF") or a fragment thereof (for example, a fragment containing at least the D' domain and the D3 domain as the binding site for FVIII) in vivo, which contributes to the stabilization or transport of FVIII in the circulating blood. Interestingly, by transplanting the cells for ex vivo gene therapy of the present invention into the body of a subject for ex vivo gene therapy together with cells that produce human vWF or a fragment thereof in vivo, the cells that produce human vWF or a fragment thereof in vivo play a role in increasing the secretion amount (raising the blood concentration) of the FVIII modifier produced from the cells for ex vivo gene therapy of the present invention into the blood.

[0112] Human vWF is a glycoprotein that is a precursor composed of 2,813 amino acid residues containing a signal peptide composed of 22 amino acid residues (its amino acid sequence is shown in SEQ ID NO: 28) and is expressed in vascular endothelial cells or bone marrow megakaryocytes. The precursor has a domain structure of SP-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK. When the signal peptide (SP) is cleaved, it is composed of a propeptide (D1-D2) composed of 741 amino acid residues and a mature subunit (D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK) composed of 2,050 amino acid residues, becoming pro-vWF. After pro-vWF forms a dimer by S-S bonding at the C-terminal side, it exerts its designated function through the formation of a multimer in which dimers are S-S bonded to each other at the N-terminal side, glycosylation modification, cleavage of the propeptide, and the like.

[0113] It can also be transplanted into the body of a subject for ex vivo gene therapy together with the cells for ex vivo gene therapy of the present invention. As cells that produce in vivo a fragment of human vWF containing at least the D' domain and the D3 domain as the binding sites for FVIII, for example, cells that produce in vivo among the domain structures of SP-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK possessed by the precursor, and have each of the SP, D', and D3 domains, and do not have each of the A2, A3, D4, B1, B2, B3, C1, and C2 domains. The A1 domain may have at most 50 amino acid residues on the N-terminal side, and may optionally have or not have each of the D1, D2, and CK domain fragments. Among them, the amino acid sequences of each of the SP, D1, D2, D', D3, A1, and CK domains are, respectively, the amino acid sequences at positions 1 to 22, positions 23 to 386, positions 387 to 763, positions 764 to 864, positions 865 to 1242, positions 1243 to 1479, and positions 2664 to 2813 in the amino acid sequence shown in SEQ ID NO: 28 possessed by the precursor.

[0114] Among the domain structures of SP-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK possessed by the precursor, as for each domain having SP, D', and D3, and lacking each domain of A2, A3, D4, B1, B2, B3, C1, and C2, the A1 domain can have up to 50 amino acid residues on the N-terminal side, and can also be a specific example of an optional domain fragment having or lacking each of D1, D2, and CK. An example can be a fragment composed of each domain of SP, D1, D2, D', D3 and 26 amino acid residues counted from the N-terminal side of the A1 domain, further 3 amino acid residues, and the CK domain (a protein composed of the amino acid sequence shown in SEQ ID NO: 29, abbreviated as: vWF1). "26 amino acid residues counted from the N-terminal side of the A1 domain" corresponds to the amino acid residues constituting the amino acid sequence at positions 1243 - 1268 in the amino acid sequence shown in SEQ ID NO: 28 possessed by the precursor, and contains multiple amino acid residues (serine, threonine) to which O-glycans are bound. "The further 3 amino acid residues" correspond to each of the amino acid residues at positions 1269, 1270, and 2663 in the amino acid sequence shown in SEQ ID NO: 28 possessed by the precursor. That is, the amino acid sequence of vWF1 is the amino acid sequence at positions 1 - 1270 and the amino acid sequence at positions 2663 - 2813 in the amino acid sequence shown in SEQ ID NO: 28 possessed by the precursor, which are linked. Additionally, among the domain structures of SP-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK possessed by the precursor, for each domain having SP, D', and D3, and lacking each domain of A2, A3, D4, B1, B2, B3, C1, and C2, the A1 domain can have up to 50 amino acid residues on the N-terminal side, and can also be an optional domain fragment having or lacking each of D1, D2, and CK. It can also be a fragment composed of each domain of SP, D1, D2, D', D3 and 26 amino acid residues counted from the N-terminal side of the A1 domain (a protein composed of the amino acid sequence shown in SEQ ID NO: 30, abbreviated as: vWF2) or a fragment composed of each domain of SP, D', D3 and 26 amino acid residues counted from the N-terminal side of the A1 domain (a protein composed of the amino acid sequence shown in SEQ ID NO: 31, abbreviated as: vWF3), etc.

[0115] Fragments of human vWF, for example, in the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, have one or more amino acid residue deletions, substitutions, additions, or insertions, and proteins containing the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and having the same biological activity as the proteins composed of the respective amino acid sequences. In addition, proteins composed of amino acid sequences having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and having the same biological activity as the proteins composed of the respective amino acid sequences. Among them, "a plurality" refers to an integer of 50 or less, preferably an integer of 30 or less, more preferably an integer of 20 or less, still more preferably an integer of 10 or less, such as 2 to 9, 2 to 7, 2 to 5, 2 to 3. The sequence identity between the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and the respective amino acid sequences is preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, still more preferably at least 99%. Furthermore, the % of identity refers to the value calculated using software (such as FASTA, DANASYS, BLAST, etc.) for calculating the identity between multiple (two) amino acid sequences with the default settings. "The same biological activity" means, for example, the same biological activity as the function of vWF (such as stabilization or transport of FVIII in circulating blood).

[0116] It can also be transplanted into the body of the subject of ex vivo gene therapy together with the cells for ex vivo gene therapy of the present invention. Cells capable of producing human vWF or its fragments in vivo can be prepared by introducing an expression vector carrying a polynucleotide encoding at least the amino acid sequence of human vWF or its fragments into cells taken from, for example, the subject of ex vivo gene therapy or a healthy person, such as adipocytes collected from adipose tissue by a known method.

[0117] As specific examples of polynucleotides having an amino acid sequence fragment encoding human vWF, there may be mentioned a polynucleotide consisting of the base sequence shown in SEQ ID NO: 32 (a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 29), a polynucleotide consisting of the base sequence shown in SEQ ID NO: 33 (a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 30), and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 34 (a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 31). However, the polynucleotides having an amino acid sequence fragment encoding human vWF are not limited to these polynucleotides, and include polynucleotides that hybridize with the complementary strands of these polynucleotides under stringent conditions and encode at least the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and proteins having the same biological activity as the proteins composed of the respective amino acid sequences. In addition, polynucleotides having at least 90%, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and still more preferably at least 99% sequence identity with the base sequences shown in SEQ ID NOs: 32 to 34, and encoding at least the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and proteins having the same biological activity as the proteins composed of the respective amino acid sequences. Among them, "hybridization under stringent conditions" includes, for example, hybridization at 30 to 50 °C, 3 to 4 × SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.2), 0.1 to 0.5% SDS for 1 to 24 hours, preferably hybridization at 40 to 45 °C, 3.4 × SSC, 0.3% SDS for 1 to 24 hours, and subsequent washing. As washing conditions, there may be mentioned, for example, conditions such as continuous washing at room temperature with a solution of 2 × SSC and 0.1% SDS, and 1 × SSC solution and 0.2 × SSC solution. However, the combinations of the above conditions are illustrative, and those skilled in the art can achieve the same stringency as above by appropriately combining the above elements or other elements (e.g., concentration, length, and GC content of the hybridization probe, reaction time of hybridization, etc.) that determine the stringency of hybridization.

[0118] As a method of transplanting, into the body of a subject for ex vivo gene therapy, cells that produce human vWF or a fragment thereof in a living body together with the ex vivo gene therapy cells of the present invention, for example, a method of transplanting a transplantation composition containing the above-described ex vivo gene therapy cells of the present invention and a pharmaceutically acceptable carrier, which further contains cells that produce human vWF or a fragment thereof in a living body, can be mentioned. However, differently from the transplantation composition containing the ex vivo gene therapy cells of the present invention and a pharmaceutically acceptable carrier, a transplantation composition containing cells that produce human vWF or a fragment thereof in a living body and a pharmaceutically acceptable carrier may be prepared, and the two transplantation compositions may be transplanted simultaneously or at regular intervals as a group. At this time, the pharmaceutically acceptable carrier in the transplantation composition containing cells that produce human vWF or a fragment thereof in a living body and a pharmaceutically acceptable carrier may be the same as the pharmaceutically acceptable carrier in the transplantation composition containing the ex vivo gene therapy cells of the present invention and a pharmaceutically acceptable carrier. In addition, similar to the transplantation composition containing the ex vivo gene therapy cells of the present invention and a pharmaceutically acceptable carrier, the transplantation composition containing cells that produce human vWF or a fragment thereof in a living body and a pharmaceutically acceptable carrier may contain various extracellular matrix components, angiogenic factors, or the like. When transplanting, into the body of a subject for ex vivo gene therapy, cells that produce human vWF or a fragment thereof in a living body together with the ex vivo gene therapy cells of the present invention, the ratio of the number of transplanted cells of the ex vivo gene therapy cells of the present invention and the cells that produce human vWF or a fragment thereof in a living body is determined from the range of, for example, 1:100 to 100:1, taking into account the number of gene-introduced copies in each cell, etc.

[0119]

Examples

[0120] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not to be construed as being limited to the following description.

[0121]

Experiment A: Investigation of Gene Transfer of Various FVIII Modifiers into Preadipocytes (Part 1)

[0122]

Experiment 1: Production of Preadipocytes into Which an FVIII Modifier Gene Has Been Transferred

[0123] (1) Production of a Lentiviral Vector Plasmid That Produces an FVIII Modifier

[0124] By synthesizing a polynucleotide with a base sequence corresponding to the amino acid sequence of a protein that binds to a signal peptide and is conjugated to each of the following 5 FVIII modifiers, with a restriction enzyme site of SbfI added at the 5'-end and MluI added at the 3'-end, and inserting it into the SbfI / MluI site of pLVSIN-EF1αNeo Vector (Takara Bio Inc., the same hereinafter), which is a SIN-type lentiviral vector, a lentiviral vector plasmid that produces an FVIII modifier was prepared. Furthermore, each base sequence was codon-optimized based on each amino acid sequence using GeneArt GeneOptimizer software (Thermo Fisher Scientific Inc.).

[0125] ……

[0126] (a) A base sequence (SEQ ID NO: 11) corresponding to the amino acid sequence (SEQ ID NO: 10, retaining substantially all of the B domain with a functional deletion of the furin recognition site) of a protein that binds to a signal peptide and is conjugated to NovoEight (Novo Nordisk Pharma A / S, Eur J Haematol. 2014 Nov;93(5):369 - 376) (ΔB), which is a recombinant B domain-deleted human FVIII preparation

[0127] (b) A base sequence (SEQ ID NO: 13) corresponding to the amino acid sequence (SEQ ID NO: 12) of a protein that binds to a signal peptide and is conjugated to an FVIII modifier (N6) in which the number of Asn residues modified with N-linked glycans in the B domain of human FVIII is 6 counted from the N-terminal side and that retains the function of the furin recognition site

[0128] (c) A base sequence (SEQ ID NO: 8) corresponding to the amino acid sequence (SEQ ID NO: 4) of a protein that binds to a signal peptide and is conjugated to an FVIII modifier (N6Δfurin) in which the number of Asn residues modified with N-linked glycans in the B domain of human FVIII is 6 counted from the N-terminal side and that has a defective function of the furin recognition site

[0129] (d) A base sequence (SEQ ID NO: 7) corresponding to the amino acid sequence (SEQ ID NO: 3) of a protein that binds to a signal peptide and is conjugated to an FVIII modifier (N5Δfurin) in which the number of Asn residues modified with N-linked glycans in the B domain of human FVIII is 5 counted from the N-terminal side and that has a defective function of the furin recognition site

[0130] (e) The number of Asn residues modified with N-linked oligosaccharides in the B domain of human FVIII is set to 5 when counted from the N-terminal side, and the function of the signal peptide binding to the furin recognition site is defective. Furthermore, the base sequence (SEQ ID NO: 9) corresponding to the amino acid sequence (SEQ ID NO: 5) of the FVIII modifier (N5Δfurin(P739T)) in which the Pro residue at position 758 is replaced with a Thr residue

[0131] ……

[0132] The differences (positions in the full-length form) in the amino acid sequences before and after the B domain of the above 5 FVIII modifiers are shown in Figure 1 . In addition, the composition of the lentiviral vector plasmid that produces N5Δfurin(P739T) is shown in Figure 2 .

[0133] (2) Production of a lentiviral vector expressing an FVIII modifier

[0134] The lentiviral vector plasmid (5.5 μg) expressing the FVIII modifier prepared in (1), serum-free DMEM (1.5 mL), Lentiviral High Titer Packaging Mix (Takara Bio Inc., 7 μL), and PEIpro transfection reagent (Avantor, 45 μL) were mixed by pipetting up and down, and left standing at room temperature for 15 minutes to prepare a transfection solution. Approximately 24 hours before transfection, the transfection solution was dropped onto Lenti-X 293T cells (Takara Bio Inc.) seeded and cultured in a 10 cm dish in 1 × 10 7 cells / 10 mL of DMEM containing 10% FBS, and the culture was continued. Approximately 24 hours after transfection, the medium was replaced with fresh DMEM containing 10 mL of 10% FBS, and the culture was further continued. The cells were cultured in a 37 °C, 5% CO2 incubator. Approximately 48 hours after transfection, the virus-containing culture supernatant was collected, centrifuged, and filtered through a filter. The lentiviral vector expressing the FVIII modifier was obtained as the supernatant.

[0135] (3) Obtaining human preadipocytes transfected with the FVIII modifier gene

[0136] 1 g of adipose tissue obtained from healthy volunteers was dispensed into 50 mL centrifuge tubes. After adding 3 mL of Hank's balanced salt solution containing 2 mg / mL collagenase (SIGMA), it was shaken at 37°C for 1 hour. After 1 hour, 10 mL of Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham (DMEM-HAM / 20% FBS, SIGMA, the same below) containing 20% FBS and 40 μg / mL gentamicin was added and stirred, followed by centrifugation at 400 g for 1 minute to remove the solution containing the precipitated fraction. Furthermore, a series of operations of adding 10 mL of DMEM-HAM / 20% FBS, stirring, and centrifuging at 400 g for 1 minute to remove the solution containing the precipitated fraction were performed twice to obtain the supernatant fraction containing preadipocytes. The supernatant fraction containing preadipocytes thus obtained was filtered through a 500 μm mesh into a new 50 mL centrifuge tube. Then, 10 mL of DMEM-HAM / 20% FBS was added to the original 50 mL centrifuge tube for washing, and the washing solution was also filtered. The filtrate thus obtained was added to a T150 flask pre-warmed at 37°C and filled with DMEM-HAM / 20% FBS, and DMEM-HAM / 20% FBS was further added in such a way that there were as few bubbles as possible remaining in the flask. Thereafter, the flask was set with the bottom surface as the culture surface (ceiling surface) and cultured in a 37°C, 5% CO2 incubator for 7 days. After 7 days, the culture medium in the flask was removed, the culture surface was washed with PBS, and then the preadipocytes were recovered by trypsin treatment. The centrifuged preadipocytes were suspended in a cell cryopreservation solution (CELLBANKER1: Takara Bio Inc., the same below) and frozen.

[0137] (4) Preparation of preadipocytes transfected with the FVIII modified gene

[0138] By thawing the preadipocytes frozen in (3), suspending them in MSF-BM / MSF-Supplement A (Shimadzu Diagnostics, the same below), centrifuging to remove the cell cryopreservation solution, then resuspending them in MSF-BM / MSF-Supplement A, placing them in a T225 flask, and culturing them in a 37°C, 5% CO2 incubator for 4 days using MSF-BM / MSF-Supplement A. The next day, the culture medium in the flask was removed, the culture surface was washed with PBS, and then the preadipocytes were recovered by trypsin treatment and centrifuged. The centrifuged preadipocytes were suspended in MSF-BM / MSF-Supplement A to measure the cell concentration, and 9.5×10 5After centrifugal sedimentation of the cells, a solution of protamine sulfate was mixed into the supernatant obtained in (2) (the lentiviral vector producing the FVIII modifier) to a concentration of 100 μg / mL, and a cell suspension with a final concentration of 4.75×10 4 cells / mL was prepared. Thus, the cell suspension for gene introduction was inoculated into a 6-well plate at 2 mL / well. From the next day, passage was performed every 3 or 4 days in a 37°C, 5% CO2 incubator using MSF-BM / MSF-Supplement A to sequentially expand the culture, and preadipocytes transfected with the FVIII modifier gene were prepared.

[0139]

Experiment 2: Evaluation of the prepared preadipocytes transfected with the FVIII modifier gene

[0140] For each of the 5 types of preadipocytes transfected with the FVIII modifier gene prepared in Experiment 1, at the time of passage on days 10 - 14 after inoculating the cell suspension into the 6-well plate in (4) of Experiment 1, the culture medium in the flask was removed, the culture surface was washed with PBS, and then a part of the recovered cells was centrifuged and suspended in a cell cryopreservation solution for freezing. After thawing the frozen cells, they were suspended in MSF-BM / MSF-Supplement A, centrifuged to remove the cell cryopreservation solution, and then resuspended in MSF-BM / MSF-Supplement A and placed in a T75 flask. Using MSF-BM / MSF-Supplement A, passage was performed every 3 or 4 days while culturing in a 37°C, 5% CO2 incubator. Passage was performed on days 8, 11, and 14 after the start of culture. At the time of passage on days 11 and 14, the culture medium in the flask was removed, the culture surface was washed with PBS, and then the recovered cells were centrifuged by trypsin treatment, suspended in MSF-BM / MSF-Supplement A, and the cell concentration was measured. 2×10 5 cells were centrifuged and then suspended in PBS for freezing and stored for use in the evaluation. In addition, a part of the culture medium removed from the flask was also frozen and stored for use in the evaluation. (1) Calculation of the average number of transfected copies of the FVIII modifier gene (Calculation method)

[0141] Thaw the frozen cells and extract DNA using the DNeasy Blood & Tissue Kit (QIAGEN) according to its protocol. Specifically, add 20 μL of Proteinase K and 4 μL of RNase A (QIAGEN) to the thawed cells and mix. After allowing it to stand at room temperature for 5 minutes, add 0.2 mL of Buffer AL and mix. Incubate at 56°C for 10 minutes. After 10 minutes, add 0.2 mL of ethanol and mix. Transfer it to a spin column, place it in a 2 mL collection tube, and centrifuge at 6,000 g for 1 minute. Place the centrifuged spin column in a new 2 mL collection tube, add 0.5 mL of Buffer AW1, and centrifuge at 6,000 g for 1 minute. Next, place the centrifuged spin column in a new 2 mL collection tube, add 0.5 mL of Buffer AW2, and centrifuge at 20,000 g for 3 minutes. Next, place the centrifuged spin column in a new 1.5 mL collection tube, add 0.2 mL of Buffer AE, allow it to stand at room temperature for 1 minute, and then centrifuge at 6,000 g for 1 minute to elute the DNA. Measure its concentration using a spectrophotometer. From the DNA extracted from the cells, use the QX200 Droplet Digital PCR System, C1000 Touch Thermal Cycler, PX1 PCR Plate Sealer, and QuantaSoft software from Bio-Rad to calculate the average copy number of the FVIII modified gene according to its manual.

[0142] (Calculation result)

[0143] The value obtained by dividing the average copy number of the FVIII modified gene calculated from the DNA extracted from the cells recovered at each passage on the 11th day and the 14th day after the start of culturing in a T75 flask by 2 is shown in Figure 3 . As is clear from Figure 3 , in the gene-introduced preadipocytes producing any one of ΔB, N6, N6Δfurin, N5Δfurin, and N5Δfurin(P739T), the average copy number is at the same level and there is no significant difference.

[0144] (2) Measurement of FVIII antigen amount in the culture medium

[0145] Thaw the frozen culture medium, and using an ELISA kit (Asserachrom FVIII, Roche, the same hereinafter), measure the amount of FVIII antigen in the culture medium according to its procedure. The amount of FVIII antigen is quantified as a relative value based on the absorbance at 450 nm (reference wavelength 570 nm) of a microplate reader from a calibration curve prepared for the amount of FVIII antigen in healthy human plasma, as a percentage value with respect to the amount of FVIII antigen in healthy human plasma as a reference (when the amount of FVIII antigen in healthy human plasma is set to 100%). The value obtained by dividing the value of the amount of FVIII antigen in the culture medium recovered at each passage on the 11th and 14th days after the start of culture in a T75 flask by 3 (number of passage days) and then dividing by 2 is shown in Figure 4 . As is clear from Figure 4 , the amount of FVIII antigen in the culture medium shows a very high value for ΔB, and the values of other FVIII modifiers are half or less of the value of ΔB with little difference.

[0146] (3) Measurement of FVIII activity in the culture medium

[0147] Thaw the frozen culture medium, and using a Coatest SP FVIII kit (CHROMOGENIX), measure the FVIII activity in the culture medium according to its procedure. The FVIII activity is quantified as a relative value based on the absorbance at 450 nm (reference wavelength 570 nm) of a microplate reader from a calibration curve prepared for the FVIII activity in healthy human plasma, as a percentage value with respect to the FVIII activity in healthy human plasma as a reference (when the FVIII activity in healthy human plasma is set to 100%). The value obtained by dividing the value of the FVIII activity in the culture medium recovered at each passage on the 11th and 14th days after the start of culture in a T75 flask by 3 (number of passage days) and then dividing by 2 is shown in Figure 5 . As is clear from Figure 5 , the FVIII activity in the culture medium shows the highest value for ΔB, followed by a high value for N5Δfurin, and then a high value for N5Δfurin(P739T). The values of N6 and N6Δfurin are less than half of the value of ΔB.

[0148] (4) Calculation of the specific activity (activity equivalent to the amount of antigen) of the FVIII modifier secreted into the culture medium The specific activity of the FVIII modifier secreted into the culture medium is calculated from the results of the measurement of the FVIII antigen amount in the culture medium in (2) and the results of the measurement of the FVIII activity of the culture medium in (3). The value obtained by dividing the value obtained by dividing the FVIII activity of the culture medium recovered at the passage on the 11th day after the start of culturing in the T75 flask by 3 by the value obtained by dividing the FVIII antigen amount in the culture medium by 3, and the value obtained by dividing the value obtained by dividing the FVIII activity of the culture medium recovered at the passage on the 14th day by 3 by the value obtained by dividing the FVIII antigen amount in the culture medium by 3, divided by 2, is shown in Figure 6 . As is clear from Figure 6 , the specific activity of the FVIII modifier secreted into the culture medium is such that N5Δfurin (P739T) shows the highest value, far higher than the values of ΔB and N6 (with significant differences). Next, the value of N5Δfurin is high, and then the value of N6Δfurin is high, and there are no significant differences compared to the values of ΔB and N6. Thus, it is known that these FVIII modifiers secreted into the culture medium from the preadipocytes transfected with the genes producing N6Δfurin, N5Δfurin, and N5Δfurin (P739T) have high specific activities.

[0149]

Experiment 3: Transplantation of preadipocytes transfected with FVIII modifier genes into NOD / SCID mice

[0150] Each of the 5 types of preadipocytes transfected with FVIII modifier genes prepared in Experiment 1 was suspended in MSF-BM / MSF-Supplement A, and inoculated into an arbitrary flask at 4×10 4 cells / cm 2 . Culturing was carried out in a 37 °C, 5% CO2 incubator using MSF-BM / MSF-Supplement A. The next day, the medium was replaced with PGM-2 (Lonza Co., the same below), and culturing was continued for another 3 days. The next day, the culture medium in the flask was removed, the culture surface was washed with PBS, and then the cells were recovered by trypsin treatment and centrifuged. After washing the cells by performing a series of operations of suspending the centrifuged cells in Ringer's solution containing 0.5% albumin at the final concentration and centrifuging 4 times, the washed cells were suspended in FiblastSpray (Kaken Pharmaceutical Co., the same below) to prepare a cell suspension containing 200 μg / mL of treflan, and an equal amount of Matrigel (Corning Co., the same below) was mixed to adjust the final concentration to 2×10 7Cell suspension for transplantation at a concentration of [number] cells / mL (final concentration of treprostinil: 100 μg / mL). The prepared cell suspension for transplantation was administered subcutaneously to the left and right dorsal sides of 7- or 8-week-old NOD / SCID mice (Jackson Laboratory, Japan) under isoflurane anesthesia using a 20G blunt needle cannula at a dose of 0.5 mL / site (2×10 7 cells / mouse).

[0151]

Experiment 4: Evaluation of cells for ex vivo gene therapy of FVIII modulator as cells producing FVIII modulator gene-transduced preadipocytes (measurement of FVIII antigen amount in the blood of transplanted mice)

[0152] (Measurement method)

[0153] On the 3rd, 7th, and 14th days after transplantation of each FVIII modulator gene-transduced preadipocyte into NOD / SCID mice in Experiment 3, blood was collected from the cheeks of the mice under isoflurane anesthesia using an animal lancet (MEDIpoint, the same below). After adding EDTA to the collected blood in a 1.5 mL centrifuge tube to a final concentration of 0.1 - 0.25% (v / v), the mixture was centrifuged at 5,000 rpm for 10 minutes at 4°C to obtain plasma specimens, which were then frozen and stored until use in the evaluation. The frozen plasma specimens were thawed, and the FVIII antigen amount in the blood of the transplanted mice was measured using an ELISA kit according to its procedure. The FVIII antigen amount was quantified based on the absorbance at 450 nm (reference wavelength 570 nm) of a plate reader and was numerically expressed as a relative value based on a calibration curve prepared for the FVIII antigen amount in healthy human plasma (percentage value when the FVIII antigen amount in healthy human plasma is set to 100%).

[0154] (Measurement results)

[0155] Shown in Figure 7 . As is clear from Figure 7 , the FVIII antigen amount in the blood of the transplanted mice was highest for N5Δfurin (P739T), far higher than the values for ΔB and N6 (significant difference). Next, the value for N5Δfurin was high, and then the value for N6Δfurin was high, both higher than the values for ΔB and N6 (the former showed a significant difference compared to the value for ΔB). Thus, it was found that gene-transduced preadipocytes producing N6Δfurin, N5Δfurin, and N5Δfurin (P739T) secreted more FVIII modulator into the blood than gene-transduced preadipocytes producing ΔB and N6.

[0156]

Experiment B: Exploration of the Introduction of Various FVIII Modifier Genes into Preadipocytes (Part 2)

[0157]

Experiment 1: Preparation of Lentiviral Vector Plasmids for Introducing FVIII Modifier Genes into Preadipocytes

[0158] (1) Preparation of Lentiviral Vector Plasmids Producing FVIII Modifiers

[0159] (f) A lentiviral vector plasmid producing N5Δfurin(P739S) was prepared by the following method. The lentiviral vector plasmid producing N5Δfurin(P739T) prepared in (e) of Experiment 1 of Experiment A was subjected to two PCR reactions using the following two primer sets (Fw1&Re1, Fw2&Re2). Using the polynucleotide composed of the base sequence corresponding to the amino acid sequence of the protein of N5Δfurin(P739S) in which the number of Asn residues modified with N-linked glycans in the B domain of human FVIII is 5 when counted from the N-terminal side, the signal peptide is defective in binding to the furin recognition site, and the 758th Pro residue is replaced with a Ser residue (SEQ ID NO: 14) as a template, two fragments of polynucleotides were obtained: one with an XbaI restriction endonuclease site at the 5'-terminal side and a P739S mutation at the 3'-terminal side, and the other with a P739S mutation at the 5'-terminal side and an MluI restriction endonuclease site at the 3'-terminal side. The linearized polynucleotide obtained by treating the two polynucleotide fragments thus obtained and the lentiviral vector plasmid producing N5Δfurin(P739T) with XbaI and MluI restriction endonucleases was ligated by In-Fusion reaction to obtain the desired lentiviral vector plasmid producing N5Δfurin(P739S).

[0160] Fw1: GTGCGGCCCC TGTATTCTAG (SEQ ID NO: 20)

[0161] Re1: CTTCTGCTCT CGATGGCATT G (SEQ ID NO: 21)

[0162] Fw2: CATCGAGAGC AGAAGCTTCA GC (SEQ ID NO: 22)

[0163] Re2: GTTGATTGTT CCAGACGCGT TC (SEQ ID NO: 23)

[0164] (g) The lentiviral vector plasmid producing N5Δfurin(P739N), which is constructed by inserting the polynucleotide corresponding to the amino acid sequence (SEQ ID NO: 15) of the FVIII modified body (N5Δfurin(P739N)) in which the number of Asn residues modified with N-type sugar chains in the B domain of human FVIII is 5 counted from the N-terminal side, the signal peptide is defective in binding to the furin recognition site, and the 758th Pro residue is replaced with an Asn residue, is prepared by the same method as the method for preparing the lentiviral vector plasmid producing N5Δfurin(P739S). The two primer sets (Fw1&Re1, Fw2&Re2) used for subjecting the lentiviral vector plasmid producing N5Δfurin(P739T) to two PCR reactions are as follows.

[0165] Fw1: GTGCGGCCCC TGTATTCTAG (SEQ ID NO: 20)

[0166] Re1: CTTCTGTTCT CGATGGCATT G (SEQ ID NO: 24)

[0167] Fw2: CATCGAGAAC AGAAGCTTCA GC (SEQ ID NO: 25)

[0168] Re2: GTTGATTGTT CCAGACGCGT TC (SEQ ID NO: 23)

[0169] (h) The lentiviral vector plasmid producing N5Δfurin(P739Q), which is constructed by inserting the polynucleotide corresponding to the amino acid sequence (SEQ ID NO: 16) of the FVIII modifier (N5Δfurin(P739Q)) into the SbfI / MluI site of the pLVSIN-EF1αNeo Vector, where the number of Asn residues modified with N-linked glycans in the B domain of human FVIII is 5 when counted from the N-terminal side, the signal peptide is defective in binding to the furin recognition site, and the Pro residue at position 758 is replaced with a Gln residue, was prepared by the same method as the method for preparing the lentiviral vector plasmid producing N5Δfurin(P739S). The two primer sets (Fw1&Re1, Fw2&Re2) used for the two PCR reactions for the lentiviral vector plasmid producing N5Δfurin(P739T) are as follows.

[0170] Fw1: GTGCGGCCCC TGTATTCTAG (SEQ ID NO: 20)

[0171] Re1: CTTCTCTGCT CGATGGCATT G (SEQ ID NO: 26)

[0172] Fw2: CATCGAGCAG AGAAGCTTCA GC (SEQ ID NO: 27)

[0173] Re2: GTTGATTGTT CCAGACGCGT TC (SEQ ID NO: 23)

[0174] (2) Preparation of lentiviral vectors producing FVIII modifiers

[0175] Using the same method as the method for preparing the lentiviral vector producing the FVIII modifier in (2) of Experiment 1 of Experiment A, a lentiviral vector producing the FVIII modifier was prepared using the lentiviral vector plasmid producing the FVIII modifier prepared in (1).

[0176] (3) Obtaining of human preadipocytes transfected with the FVIII modifier gene

[0177] Using the same method as the method for obtaining human preadipocytes transfected with the FVIII modifier gene in (3) of Experiment 1 of Experiment A, preadipocytes were obtained from adipose tissue obtained from healthy human volunteers, suspended in a cell cryopreservation solution, and frozen.

[0178] (4) Preparation of human preadipocytes transfected with the FVIII modifier gene

[0179] After thawing the cryopreserved preadipocytes in (3), suspending them in MSF-BM / MSF-Supplement A, centrifuging to remove the cell cryopreservation solution, and then resuspending them in MSF-BM / MSF-Supplement A, they were placed in a T150 flask and cultured overnight in a 37°C, 5% CO2 incubator using MSF-BM / MSF-Supplement A. The next day, the culture medium in the flask was removed, the culture surface was washed with PBS, and the preadipocytes were recovered by trypsin treatment and centrifuged. The centrifuged preadipocytes were resuspended in MSF-BM / MSF-Supplement A to measure the cell concentration, and 1.25×10 5 cells were centrifuged and then resuspended in a solution in which protamine sulfate was mixed with the supernatant (lentiviral vector producing the FVIII modifier) obtained in (2) at a concentration of 100 μg / mL to prepare a cell suspension with a final concentration of 2.5×10 4 cells / mL. Thus, the cell suspension for gene transfer operation was inoculated into T25 flasks at 5 mL / flask, and from the next day, using MSF-BM / MSF-Supplement A, subculture was performed every 4 days while successively expanding the culture in a 37°C, 5% CO2 incubator. On the 8th day after the gene transfer operation in Experiment 1, the second gene transfer operation was performed in the same manner as above, and then subculture was performed every 3 days while successively expanding the culture. Furthermore, on the 6th day after the second gene transfer operation (the 14th day after the first gene transfer operation), after performing the gene transfer operation in the same manner as above, FVIII modifier gene-transferred preadipocytes were prepared by successively expanding the culture while performing subculture every 3 or 4 days (the total number of gene transfer operations was 3 times).

[0180]

Experiment 2: Evaluation of the prepared FVIII modifier gene-transferred preadipocytes

[0181] For each of the three types of FVIII modifier gene-transferred preadipocytes prepared in Experiment 1, subculture was performed on the 25th and 29th days after the first gene transfer operation in (4) of Experiment 1. At the time of subculture on the 29th day, the culture medium in the flask was removed, the culture surface was washed with PBS, and the recovered cells were centrifuged by trypsin treatment, resuspended in MSF-BM / MSF-Supplement A to measure the cell concentration, and 2×10 5 cells were centrifuged and then resuspended in PBS and frozen for storage until use in the evaluation. In addition, a part of the culture medium removed from the flask was also frozen for storage until use in the evaluation.

[0182] Calculation of the average number of introduced copies of the FVIII modifier gene

[0183] Using the same method as the method for calculating the average number of introduced copies of the FVIII modifier gene in (1) of Experiment 2 of Experiment A, the average number of introduced copies of the FVIII modifier gene was calculated from the DNA extracted from the cells recovered at passage on the 29th day from the day of the first gene introduction operation. The results are shown in Figure 8 (Each of N5Δfurin(P739S) is expressed as P739S, N5Δfurin(P739N) is expressed as P739N, and N5Δfurin(P739Q) is expressed as P739Q. The same applies hereinafter). In addition, in Figure 8 , using each of the lentiviral vector plasmids producing ΔB, the lentiviral vector plasmid producing N5Δfurin, the lentiviral vector plasmid producing N5Δfurin(P739T), and the lentiviral vector plasmid producing N6Δfurin prepared in (1) of Experiment 1 of Experiment A, the results of introducing 4 types of FVIII modifier genes into preadipocytes prepared by the same method as the methods described in (2) to (4) of Experiment 1 were also comprehensively shown (N5Δfurin(P739T) is expressed as P739T, and the same applies hereinafter). As is clear from Figure 8 , in the preadipocytes into which any one of ΔB, N5Δfurin, N5Δfurin(P739T), N5Δfurin(P739S), N5Δfurin(P739N), N5Δfurin(P739Q), and N6Δfurin was introduced, the average number of introduced copies was also of the same level and there was no significant difference.

[0184] (2) Measurement of the amount of FVIII antigen in the culture medium

[0185] Using the same method as the method for measuring the amount of FVIII antigen in the culture medium in (2) of Experiment 2 of Experiment A, the amount of FVIII antigen in the culture medium recovered at passage on the 29th day from the day of the first gene introduction operation was measured. The results are shown in Figure 9 . As is clear from Figure 9 , the amount of FVIII antigen in the culture medium was a very high value for ΔB, and the values of the other FVIII modifiers were half or less of the value of ΔB and there was no significant difference. (3) Measurement of the FVIII activity of the culture medium

[0186] Thaw the frozen culture medium, and measure the FVIII activity of the culture medium according to the procedure using the Revohem FVIII synthesis matrix kit (Sysmex Corporation). The FVIII activity is numerically expressed as a relative value based on the absorbance of a fully automated blood coagulation measurement device (CS-1600, Sysmex Corporation) from a calibration curve prepared for the FVIII activity of healthy human plasma, with the FVIII activity of healthy human plasma as the reference (the % value when the FVIII activity of healthy human plasma is set to 100%). The FVIII activity of the culture medium recovered at the passage on the 29th day from the date of the first gene transfer operation is shown in Figure 10 . As is clear from Figure 10 , the FVIII activity of the culture medium is such that N5Δfurin (P739N) and N5Δfurin (P739Q) show values as high as those of ΔB.

[0187] (4) Calculation of the specific activity (activity equivalent to the antigen amount) of the FVIII modifier secreted into the culture medium Calculate the specific activity of the FVIII modifier secreted into the culture medium from the results of the measurement of the FVIII antigen amount in the culture medium in (2) and the results of the measurement of the FVIII activity of the culture medium in (3). The results are shown in Figure 11 . As is clear from Figure 11 , the specific activity of the FVIII modifier secreted into the culture medium is such that N5Δfurin (P739T), N5Δfurin (P739S), N5Δfurin (P739N), and N5Δfurin (P739Q) show high values, far higher than the value of ΔB (there is a significant difference). In addition, the values are higher than those of N5Δfurin (there is a significant difference except for N5Δfurin (P739S)). Thus, it is known that FVIII modifiers in which the Pro residue at position 758 in the amino acid sequence of N5Δfurin composed of the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3 is replaced with a polar uncharged amino acid residue such as a Thr residue, a Ser residue, an Asn residue, or a Gln residue have a high specific activity.

[0188]

Experiment C: Investigation of the mixed transplantation of preadipocytes transfected with the FVIII modifier gene and preadipocytes transfected with the human vWF fragment gene

[0189]

Experiment 1: Production of preadipocytes transfected with the human vWF fragment gene

[0190] (1) Production of a lentiviral vector plasmid that produces the human vWF fragment

[0191] By synthesizing a polynucleotide corresponding to the amino acid sequence (SEQ ID NO: 29) of a fragment (vWF1) composed of the domains SP, D1, D2, D', D3 and 26 amino acid residues counted from the N-terminal side of the A1 domain and further 3 amino acid residues and the CK domain in the domain structure of the precursor of human vWF, SP-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK, and adding a SbfI restriction enzyme site at the 5'-end and an MluI restriction enzyme site at the 3'-end, and inserting it into the SbfI / MluI site of the pLVSIN-EF1αNeo Vector, a lentiviral vector plasmid producing vWF1 was prepared. Similarly, a polynucleotide composed of a base sequence (SEQ ID NO: 33) corresponding to the amino acid sequence (SEQ ID NO: 30) of a fragment (vWF2) composed of the domains SP, D1, D2, D', D3 and 26 amino acid residues counted from the N-terminal side of the A1 domain was inserted into the SbfI / MluI site of the pLVSIN-EF1αNeo Vector, and a lentiviral vector plasmid producing vWF2 was prepared. A polynucleotide composed of a base sequence (SEQ ID NO: 34) corresponding to the amino acid sequence (SEQ ID NO: 31) of a fragment (vWF3) composed of the domains SP, D', D3 and 26 amino acid residues counted from the N-terminal side of the A1 domain was inserted into the SbfI / MluI site of the pLVSIN-EF1αNeo Vector to prepare a lentiviral vector plasmid producing vWF3. Furthermore, the base sequence of vWF1 uses the corresponding base sequence in the base sequence of human vWF (SEQ ID NO: 35). The base sequences of vWF2 and vWF3 use codon-optimized base sequences. The composition of the lentiviral vector plasmid producing vWF3 is shown in Figure 12 .

[0192] (2) Preparation of Lentiviral Vectors Producing Human vWF Fragments

[0193] Using the same method as the method for preparing the lentiviral vector producing the FVIII modifier in (2) of Experiment 1 of Experiment A, a lentiviral vector producing a human vWF fragment was prepared using the lentiviral vector plasmid producing the human vWF fragment prepared in (1).

[0194] (3) Obtaining of Human Preadipocytes into Which the Human vWF Fragment Gene was Introduced

[0195] Using the same method as the method for obtaining human preadipocytes transfected with the FVIII modifier gene in Experiment 1 of Experiment A, preadipocytes were obtained from adipose tissue obtained from healthy human volunteers, suspended in a cell cryopreservation solution, and frozen.

[0196] (4) Preparation of human vWF fragment gene-transfected preadipocytes

[0197] The preadipocytes frozen in (3) were thawed, suspended in MSF-BM / MSF-Supplement A, centrifuged to remove the cell cryopreservation solution, then resuspended in MSF-BM / MSF-Supplement A, placed in a T225 flask, and cultured in an incubator at 37 °C and 5% CO2 for 4 days using MSF-BM / MSF-Supplement A. The next day, the culture medium in the flask was removed, the culture surface was washed with PBS, and then the preadipocytes were recovered by trypsin treatment and centrifuged. The centrifuged preadipocytes were suspended in MSF-BM / MSF-Supplement A to measure the cell concentration, and 2.5×10 5 cells were centrifuged and then suspended in a solution of protamine sulfate mixed with the supernatant obtained in (2) (lentiviral vector producing human vWF fragment) at a concentration of 100 μg / mL to prepare a cell suspension with a final concentration of 5.0×10 4 cells / mL. Thus, the cell suspension for gene transfer operation was inoculated into T25 flasks at 5 mL / flask, and from the next day, it was cultured in an incubator at 37 °C and 5% CO2 using MSF-BM / MSF-Supplement A. After the second gene transfer operation was performed in the same manner as above on the second day after the gene transfer operation, subculture was performed every 3 days while successively expanding the culture. Furthermore, on the sixth day after the second gene transfer operation (the eighth day after the first gene transfer operation), after the gene transfer operation was performed in the same manner as above, subculture was performed every 3 or 4 days while successively expanding the culture to prepare vWF1 gene-transfected preadipocytes. In addition, after the second gene transfer operation was performed in the same manner as above on the second day after the gene transfer operation, the cells were cultured. Furthermore, on the third day after the second gene transfer operation (the fifth day after the first gene transfer operation), after the gene transfer operation was performed in the same manner as above, subculture was performed every 3 or 4 days while successively expanding the culture to prepare vWF2 gene-transfected preadipocytes and vWF3 gene-transfected preadipocytes (the total number of gene transfer operations for any gene-transfected preadipocytes was 3 times).

[0198]

Experiment 2: Evaluation of mixed transplantation of human vWF fragment gene-introduced preadipocytes and FVIII modifier gene-introduced preadipocytes (measurement of FVIII antigen amount in the blood of transplanted mice)

[0199] (Measurement method)

[0200] Using the lentiviral vector plasmid producing N5Δfurin(P739T) prepared in (1) of Experiment 1 of Experiment A, the lentiviral vector producing N5Δfurin(P739T) prepared by the same method as described in (2) of Experiment 1 of Experiment A, and the human preadipocytes obtained in (3) of Experiment 1, the N5Δfurin(P739T) gene was introduced into preadipocytes by the same method as described in (4) of Experiment 1 (however, for the gene introduction operation, a total of 3 times were performed on the 2nd and 7th days from that day after the first gene introduction operation (no passage during the 5th day from the day of the second gene introduction operation)) and each of the 3 kinds of human vWF fragment gene-introduced preadipocytes prepared in Experiment 1 was suspended in MSF-BM / MSF-Supplement A, and inoculated at 4×10 4 cells / cm 2 in any flask, and cultured in an MSF-BM / MSF-Supplement A in a 37 °C, 5% CO2 incubator. The next day, the medium was changed to PGM-2, and further cultured for 3 days. The next day, after removing the culture solution in the flask, the culture surface was washed with PBS, then trypsinized to recover the cells, and centrifuged. After washing the cells by performing a series of operations of centrifuging the cells suspended in Ringer's solution containing albumin at a final concentration of 0.5% 4 times, the washed cells were suspended in a mixed solution of Fiblast Spray and Medgel (Nitta Gelatin Inc.) to prepare cell suspensions of each cell with a concentration of 8×10 7 cells / mL containing 200 μg / mL trevimine and 8 mg / mL Medgel. Next, by mixing the prepared N5Δfurin(P739T) gene-introduced preadipocyte suspension and human vWF fragment gene-introduced preadipocyte suspension in equal amounts, a mixed suspension of N5Δfurin(P739T) gene-introduced preadipocytes and human vWF fragment gene-introduced preadipocytes was prepared, and then an equal amount of Matrigel was mixed to prepare a cell suspension for transplantation with a final concentration of 2×10 7 cells / mL (the final concentration of trevimine is 100 μg / mL, and the final concentration of Medgel is 4 mg / mL). The prepared cell suspension for transplantation was administered using a 20G blunt needle cannula at 0.5 mL / position (2×10 7Cells / mouse) subcutaneously into the left and right dorsal regions of 6-week-old NOD / SCID mice (CLEA Japan Inc., the same applies hereinafter) under isoflurane anesthesia. Blood was collected from the cheeks of the mice on days 3, 7, 14, 21, and 28 after transplantation of the cell suspension containing preadipocytes transfected with the FVIII modifier gene and preadipocytes transfected with the human vWF fragment gene into NOD / SCID mice under isoflurane anesthesia using an animal lancet. The FVIII antigen amount in the blood of the transplanted mice was measured by the same method as the method for measuring the FVIII antigen amount in the blood of the transplanted mice in Experiment 4 of Experiment A.

[0201] (Measurement results)

[0202] Shown in Figure 13 . In addition, in Figure 13 , a cell suspension for transplantation containing only preadipocytes transfected with the N5Δfurin (P739T) gene at a final concentration of 2×10 7 cells / mL was prepared by the same method as above, and administered subcutaneously at 0.5 mL / position (2×10 7 cells / mouse), and the results are also comprehensively shown. As is clear from Figure 13 , the FVIII antigen amount in the blood of the transplanted mice was higher when preadipocytes transfected with the N5Δfurin (P739T) gene were co-transplanted with preadipocytes transfected with the vWF3 gene, when only preadipocytes transfected with the N5Δfurin (P739T) gene were transplanted, and when co-transplanted with preadipocytes transfected with the vWF1 gene, than when co-transplanted with preadipocytes transfected with the vWF2 gene (there were significant differences compared to any case). Thus, it was found that by co-transplanting preadipocytes transfected with the human vWF fragment gene and preadipocytes transfected with the FVIII modifier gene, the secretion amount of the FVIII modifier into the blood was increased.

[0203]

Experiment 3: Evaluation of the scaffold material (extracellular matrix component) when co-transplanting preadipocytes transfected with the human vWF fragment gene and preadipocytes transfected with the FVIII modifier gene (measurement of the FVIII antigen amount in the blood of transplanted mice)

[0204] (Measurement method)

[0205] The preadipocytes transfected with the N5Δfurin (P739T) gene described in Experiment 2 and the preadipocytes transfected with the vWF3 gene prepared in Experiment 1 were each cultured, recovered, centrifuged, and washed by the same method as described in Experiment 2, and then suspended in Ringer's solution containing 0.5% albumin at a final concentration to prepare a cell concentration of 2×10 8A cell suspension of cells / mL. Next, by mixing the prepared N5Δfurin(P739T) gene-introduced preadipocyte suspension and vWF3 gene-introduced preadipocyte suspension in equal amounts, a mixed suspension of N5Δfurin(P739T) gene-introduced preadipocytes and vWF3 gene-introduced preadipocytes was prepared, and then 1.2% or 2.0% of terminal collagen (same as that of Gaoyan Company below) was mixed in equal amounts to adjust the final concentration of each cell to 5×10 7 cells / mL of the cell suspension for transplantation (the final concentration of terminal collagen is 0.6% or 1.0%). The prepared cell suspension for transplantation was administered subcutaneously on the left and right backs of 6-week-old NOD / SCID mice under isoflurane anesthesia using a 20G blunt needle cannula at 0.2 mL / position (2×10 7 cells / mouse) for each of the two types of cells. Blood was collected from the cheeks of the mice on days 3, 7, 14, 21, 28, 35, 42, 49, and 56 from the transplantation date of the cell suspension for transplantation containing FVIII-modified gene-introduced preadipocytes and vWF3 gene-introduced preadipocytes to NOD / SCID mice under isoflurane anesthesia using an animal lancet, and the FVIII antigen amount in the blood of the transplanted mice was measured by the same method as that for measuring the FVIII antigen amount in the blood of the transplanted mice in Experiment 4 of Experiment A.

[0206] (Measurement results)

[0207] Shown in Figure 14 . In addition, in Figure 14 , by the same method as above, a cell suspension for transplantation containing only N5Δfurin(P739T) gene-introduced preadipocytes with a final concentration of 5×10 7 cells / mL (the final concentration of terminal collagen is 0.6%) was prepared and administered subcutaneously on the left and right backs at 0.2 mL / position (2×10 7 cells / mouse), and the results are also comprehensively shown. As shown in Figure 14It was clear that the amount of FVIII antigen in the blood of the transplanted mice was higher when the transplantation cell suspension containing N5Δfurin(P739T) gene-introduced preadipocytes and vWF3 gene-introduced preadipocytes was made to contain telopeptide collagen than when the transplantation cell suspension containing only N5Δfurin(P739T) gene-introduced preadipocytes was made to contain telopeptide collagen. The value when the transplantation cell suspension containing N5Δfurin(P739T) gene-introduced preadipocytes and vWF3 gene-introduced preadipocytes was made to contain telopeptide collagen at a final concentration of 1.0% was higher over the long term (also significantly different from the values in any case) than the value when the transplantation cell suspension containing only N5Δfurin(P739T) gene-introduced preadipocytes was made to contain telopeptide collagen at a final concentration of 0.6%, and was higher than the value when the transplantation cell suspension containing N5Δfurin(P739T) gene-introduced preadipocytes and vWF3 gene-introduced preadipocytes was made to contain telopeptide collagen at a final concentration of 0.6%. Thus, it was found that when telopeptide collagen was used as the scaffold material for co-transplanting human vWF fragment gene-introduced preadipocytes and FVIII modifier gene-introduced preadipocytes, the secretion amount of the FVIII modifier into the blood increased.

[0208]

Experiment 4: Evaluation of the transplantation site when co-transplanting human vWF fragment gene-introduced preadipocytes and FVIII modifier gene-introduced preadipocytes (measurement of the amount of FVIII antigen in the blood of transplanted mice)

[0209] (Measurement method)

[0210] The N5Δfurin(P739T) gene-introduced preadipocytes described in Experiment 2 and the vWF3 gene-introduced preadipocytes prepared in Experiment 1 were each cultured, recovered, centrifuged, and washed by the same method as described in Experiment 2, and then suspended in Ringer's solution containing albumin at a final concentration of 0.5% to prepare cell suspensions of each cell at a concentration of 2×10 8 cells / mL. Next, the prepared N5Δfurin(P739T) gene-introduced preadipocyte suspension and vWF3 gene-introduced preadipocyte suspension were mixed in equal amounts to prepare a mixed suspension of N5Δfurin(P739T) gene-introduced preadipocytes and vWF3 gene-introduced preadipocytes, and then an equal amount of 2.0% telopeptide collagen was mixed to prepare a transplantation cell suspension at a final concentration of 5×10 7 cells / mL (final concentration of telopeptide collagen: 1.0%). The prepared transplantation cell suspension was administered subcutaneously to the left and right backs of 6-week-old NOD / SCID mice under isoflurane anesthesia using a 20G blunt needle cannula, or to the left and right mouse groins using a 25G injection needle at 0.2 mL / position (2×107 cells / mouse). From the transplantation day of the cell suspension containing preadipocytes transfected with the FVIII modifier gene and preadipocytes transfected with the vWF3 gene into NOD / SCID mice, blood was collected from the cheeks of the mice on days 3, 7, 14, 21, 28, 35, 42, 49, and 56 under isoflurane anesthesia using an animal lancet, and the amount of FVIII antigen in the blood of the transplanted mice was measured by the same method as the method for measuring the amount of FVIII antigen in the blood of the transplanted mice in Experiment 4 of Experiment A.

[0211] (Measurement results)

[0212] Shown in Figure 15 . In addition, in Figure 15 , a cell suspension for transplantation containing only preadipocytes transfected with the N5Δfurin(P739T) gene at a final concentration of 5×10 7 cells / mL (final concentration of tropocollagen 1.0%) was prepared by the same method as above, and administered at 0.2 mL / position in the inguinal regions of the left and right mice (2×10 7 cells / mouse), and the results are also comprehensively shown. As is clear from Figure 15 , the amount of FVIII antigen in the blood of the transplanted mice was higher when preadipocytes transfected with the N5Δfurin(P739T) gene and preadipocytes transfected with the vWF3 gene were mixed and transplanted in the inguinal region of the mouse, or when only preadipocytes transfected with the N5Δfurin(P739T) gene were transplanted into the inguinal region of the mouse, than when preadipocytes transfected with the N5Δfurin(P739T) gene and preadipocytes transfected with the vWF3 gene were mixed and transplanted subcutaneously on the back (there were significant differences compared to any case). Thus, it was found that when the transplantation site was the inguinal region of the mouse when preadipocytes transfected with the human vWF fragment gene and preadipocytes transfected with the FVIII modifier gene were mixed and transplanted, the secretion amount of the FVIII modifier into the blood increased.

[0213] [Experiment 5: Evaluation of the ratio of the number of transplanted cells of both when preadipocytes transfected with the human vWF fragment gene and preadipocytes transfected with the FVIII modifier gene are mixed and transplanted (measurement of the amount of FVIII antigen in the blood of transplanted mice)]

[0214] (Measurement method)

[0215] The preadipocytes transfected with the N5Δfurin(P739T) gene described in Experiment 2 and the preadipocytes transfected with the vWF3 gene prepared in Experiment 1 were cultured, recovered, centrifuged, and washed by the same method as the method described in Experiment 2, and then suspended in Ringer's solution containing albumin at a final concentration of 0.5% to prepare cell suspensions of the respective cells with the cell concentrations described below.

[0216] (Suspension of preadipocytes transfected with N5Δfurin(P739T) gene)

[0217] ·5×10 7 cells / mL

[0218] ·3.3×10 7 cells / mL

[0219] (Suspension of preadipocytes transfected with vWF3 gene)

[0220] ·1×10 8 cells / mL

[0221] ·5×10 7 cells / mL

[0222] ·2.5×10 7 cells / mL

[0223] Next, by mixing any one of the prepared suspension of preadipocytes transfected with N5Δfurin(P739T) gene and any one of the suspension of preadipocytes transfected with vWF3 gene in equal amounts, a mixed suspension of preadipocytes transfected with N5Δfurin(P739T) gene and preadipocytes transfected with vWF3 gene was prepared, and then 1.6% of telopeptide collagen in equal amounts was mixed to prepare the final concentration of each cell to be the following 3 kinds of cell suspensions for transplantation (final concentration of telopeptide collagen 0.8%).

[0224] (1) Preadipocytes transfected with N5Δfurin(P739T) gene at 1.25×10 7 cells / mL, preadipocytes transfected with vWF3 gene at 6.25×10 6 cells / mL

[0225] (2) Preadipocytes transfected with N5Δfurin(P739T) gene at 1.25×10 7 cells / mL, preadipocytes transfected with vWF3 gene at 1.25×10 7 cells / mL

[0226] (3) Preadipocytes transfected with N5Δfurin(P739T) gene at 8.25×10 6 cells / mL, preadipocytes transfected with vWF3 gene at 2.5×10 7 cells / mL

[0227] Each of the three prepared cell suspensions for transplantation was administered to the left and right inguinal regions of 6-week-old NOD / SCID mice under isoflurane anesthesia using a 25G injection needle. For the cell suspension for transplantation (1), it was administered at 0.4 mL / position (preadipocytes transfected with the N5Δfurin(P739T) gene at 1×10 7 cells / mouse, preadipocytes transfected with the vWF3 gene at 5×10 6 cells / mouse). For the cell suspension for transplantation (2), it was also administered at 0.4 mL / position (1×10 7 cells / mouse for each of the two types of cells). For the cell suspension for transplantation (3), it was administered at 0.6 mL / position (preadipocytes transfected with the N5Δfurin(P739T) gene at 1×10 7 cells / mouse, preadipocytes transfected with the vWF3 gene at 3×10 7 cells / mouse). Blood was collected from the cheeks of the mice on days 3, 7, 14, 21, 28, 35, 42, 49, and 56 after transplantation of the cell suspensions for transplantation containing preadipocytes transfected with the FVIII modifier gene and preadipocytes transfected with the vWF3 gene under isoflurane anesthesia using an animal lancet. The amount of FVIII antigen in the blood of the transplanted mice was measured by the same method as that for measuring the amount of FVIII antigen in the blood of the transplanted mice in Experiment 4 of Experiment A.

[0228] (Measurement results)

[0229] Shown in Figure 16 . As is clear from Figure 16 , the amount of FVIII antigen in the blood of the transplanted mice was higher when the ratio of the number of transplanted cells of preadipocytes transfected with the vWF3 gene to the number of transplanted cells of preadipocytes transfected with the N5Δfurin(P739T) gene was higher. The value when the cell suspension for transplantation (3) was administered was higher than the value when the cell suspension for transplantation (2) was administered and higher than the value when the cell suspension for transplantation (1) was administered (there were also significant differences compared to any case). Thus, it was found that when preadipocytes transfected with the human vWF fragment gene and preadipocytes transfected with the FVIII modifier gene were co-transplanted, increasing the ratio of the number of transplanted cells of preadipocytes transfected with the human vWF fragment gene to the number of transplanted cells of preadipocytes transfected with the FVIII modifier gene increased the secretion amount of the FVIII modifier into the blood.

[0230]

Industrial Applicability

[0231] The present invention has industrial applicability in that it can provide new ex vivo gene therapy cells that produce the FVIII modifier.

Claims

1. A cell for ex vivo gene therapy, which produces a modified human FVIII composed of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1, wherein the modified human FVIII: The B domain (amino acid sequence at positions 760 to 1667) is altered, The number of Asn residues modified with N-linked glycans is 4 to 6 when counted from the N-terminal side, and The function of the furin recognition site is defective.

2. The cell for ex vivo gene therapy according to claim 1, wherein the FVIII modified body is any one of the following (a) to (c): (a) A protein composed of the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2, (b) A protein composed of an amino acid sequence containing one or more deletions, substitutions, additions or insertions of amino acid residues in the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2 and having the same biological activity as the protein of (a), (c) A protein composed of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1607 in the amino acid sequence shown in SEQ ID NO: 2 and having the same biological activity as the protein of (a).

3. The cell for ex vivo gene therapy according to claim 1, wherein the FVIII modified body is any one of the following (a) to (c): (a) A protein composed of the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3, (b) A protein composed of an amino acid sequence containing one or more deletions, substitutions, additions or insertions of amino acid residues in the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3 and having the same biological activity as the protein of (a), (c) A protein composed of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 3 and having the same biological activity as the protein of (a).

4. The cell for ex vivo gene therapy according to claim 1, wherein the FVIII modified body is any one of the following (a) to (c): (a) A protein composed of the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4, (b) A protein composed of an amino acid sequence containing one or more deletions, substitutions, additions or insertions of amino acid residues in the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4 and having the same biological activity as the protein of (a), (c) A protein composed of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1670 in the amino acid sequence shown in SEQ ID NO: 4 and having the same biological activity as the protein of (a).

5. The cell for ex vivo gene therapy according to claim 1, wherein the FVIII modifier is that the Pro residue at position 758 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with another amino acid residue.

6. The cell for ex vivo gene therapy according to claim 5, wherein the other amino acid residue is a polar uncharged amino acid residue selected from Thr residue, Ser residue, Asn residue, and Gln residue.

7. The cell for ex vivo gene therapy according to claim 5, wherein the FVIII modifier is any one of the following (a) to (c): (a) A protein consisting of the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5; (b) A protein consisting of an amino acid sequence containing one or more deletions, substitutions, additions, or insertions of amino acid residues in the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5 and having the same biological activity as the protein in (a); (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 5 and having the same biological activity as the protein in (a).

8. The cell for ex vivo gene therapy according to claim 5, wherein the FVIII modifier is any one of the following (a) to (c): (a) A protein consisting of the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14; (b) A protein consisting of an amino acid sequence containing one or more deletions, substitutions, additions, or insertions of amino acid residues in the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14 and having the same biological activity as the protein in (a); (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 14 and having the same biological activity as the protein in (a).

9. The cell for ex vivo gene therapy according to claim 5, wherein the FVIII modifier is any one of the following (a) to (c): (a) A protein consisting of the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15; (b) A protein consisting of an amino acid sequence containing one or more deletions, substitutions, additions, or insertions of amino acid residues in the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15 and having the same biological activity as the protein in (a); (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence from positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 15 and having the same biological activity as the protein in (a).

10. The ex vivo gene therapy cell according to claim 5, wherein the FVIII modifier is any one of the following (a) to (c): (a) A protein consisting of the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16, (b) A protein consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, added or inserted in the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16 and having the same biological activity as the protein of (a), (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence at positions 20 to 1652 in the amino acid sequence shown in SEQ ID NO: 16 and having the same biological activity as the protein of (a).

11. The ex vivo gene therapy cell according to claim 1, wherein the cell is an adipocyte.

12. A transplantation composition comprising the ex vivo gene therapy cell according to claim 1 and a pharmaceutically acceptable carrier.

13. The transplantation composition according to claim 12, further comprising an extracellular matrix component.

14. The transplantation composition according to claim 12, further comprising an angiogenesis factor.

15. A method for producing an ex vivo gene therapy cell, which produces an ex vivo gene therapy cell by introducing an expression vector carrying a polynucleotide encoding an amino acid sequence of a modified human FVIII consisting of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1 into a cell that can be transplanted into the body of a subject for ex vivo gene therapy, wherein the modified human FVIII: The B domain (amino acid sequence at positions 760 to 1667) is altered, The number of Asn residues modified with N-linked glycans is 4 to 6 from the N-terminal side, and The function of the furin recognition site is defective.

16. The method for producing an ex vivo gene therapy cell according to claim 15, wherein the expression vector is a viral vector.

17. The method for producing an ex vivo gene therapy cell according to claim 16, wherein the viral vector is a lentiviral vector.

18. An expression vector for producing an ex vivo gene therapy cell, which carries a polynucleotide encoding an amino acid sequence of a modified human FVIII consisting of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1, and the modified human FVIII: The B domain (amino acid sequence at positions 760 to 1667) is altered, The number of Asn residues modified with N-linked glycans is 4 to 6 from the N-terminal side, and The function of the furin recognition site is defective.

19. An FVIII modifier, which is a modified human FVIII consisting of the amino acid sequence at positions 20 to 2351 in the amino acid sequence shown in SEQ ID NO: 1, wherein The B domain (amino acid sequence at positions 760 to 1667) is altered, The number of Asn residues modified with N-linked glycans is 4 to 6 from the N-terminal side, and, Functional defect in the recognition site of furin protease, and further, the Pro residue at position 758 is replaced with another amino acid residue.

20. The FVIII modifier according to claim 19, wherein the other amino acid residue is a polar uncharged amino acid residue selected from Thr residue, Ser residue, Asn residue, and Gln residue.

21. A polynucleotide encoding at least the amino acid sequence of the FVIII modifier according to claim 19 or 20.

22. The transplantation composition according to any one of claims 12 to 14, further comprising cells that produce human vWF and / or its fragment in a living body.

23. Cells that produce a fragment of human vWF in a living body.

24. The cells according to claim 23, wherein the fragment of human vWF contains at least the D' domain and D3 domain as the binding site for FVIII.

25. The cells according to claim 23, wherein the fragment of human vWF has the domain structure of SP-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK possessed by the precursor, has each domain of SP, D', and D3, does not have each domain of A2, A3, D4, B1, B2, B3, C1, and C2, and the A1 domain may have at most 50 amino acid residues on the N-terminal side, and optionally has or does not have each domain of D1, D2, and CK.

26. The cells according to claim 24, wherein the fragment of human vWF is any one of the following (a) to (c): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 29, (b) A protein consisting of an amino acid sequence containing one or more deletions, substitutions, additions, or insertions in the amino acid sequence shown in SEQ ID NO: 29 and having the same biological activity as the protein in (a), (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 29 and having the same biological activity as the protein in (a).

27. The cells according to claim 24, wherein the fragment of human vWF is any one of the following (a) to (c): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 30, (b) A protein consisting of an amino acid sequence containing one or more deletions, substitutions, additions, or insertions in the amino acid sequence shown in SEQ ID NO: 30 and having the same biological activity as the protein in (a), (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 30 and having the same biological activity as the protein in (a).

28. The cells according to claim 24, wherein the fragment of human vWF is any one of the following (a) to (c): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 31, (b) A protein consisting of an amino acid sequence with one or more amino acid residue deletions, substitutions, additions, or insertions in the amino acid sequence shown in SEQ ID NO: 31 and having the same biological activity as the protein in (a). (c) A protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 31 and having the same biological activity as the protein in (a).

29. An agent for increasing the secretion amount of the FVIII modifier into the blood, wherein the FVIII modifier is produced by transplanting the ex vivo gene therapy cells according to claim 1 into the body of a subject for ex vivo gene therapy, and wherein the agent contains cells that produce human vWF and / or its fragment in vivo as an active ingredient.

30. A transplantation composition containing cells that produce human vWF and / or its fragment in vivo and a pharmaceutically acceptable carrier.

31. A group of transplantation compositions containing the transplantation composition according to claim 12 and the transplantation composition according to claim 30.

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