Bornavirus vector gene modification of cells and cell therapy drug using cells

Abstract

Disclosed are: a bornavirus vector that includes a nucleic acid that encodes a growth factor; a recombinant virus that includes RNA that is encoded by the bornavirus vector; cells into which the nucleic acid has been introduced by infection with the recombinant virus; and a pharmaceutical composition that includes the cells.

Description

Genetic modification of cells using Borna virus vectors and cell therapy using these cells

[0001] The present invention relates to a bornavirus vector, a recombinant virus containing RNA encoded by the bornavirus vector, a cell infected with the recombinant virus, and a pharmaceutical composition containing the cell.

[0002] Borna disease virus (hereinafter referred to as BDV) is a virus belonging to the order Mononegavirales that has a genome of non-segmented, negative-strand, single-stranded RNA and is characterized by its neuronal tropism. Furthermore, although BDV replicates in the cell nucleus, it is characterized by its non-cytopathic, long-term persistent infection and an extremely wide range of infectable hosts (see Non-Patent Documents 1-7).

[0003] As a technique for introducing foreign genes using such BDV, Patent Document 1 reports a viral vector characterized by comprising (a) a recombinant viral cDNA in which an arbitrary foreign gene has been inserted into the translation region of the G gene of the cDNA encoding the Borna disease virus genome, (b) a cDNA encoding a ribozyme, and (c) a promoter sequence, with (b) positioned upstream and downstream of (a), and (a) and (b) positioned downstream of (c).

[0004] Furthermore, Patent Document 2 reports a viral vector characterized by comprising: (a) a cDNA of a recombinant viral RNA having at least the N gene, X gene, P gene, and L gene in the Borna disease virus genome in the same order as in the Borna disease virus genome, with a foreign gene inserted into the untranslated region linked downstream of the translated region of the P gene; (b) DNA encoding a ribozyme; and (c) a promoter sequence, with (b) located upstream and downstream of (a), and (a) and (b) located downstream of (c).

[0005] Patent Document 3 reports a technology that can introduce foreign genes into cells very efficiently, in which a viral vector is characterized by containing cDNA of recombinant viral RNA having a sequence in which the G gene in the Borna disease virus genome has been disrupted and the G gene in the avian Borna virus genome has been inserted.

[0006] Japanese Patent Application Publication No. 2010-22338 International Publication No. 2010 / 113647 International Publication No. 2018 / 168586

[0007] Microbiol Immunol. 2020 Sep;64(9):602-609Sci Rep. 2020 Apr 3;10(1):5890Front Microbiol. 2019 Nov 6;10:2485.Mol Ther Methods Clin Dev. 2019 May 28;14:47-55Microbiol Immunol. 2017 Sep;61(9):380-386.Sci Rep. 2016 May 18;6:26154J Virol. 2011 Dec;85(23):12170-8.

[0008] An object of the present invention is to provide a bornavirus vector that has improved proliferation properties, engraftment efficiency, and target gene expression in transplanted cells.

[0009] Furthermore, the present invention aims to provide a recombinant virus containing RNA encoded by the bornavirus vector, a cell infected with the recombinant virus, and a pharmaceutical composition containing the cell.

[0010] As a result of extensive research to achieve the above-mentioned objective, the inventors discovered that when a target gene was introduced into cells using a borna virus vector carrying a growth factor gene and the cells were then transplanted into an animal, the proliferation and engraftment of the transplanted stem cells, as well as the expression of the target gene, were improved, resulting in high medicinal efficacy.

[0011] The present invention was completed based on these findings and after further investigation, and provides the following bornavirus vector, a recombinant virus containing RNA encoded by the bornavirus vector, cells infected with the recombinant virus, and a pharmaceutical composition containing the cells.

[0012] Item 1. A bornavirus vector having a nucleic acid encoding a growth factor. Item 2. The bornavirus vector according to Item 1, further comprising a gene of interest. Item 3. The bornavirus vector according to Item 1 or 2, wherein the growth factor is an NTF (neurotrophic factor). Item 4. The bornavirus vector according to Item 1 or 2, wherein the growth factor is selected from the group consisting of BDNF, CXCR4, FGF2, GDNF, HGF, IGF1, Neurogenin 1, Notch1 domain, PDGFA, PDGFB, TGFB1, and VEGFA. Item 5. The bornavirus vector according to any one of Item 2 to 4, wherein the gene of interest is a gene encoding an antibody or an antibody fragment. Item 6. The antibody fragment is selected from the group consisting of Fab, Fab', F(ab') ２, Fv, scFv, scFab, dsFv, ds-scFv, diabody, triabody, tetrabody, or minibody. Item 7. The bornavirus vector of Item 5 or 6, wherein the antibody or antibody fragment is a humanized antibody or antibody fragment. Item 8. The bornavirus vector of any one of Items 5 to 7, wherein the antibody or antibody fragment has binding activity to a mutant SOD1 protein. Item 9. The bornavirus vector of any one of Items 1 to 8, comprising: (a) cDNA of a recombinant viral RNA having at least the N gene, X gene, P gene, and L gene in the Borna disease virus genome in the same order as in the Borna disease virus genome, and having a sequence in which the nucleic acid is inserted into an untranslated region linked downstream of the translated region of the P gene, (b) DNA encoding a ribozyme, and (c) a promoter sequence, wherein (b) is located upstream and downstream of (a), and (a) and (b) are located downstream of (c). Item 10. The bornavirus vector according to Item 9, wherein (a) is a cDNA of a recombinant viral RNA having at least the N gene, X gene, P gene, and L gene in the Borna disease virus genome in the same order as in the Borna disease virus genome, with the nucleic acid and the gene of interest inserted into the untranslated region linked downstream of the translated region of the P gene. Item 11. A recombinant virus comprising RNA encoded by the bornavirus vector according to any one of Item 1 to 10. Item 12. A cell into which the nucleic acid has been introduced by infection with the recombinant virus according to Item 11. Item 13. The cell according to Item 12, wherein the cell is an oligodendrocyte precursor cell, a glial neural precursor cell, a dopaminergic neural precursor cell, a microglia, a neural precursor cell, a neural stem cell, a mesenchymal stem cell, a Muse cell, an iPS cell, or an ES cell. Item 14. A pharmaceutical composition comprising the cell according to Item 13. Item 15. Item 16. A method for treating a disease associated with the accumulation of mutant SOD1, such as amyotrophic lateral sclerosis, comprising administering an effective amount of the cell of Item 12 or 13 to a mammal.Item 17. Use of the cell according to Item 12 or 13 in the production of a pharmaceutical composition for treating and / or preventing a disease in which mutant SOD1 accumulates, such as amyotrophic lateral sclerosis.

[0013] By using the bornavirus vector of the present invention to produce a recombinant virus containing RNA encoded by the bornavirus vector, and then infecting cells with the recombinant virus and transplanting the cells, it is possible to improve cell proliferation, engraftment efficiency, and target gene expression. Furthermore, it is possible to maintain target gene expression for a long period of time. As a result, the present invention makes it possible to achieve greater and more durable efficacy than conventional cell therapies.

[0014] 1 is a graph showing the change in mesenchymal stem cell cell count (cell count / well) over time after mesenchymal stem cells were transfected with a viral vector encoding an NTF and scFv or GFP. The NTFs are (A) BDNF, (B) FGF2, (C) HGF, (D) IGF1, and (E) VEGFA. Mock: MSCs not transfected with vector; data are mean ± SD, n = 3. This graph shows the results of measuring the amount of scFv in serum collected from wild-type SD rats transplanted with MSCs transfected with Borna virus vectors. The vertical axis represents the relative amount of scFv in serum, with the value on day 2 of administration taken as 100. The genes encoded by the vectors used in each group are as follows: Control: scFv, BDNF: BDNF-scFv, FGF2: FGF2-scFv, HGF: HGF-scFv, IGF1: IGF1-scFv. Data are mean ± SD, n = 3. Figure 1 is a graph showing the results of measuring the amount of scFv in tissues (cerebral cortex tissue, cerebellum tissue, and spinal fluid) collected from wild-type SD rats transplanted with MSCs transduced with Borna virus vectors. The vertical axis represents the relative amount of scFv in the tissues, with the value for the scFv group set to 100. Control represents a group that used MSCs not transduced with a vector. The genes encoded by the vectors used in each group are as follows: scFv: scFv, scFv + BDNF: BDNF-scFv, scFv + HGF: HGF-scFv. Data are mean ± SD, n = 4. Figure 1 is a graph showing the results of measuring the amount of scFv in serum collected from immunodeficient rats F344 / NJcl-rnu / rnu transplanted with MSCs transduced with Borna virus vectors. The vertical axis represents the relative amount of scFv in the tissues, with the value on day 3 of administration set to 1. Mock is a group that used MSCs without vector introduction. The genes encoded by the vectors used in each group are as follows: Control: scFv, BDNF: BDNF-scFv, HGF: HGF-scFv. Data are mean ± SD, n = 3. This graph shows the results of measuring weight changes (g) in ALS model mice transplanted with MSCs introduced with a Borna virus vector.Non-treat ALS: A group of ALS model mice administered with culture medium only, Non-treat WT: A group of wild-type mice administered with culture medium only, REVec-MSC ALS group: A group of ALS model mice transplanted with MSCs transduced with HGF-scFv. Data are mean ± SD, n = 6. This graph shows the results of measuring pNF-H (ng / mL) in ALS model mice transplanted with MSCs transduced with Borna virus vector. Non-treat / WT: A group of wild-type mice administered with culture medium only, Non-treat / ALS: A group of ALS model mice administered with culture medium only, MSC / ALS: A group of ALS model mice transplanted with MSCs not transduced with vector, REVec-MSC / ALS: A group of ALS model mice transplanted with MSCs transduced with HGF-scFv vector. n = 6.

[0015] Hereinafter, embodiments of the present invention will be described in detail.

[0016] In the present invention, "nucleic acid" means RNA or DNA. In the present invention, unless otherwise specified, "gene" includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof. Furthermore, in the present invention, "gene" refers to regulatory regions, coding regions, exons, and introns without distinction, unless otherwise specified.

[0017] In the present invention, the terms "nucleic acid," "nucleotide," and "polynucleotide" are synonymous and include both DNA and RNA, which may be double-stranded or single-stranded.

[0018] In this specification, "SOD1" refers to a protein unless otherwise specified, but when it is appropriate to interpret it as a gene, it refers to a gene.

[0019] The RefSeq IDs shown below are registered on the NCBI website.

[0020] The bornavirus vector of the present invention is characterized by having a nucleic acid encoding a growth factor (hereinafter sometimes referred to as "the nucleic acid of the present invention").

[0021] Furthermore, the bornavirus vector of the present invention can further comprise a gene of interest in addition to the nucleic acid encoding the growth factor.

[0022] Growth factors are a general term for endogenous proteins that promote the proliferation and differentiation of specific cells in the body. Examples of growth factors include hepatocyte growth factor (HGF), nerve growth factor (NGF), neurotrophic factors, epidermal growth factor (EGF), milk-derived growth factor, fibroblast growth factor (FGF), brain-derived fibroblast growth factor, acidic fibroblast growth factor, basic fibroblast growth factor (FGF2), platelet-derived growth factors (PDGFA, PDGFB, PDGFC, PDGFD), platelet basic protein, connective tissue activating peptide, insulin-like growth factor (IGF1, IGF2), colony-stimulating factor, erythropoietin, thrombopoietin, T-cell growth factor, interleukins (e.g., interleukin 2, 3, 4, 5, 7, 9, 11, 15), B-cell growth factor, cartilage-derived growth factor, bone-derived growth factor, skeletal growth factor, endothelial cell ... Cell-derived growth factor, eye-derived growth factor, testis-derived growth factor, Sertoli cell-derived growth factor, mammary gland stimulating factor, spinal cord-derived growth factor, macrophage-derived growth factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, recycled mesenchymal growth factor, transforming growth factor-α, transforming growth factor-β (TGFB1, TGFB2, TGFB3), heparin-binding EGF-like growth factor, amphiregulin, SDGF, betacellulin, epiregulin, neuregulin 1, 2, 3, vascular endothelial growth factor (VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, PIGF1, PIGF2), neurotrophin, neurogenin (neurogenin 1, neurogenin 2, neurogenin 3), BDNF, CXCR4, Notch1 domain, NT-3, NT-4, NT-5, NT-6, NT-7, glial cell line-derived neurotrophic factor (GDNF), stem cell factor, midkine, pleiotrophin, ephrin, angiopoietin, activin, tumor necrosis factor, interferons, and the like.

[0023] Among the growth factors, NTFs (neurotrophic factors) are preferred, and those selected from the group consisting of BDNF, CXCR4, FGF2, GDNF, HGF, IGF1, Neurogenin1, Notch1 domain, PDGFA, PDGFB, TGFB1, and VEGFA are more preferably used. "Neurotrophic factors (NTFs)" refer to cellular factors that act on the central nervous system, including neuronal proliferation (growth), differentiation, functional maintenance, and survival effects.

[0024] The above-mentioned growth factors are usually derived from animals, preferably mammals, and particularly preferably humans. The amino acid and nucleotide sequences of the above-mentioned growth factors are publicly known and can be obtained from public databases such as GenBank. In the present invention, the amino acid and nucleotide sequences of the above-mentioned growth factors include degenerates and variants thereof, even if they are not registered in the above-mentioned databases. Desirably, variants encode proteins with biological activity equivalent to that of proteins consisting of the above-mentioned amino acid sequences. Proteins with equivalent biological activity can be inferred, for example, by comparison with the amino acid sequences of the same proteins derived from other organisms. Variants broadly include modifications obtained by substituting, adding, deleting, or inserting one or more amino acids in the amino acid sequences registered in the above-mentioned databases. Furthermore, the secretion signal of the growth factor gene may be the sequence of the original gene, or any modified signal sequence (e.g., the secretion signal of another gene) can also be used.

[0025] By introducing nucleic acids encoding growth factors into cells using a borna virus vector, it is possible to improve the proliferation and engraftment of transplanted stem cells and improve the expression of the target gene (the expression level and persistence of expression of the target gene). Furthermore, even when the target gene is not inserted and a growth factor is used alone, it is possible to improve the proliferation and engraftment of transplanted stem cells, and it is possible to obtain medicinal efficacy and persistence based on the action of the growth factor.

[0026] The type and length of the target gene are not particularly limited, and any desired gene can be used depending on the purpose. For example, cDNA of a gene encoding a protein or peptide; cDNA encoding siRNA, short hairpin RNA (shRNA), microRNA (miRNA), or antisense RNA; cDNA encoding an RNA aptamer; cDNA encoding a circular RNA, etc., can be used. Specific examples include genes encoding enzymes that degrade proteins that cause nervous system disorders, genes encoding antibodies or antibody fragments that bind to proteins that cause nervous system disorders, genes encoding nucleic acids that function to inhibit the expression of such proteins, and genes encoding brain substances (e.g., serotonin, dopamine, somatostatin, neprilysin, etc.). The viral vector of the present invention can use one or more target genes. In this case, the same or different target genes can be inserted in tandem into a single viral vector.

[0027] The target gene particularly includes a gene encoding an antibody or an antibody fragment. An antibody fragment refers to a portion of an antibody that retains all or part of the binding activity of the antibody from which the antibody fragment is derived. Examples of antibody fragments include Fab, Fab', F(ab') and the like. ２ , Fv, scFab, scFv, dsFv, ds-scFv, diabody, triabody, tetrabody, minibody, etc. The diabody, triabody, tetrabody, and minibody may be of the same type or a combination of different types.

[0028] The antibody fragment is preferably an scFv. An scFv is an antibody fragment in which Fvs consisting of a heavy chain variable region and a light chain variable region are linked by an appropriate peptide linker. As an scFv, one in which VH and VL are linked in any order can be used.

[0029] The above-mentioned antibody or antibody fragment may be derived from any animal, including mouse, rat, guinea pig, rabbit, cow, goat, or sheep, and is not particularly limited. The isotype of the antibody or antibody fragment of the present invention is also not particularly limited, and may be any of IgG (IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE, and IgM. Furthermore, any functional peptide domain (e.g., a signal sequence, a blood-brain barrier-permeable peptide), etc., may be added to the N-terminus and / or C-terminus of the above-mentioned antibody or antibody fragment.

[0030] The sequences of the framework regions (FR regions) in the variable regions of the above-mentioned antibodies or antibody fragments are not particularly limited, and any sequences can be used as long as the antibody fragment has binding activity to the target target. The above-mentioned antibodies or antibody fragments are preferably humanized antibodies or antibody fragments in which the framework regions are derived from humans. Such humanized antibodies or antibody fragments can be produced by known methods.

[0031] The above-mentioned antibody or antibody fragment particularly includes those having binding ability to mutant SOD1 protein.

[0032] Superoxide dismutase (SOD) is an enzyme that catalyzes the dismutation of superoxide anions into oxygen and hydrogen peroxide (EC 1.15.1.1). In humans, there are three forms of SOD: SOD1, which exists in the cytoplasm and is usually a dimer; SOD2, which exists in mitochondria; and SOD3, which exists extracellularly and is usually a tetramer. SOD1 and SOD3 contain copper and zinc in their reaction centers, while SOD2 contains manganese in its reaction center.

[0033] The SOD1 of the present invention is generally derived from an animal, preferably from a mammal, and particularly preferably from a human.

[0034] The amino acid sequence of human SOD1 protein has been registered as RefSeq Accession No. NP_000445 and is shown in SEQ ID NO: 6. The gene encoding human SOD1 protein has been registered as RefSeq Accession No. NM_000454 and its nucleotide sequence is shown in SEQ ID NO: 7.

[0035] The mutant SOD1 of the present invention refers to SOD1 that has been folded into a structure different from its native structure due to a mutation in the protein caused by amino acid substitution, deletion, insertion, or addition, and particularly refers to SOD1 having a pathogenic structure associated with amyotrophic lateral sclerosis (ALS). The mutant SOD1 may be caused by a mutation in the protein sequence or by other modifications. Furthermore, the mutant SOD1 may be SOD1 that has either a sporadic mutation or a familial mutation, and particularly refers to SOD1 that has a familial mutation.

[0036] It is desirable that the above antibody or antibody fragment specifically binds to mutant SOD1 and does not bind to wild-type SOD1 having a normal structure.

[0037] Specific examples of antibodies or antibody fragments that have binding affinity to mutant SOD1 proteins include antibodies or antibody fragments (hereinafter sometimes referred to as "antibody or antibody fragment (A)") that comprise a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence of GFSLNTSGMG (SEQ ID NO: 1), a heavy chain CDR2 consisting of the amino acid sequence of IWWDDDK (SEQ ID NO: 2), and a heavy chain CDR3 consisting of the amino acid sequence of ARLGYAMDY (SEQ ID NO: 3), each of which may have up to three amino acid substitutions; and / or a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence of QNVGTN (SEQ ID NO: 4), a light chain CDR2 consisting of the amino acid sequence of SAS, and a light chain CDR3 consisting of the amino acid sequence of QQYYIYPYT (SEQ ID NO: 5), each of which may have up to three amino acid substitutions.

[0038] The antibody or antibody fragment (A) preferably comprises a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence of GFSLNTSGMG (SEQ ID NO: 1), a heavy chain CDR2 consisting of the amino acid sequence of IWWDDDK (SEQ ID NO: 2), and a heavy chain CDR3 consisting of the amino acid sequence of ARLGYAMDY (SEQ ID NO: 3), each of which may have up to three amino acid substitutions; and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence of QNVGTN (SEQ ID NO: 4), a light chain CDR2 consisting of the amino acid sequence of SAS, and a light chain CDR3 consisting of the amino acid sequence of QQYYIYPYT (SEQ ID NO: 5), each of which may have up to three amino acid substitutions.

[0039] The amino acid substitutions are made so as to maintain the binding ability of the antibody or antibody fragment to mutant SOD1. The above phrase "up to three amino acid substitutions may be made" means that up to three amino acids in total may be substituted in the heavy chain variable region or light chain variable region, and the amino acid substitutions are preferably made in the CDRs.

[0040] The number of amino acid substitutions is preferably 2 or less, more preferably 1 or less, and even more preferably 0. When amino acids are substituted, it is thought that the activity of the original antibody fragment is more likely to be maintained if they are substituted with amino acids having similar properties. Techniques for substituting amino acids in a specific amino acid sequence are known.

[0041] The antibody or antibody fragment (A) can recognize a wide variety of mutant SOD1 proteins regardless of the amino acid substitution site, and is therefore widely used as an antibody or antibody fragment for the prevention and treatment of ALS caused by mutations in the SOD1 gene, such as A4V, C6G, H46R, G85R, G93A, C111Y, I112T, and I113T mutant SOD1 proteins.

[0042] The nucleic acid of the present invention is characterized in that it encodes a growth factor. The nucleic acid may have sequences added to the untranslated region of the transcribed mRNA to improve translation and RNA stability. The nucleic acid, target gene, etc. can be produced by standard methods such as PCR, chemical synthesis, and biochemical cleavage / recombination. Examples of chemical synthesis methods include the phosphoramidite method and the H-phosphonate method. The chemical synthesis method can be performed, for example, using a commercially available automated nucleic acid synthesizer.

[0043] The bornavirus vector of the present invention is used to produce a growth factor (and a nucleic acid or protein encoded by the gene of interest), and is characterized by having the nucleic acid (and gene of interest). The bornavirus vector of the present invention may also contain genes other than the nucleic acid and gene of interest. The bornavirus vector is not particularly limited, and a wide variety of known bornavirus vectors can be used (see, for example, WO 2010 / 113647, JP 2018-148865, Microbiol Immunol 2017 Sep; 61(9): 380-386, J Virol. 2011 Dec; 85(23): 12170-8, Sci Rep. 2016 May 18; 6: 26154, etc.). The nucleic acid and gene of interest can be inserted into the vector by known methods. Borna virus vectors exist as extrachromosomal RNA (episomal) in the cell nucleus and can maintain semi-persistent infection by localizing to the chromosome even in dividing and proliferating cells. They also constantly express N, P, X, and L proteins (derived from the virus) and growth factors (and target genes) within the cells.

[0044] The BDV genome encodes at least six proteins: nucleoprotein (N protein), X protein, phosphoprotein (P protein), matrix protein (M protein), surface glycoprotein (G protein), and RNA-dependent RNA polymerase (L protein). The BDV genome has the N, X, P, M, G, and L genes in this order from the 3' end. In this specification, the viral RNAs encoding the G protein, M protein, N protein, P protein, and L protein are referred to as the G gene, M gene, N gene, P gene, and L gene, respectively.

[0045] Bornavirus vectors and methods for preparing them, recombinant viruses containing RNA encoded by bornavirus vectors and methods for preparing them, and methods for infecting cells with the viruses are well known, and can be carried out with reference to, for example, WO 2010 / 113647, JP 2018-148865 A, Microbiol Immunol 2017 Sep; 61 (9): 380-386, J Virol. 2011 Dec; 85 (23): 12170-8, Sci Rep. 2016 May 18; 6: 26154, etc.

[0046] Specific examples of Borna virus vectors of the present invention include those comprising: (a) a cDNA of a recombinant viral RNA having at least the N gene, X gene, P gene, and L gene in the Borna disease virus genome in the same order as in the Borna disease virus genome, with a nucleic acid of the present invention inserted into the untranslated region linked downstream of the translated region of the P gene; (b) DNA encoding a ribozyme; and (c) a promoter sequence, with (b) located upstream and downstream of (a), and (a) and (b) located downstream of (c).

[0047] Other specific examples of Borna virus vectors of the present invention include those that contain (a) cDNA of a recombinant viral RNA having at least the N gene, X gene, P gene, and L gene in the Borna disease virus genome in the same order as in the Borna disease virus genome, with the nucleic acid of the present invention and a gene of interest inserted in the untranslated region linked downstream of the translated region of the P gene, (b) DNA encoding a ribozyme, and (c) a promoter sequence, with (b) located upstream and downstream of (a), and (a) and (b) located downstream of (c).

[0048] As such (a) cDNA of recombinant viral RNA, preferred is a cDNA of recombinant viral RNA that encodes a sequence in which the nucleic acid of the present invention (and the target gene) is inserted into the untranslated region linked downstream of the translated region of the P gene in the BDV genome or in a sequence in which either the G gene or the M gene or both of the G gene and the M gene have been disrupted in the BDV genome.

[0049] Among these, (a) as the cDNA of recombinant viral RNA, a cDNA of recombinant viral RNA having a sequence in which either or both of the G gene and the M gene in the BDV genome are disrupted, and having a sequence in which the nucleic acid of the present invention (and the gene of interest) is inserted in the untranslated region linked downstream of the translated region of the P gene is more preferred. Even more preferred is a cDNA of recombinant viral RNA having a sequence in which the G gene in the BDV genome is disrupted, and having a sequence in which the nucleic acid of the present invention (and the gene of interest) is inserted in the untranslated region linked downstream of the translated region of the P gene. By disrupting the G gene, the recombinant virus produced from the viral vector does not spread to cells other than those into which it has been introduced, thereby reducing pathogenicity and making it safer.

[0050] In addition, (a) as the cDNA of the recombinant viral RNA, it is also possible to use the cDNA of a recombinant virus in which the nucleic acid of the present invention (and the target gene) has been inserted into the untranslated region between the translation region of the P gene and the translation region of the M gene of the cDNA encoding the BDV genome.

[0051] The BDV genome may be a gene of a virus belonging to the Bornaviridae family or a mutant strain thereof. Examples of viruses belonging to the Bornaviridae family include He80, H1766, Strain V, and huP2br. Examples of mutant strains include No / 98, Bo / 04w, and HOT6.

[0052] (a) As the cDNA of the recombinant viral RNA, a cDNA of a recombinant BDV genome having a sequence in which the G gene in the BDV genome is disrupted and the G gene in the avian Bornavirus genome gene is inserted may be used (see JP 2018-148865 A). This allows for higher productivity and recovery rate of the resulting recombinant virus and efficient introduction of the nucleic acid of the present invention.

[0053] (a) In the cDNA of the recombinant viral RNA, the nucleic acid of the present invention may be located either upstream or downstream of the target gene, and two or more target genes may be located in tandem.

[0054] The ribozyme of the (b) ribozyme-encoding DNA may be any DNA having a sequence capable of cleaving the nucleic acid of the present invention (and the target gene) transcribed from the cDNA of the (a) recombinant viral RNA, such as a cDNA encoding a ribozyme such as a hammerhead ribozyme (HamRz), a hepatitis δ virus ribozyme (HDVRz), a hairpin ribozyme, or an artificial ribozyme. Among these, hammerhead ribozyme (HamRz) and a hepatitis δ virus ribozyme (HDVRz) are preferred. Furthermore, it is particularly preferred that the (b1) HamRz-encoding DNA (preferably cDNA) sequence is located upstream of the (a) recombinant BDV genome cDNA, i.e., at the 3'-end, and the (b2) HDVRz-encoding DNA (preferably cDNA) sequence is located downstream of the (a), i.e., at the 5'-end. This improves the expression efficiency of the nucleic acid of the present invention (and the target gene) in cells.

[0055] (c) Examples of promoter sequences include RNA polymerase II promoter sequences, RNA polymerase I promoter sequences, and T7 polymerase promoter sequences, with RNA polymerase II promoter sequences being preferred. Examples of RNA polymerase II promoters include the CMV promoter and the CAGGS promoter. Among these, the CAGGS promoter is particularly preferred.

[0056] The bornavirus vector of the present invention may further comprise one or more nucleic acid sequences encoding a part or all of the SV40 virus replication origin / promoter region sequence, an enhancer, an activator (e.g., a trans-acting factor), a chaperone, or a processing protease. The viral vector of the present invention may be either linear or circular DNA, and is preferably circular when introduced into a cell.

[0057] The recombinant virus of the present invention is characterized by containing RNA encoded by the above-mentioned bornavirus vector. The recombinant virus of the present invention is preferably a recombinant virus containing RNA encoded by the above-mentioned bornavirus vector, as well as BDV N protein, BDV P protein, and BDV L protein. By containing the above-mentioned recombinant viral RNA, the recombinant virus of the present invention is a persistently infective recombinant virus that can persistently express a growth factor (and a nucleic acid or protein encoded by a target gene) after infecting a cell. The recombinant virus may optionally contain BDV G protein or an outer coat protein of another virus, and may further contain BDV M protein.

[0058] The recombinant virus of the present invention can be produced, for example, by a method comprising the steps of introducing into in vitro cells a plasmid or group of plasmids expressing the BDV N, P, and L genes as a helper plasmid together with the above-mentioned bornavirus vector, and culturing the cells into which the bornavirus vector and helper plasmid have been introduced to produce the recombinant virus. When a G- and M-gene-deficient bornavirus vector is used, it is preferable to introduce into in vitro cells a plasmid expressing the viral coat gene and a plasmid expressing the BDV M gene together with the above-mentioned plasmid or group of plasmids expressing the BDV N, P, and L genes. With regard to the N and P genes, helper plasmids into which the gene sequences of not only borna disease virus 1 (BoDV-1) but also borna disease virus 2 (BoDV-2) have been inserted may be used. Furthermore, with regard to the M gene and G gene, helper plasmids into which gene sequences of not only borna disease virus 1 but also parrot bornavirus, canary bornavirus, or waterfowl bornavirus belonging to the Bornaviridae family are inserted may also be used. Specific examples include Variegated Squirrel Bornavirus (VSBV), Parrot Bornavirus 4 (PaBV-4), Parrot Bornavirus 5 (PaBV-5), Munia Bornavirus 1 (MuBV-1), Aquatic Bird Bornavirus 1 (ABBV-1), Estrilid Finch Bornavirus-1 (EsBV-1), and Canary Bornavirus 2 (CnBV-2).

[0059] The cells of the present invention are those into which the nucleic acid of the present invention (and the gene of interest) has been introduced by infection with the recombinant virus. By infecting cells with the recombinant virus of the present invention, the nucleic acid of the present invention (and the gene of interest) incorporated into the virus can be introduced into the cells.

[0060] The cells of the present invention are not particularly limited and include, for example, oligodendrocyte precursor cells, neural stem cells, mesenchymal stem cells (e.g., mesenchymal stem cells derived from bone marrow, dental pulp, or adipose tissue), etc. In addition to oligodendrocyte precursor cells, neural stem cells, and mesenchymal stem cells, cells to be infected with the recombinant virus include pluripotent stem cells such as iPS cells (induced pluripotent stem cells), ES cells (embryonic stem cells), and Muse cells, as well as glial neural precursor cells, dopaminergic neural precursor cells, microglia, and neural precursor cells. Borna virus vectors have high transduction efficiency into stem cells and can induce differentiation while maintaining gene expression. Therefore, cells obtained by infecting pluripotent stem cells such as iPS cells, ES cells, and Muse cells with the recombinant virus of the present invention and then inducing differentiation into oligodendrocyte precursor cells, neural stem cells, mesenchymal stem cells, etc. also maintain expression of growth factors (and nucleic acids or proteins encoded by the target gene). Therefore, the cells of the present invention also include cells obtained by inducing differentiation from pluripotent stem cells infected with the above-mentioned recombinant virus (e.g., oligodendrocyte precursor cells, neural stem cells, neural precursor cells, mesenchymal stem cells, etc.). Examples of the cells include mammalian cells such as human, monkey, mouse, rat, dog, cat, and rabbit, with human cells being preferred. Furthermore, the cells may be either autologous or allogeneic.

[0061] The cells of the present invention have improved proliferation and engraftment ability as well as improved expression of the target gene (expression level and persistence of expression of the target gene) due to the introduction of nucleic acid encoding a growth factor by a borna virus vector.

[0062] A desirable method for infecting cells with a recombinant virus in vitro is to prepare a dilution by serially diluting the recombinant virus with a culture medium such as Dulbecco's modified Eagle's method or phosphate-buffered saline, etc. The concentration of the recombinant virus in the dilution may be appropriately selected depending on the type of cells to be infected with the virus, and the recombinant virus is used to infect cells.

[0063] The pharmaceutical composition of the present invention is administered to mammals including humans, and is characterized by containing the above-mentioned cells. It can be used for the treatment and / or prevention of diseases in which mutant SOD1 accumulates, such as amyotrophic lateral sclerosis.

[0064] The pharmaceutical composition of the present invention may optionally contain biologically acceptable carriers, excipients, etc. depending on the form of use. The pharmaceutical composition of the present invention can be prepared according to conventional methods. For example, it can be administered intranasally as a spray or parenterally in the form of an injection such as a sterile solution or suspension with water or other pharmaceutically acceptable liquid. The pharmaceutical composition of the present invention can also be used in combination with other known low-molecular-weight drugs.

[0065] The amount of cells, which are the active ingredient in the pharmaceutical composition of the present invention, is appropriately selected depending on the dosage form, route of administration, etc., and is usually 1.0 x 10 ３ ~1.0 x 10 １０ cells, preferably 1.0 x 10 ４ ~1.0 x 10 ９ It is about cells.

[0066] The pharmaceutical composition of the present invention may be administered locally or systemically. There are no particular limitations on the administration method. Examples of administration routes include intrathecal, intracerebral parenchymal, or intravenous injection or infusion, and intranasal administration. Oligodendrocyte precursor cells and neural stem cells can be administered intrathecally or intracerebral parenchyma, while mesenchymal stem cells can be administered intrathecally, intracerebral parenchyma, intravenously, or intranasally. The dosage of the pharmaceutical composition of the present invention can ultimately be appropriately determined at the discretion of a physician, taking into consideration the type of dosage form, administration method, the patient's age and weight, and the patient's symptoms, etc.

[0067] The pharmaceutical composition of the present invention can be used as a gene delivery vector for the treatment of cranial nervous system diseases, and can also be used as a vaccine against chronic liver diseases, tumors, infectious diseases, etc. Examples of cranial nervous system diseases include amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, multiple sclerosis, schizophrenia, autism, and other functional psychiatric disorders. When the target gene in the pharmaceutical composition of the present invention is a gene encoding an enzyme that degrades a protein that causes a cranial nervous system disease, a gene encoding an antibody or antibody fragment that binds to a protein that causes a cranial nervous system disease, or a gene encoding a nucleic acid that has the function of suppressing the expression of the protein, the administered cells can degrade the causative protein or suppress the expression of the causative protein, thereby preventing or treating the disease. Furthermore, in diseases caused by decreased secretion of brain substances (e.g., serotonin, dopamine, somatostatin, neprilysin, etc.), the administered cells can produce the brain substance, thereby preventing or treating the disease. When administered cells secrete an antibody or antibody fragment that binds to a mutant SOD1 protein, the pharmaceutical composition of the present invention can degrade and remove misfolded SOD1, and therefore can be used to treat diseases in which mutant SOD1 accumulates. Diseases in which mutant SOD1 accumulates include, but are not limited to, amyotrophic lateral sclerosis.

[0068] By using the bornavirus vector of the present invention to produce a recombinant virus containing RNA encoded by the bornavirus vector, and then infecting cells with the recombinant virus and transplanting the cells, a nucleic acid encoding a growth factor can be introduced into the transplanted cells, thereby improving cell proliferation, engraftment efficiency, and target gene expression. Furthermore, target gene expression can be maintained for an extended period of time. As a result, the present invention makes it possible to achieve greater and more sustained efficacy than conventional cell therapies.

[0069] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present invention encompasses all arbitrary combinations of the constituent elements described in this specification.

[0070] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present invention may be combined in any way to specify the subject matter encompassed by the present invention, i.e., the present invention encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0071] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0072] <Experimental Method> Anti-SOD1 Antibody The amino acid and nucleotide sequences of VH and VL of the anti-SOD1 antibody (scFv) that has binding ability to mutant SOD1 proteins and was used in the following experiments are shown below. The underlined parts are CDR1, CDR2, and CDR3, respectively.VH (amino acid sequence) QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGMGVGWIRQPSGRVLEWLAHIWWDDDKRYNPALKSRLTISKDTSTNQVFLKIASVDTTDTATYYCARLGYAMDYWGHGTSVTVS (SEQ ID NO: 8) VL (amino acid sequence) DIVMTQSQKFMSTSLGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYYIYPYTFGGGTKLEI (SEQ ID NO: 9) VH (nucleotide sequence) CAGGTTACTCTGAAGGAGTCTGGCCCTGGGATACTGCAGCCCTCACAGACCCTGAGTCTGACTTGTTCCTTCTCTGGATTTTCACTGAACACCTCTGGTATGGTGTAGGCTGGATTCGTCAGCCTTCCGGGAGGGTTCTGGAGTGGCTGGCTCACATCTGGTGGGATGATGATAAGC GCTATAACCCTGCCCTGAAGAGCCGACTGACCATCTCTAAGGATACCTCCACCAACCAGGTATTCCTCAAGATCGCCAGTGTGGACACCACCGATACTGCTACATACTACTGTGCTCGTCTTGGCTATGCTATGGACTACTGGGGTCACGGAACCTCAGTCACCGTCTCC (SEQ ID NO: 10) VL (base sequence) GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACCTCACTTGGAGACCGAGTCTCAGTCACCTGCAAGGCTTCCCAGAATGTGGGTACAAATGTAGCCTGGTATCAGCAGAAACCAGGACAGTCTCCTAAGGCACTGATTTACTCGGCTTCCTACCGTT ACTCCGGAGTCCCTGATCGTTTCACAGGTAGTGGATCTGGAACAGATTTCACCCTGACCATCAGCAATGTTCAGTCTGAAGATTTGGCAGAGTATTTCTGCCAGCAATATTACATCTATCCGTACACGTTCGGAGGAGGAACCAAGCTGGAAATT (SEQ ID NO: 11).

[0073] Preparation of pCAG-FctHe80 p / lΔMG scFv plasmid with anti-SOD1 antibody gene inserted The pCAG-FctHe80 p / lΔMG scFv plasmid was prepared using a procedure similar to that described by Fujino K et al. (Microbiol Immunol. 2017 Sep; 61(9): 380-386. doi: 10.1111 / 1348-0421.12505.). This plasmid contains a BstBI and PacI site cassette between the P and L genes as a foreign gene insertion cassette. Using the anti-SOD1 antibody scFv cDNA as a template, primers and PrimeSTAR™ MAX DNA polymerase (Takara Bio Inc.) were used to perform PCR (98°C 10 seconds, 55°C 5 seconds, 72°C 5 seconds, 35 cycles). The PCR product was then recombinantly transformed with the pCAG-FctHe80 p / lΔMG plasmid using BstBI and PacI to obtain the pCAG-FctHe80 p / lΔMG scFv plasmid. The scFv here contained Myc and HiBit tags.

[0074] Preparation of pCAG-FctHe80 p / lΔMG NTF tandem vector plasmid with growth factor gene and anti-SOD1 antibody gene inserted The pCAG-FctHe80 p / lΔMG BstBI-AsisI + SbfI-PacI plasmid was prepared by inserting the S3 and T2 sequences and AsisI and SbfI sites as a foreign gene insertion cassette between the BstBI and PacI cassettes of the pCAG-FctHe80 p / lΔMG plasmid prepared above. This plasmid contains a BstBI, AsisI site cassette and an SbfI, PacI site cassette between the P gene and the L gene as a foreign gene insertion cassette.

[0075] Next, the anti-SOD1 antibody gene or the GFP gene was inserted into a cassette containing SbfI and PacI sites to prepare the pCAG-FctHe80 p / lΔMG Empty-scFv and pCAG-FctHe80 p / lΔMG Empty-GFP vector plasmids. PCR was performed using primers with the anti-SOD1 antibody scFv cDNA or the GFP gene (Promega) cDNA as a template (98°C for 10 seconds, 55°C for 5 seconds, 72°C for 10 seconds, 30 cycles). The PCR product was recombined with the pCAG-FctHe80 p / lΔMG BstBI-AsisI+SbfI-PacI plasmid using SbfI and PacI to obtain pCAG-FctHe80 p / lΔMG Empty-scFv and pCAG-FctHe80 p / lΔMG Empty-GFP, respectively.

[0076] Subsequently, each growth factor gene was inserted into a cassette containing BstBI and AsisI sites to create the pCAG-FctHe80 p / lΔMG NTF-scFv and pCAG-FctHe80 p / lΔMG NTF-GFP tandem vector plasmids. PCR (98°C for 10 seconds, 55°C for 5 seconds, 72°C for 10 seconds, 30 cycles) was performed using primers and cDNAs of human BDNF, FGF2, HGF, IGF1, and VEGFA as templates. The PCR product was recombined with pCAG-FctHe80 p / lΔMG Empty-scFv and pCAG-FctHe80 p / lΔMG Empty-GFP plasmids using BstBI and AsisI to obtain pCAG-FctHe80 p / lΔMG NTF-scFv and pCAG-FctHe80 p / lΔMG NTF-GFP vector plasmids, respectively.

[0077] - Preparation of M and G gene-deficient recombinant viruses pCAG-FctHe80 p / lΔMG scFv, pCAG-FctHe80 p / lΔMG NTF-scFv, or pCAG-FctHe80 p / lΔMG NTF-GFP and helper plasmids (N gene expression plasmid, P gene expression plasmid, L gene expression plasmid, M gene expression plasmid, and G gene expression plasmid: pcN, pCXN2-P, pcL, pcM, and pcG, respectively) were transfected into 293T cells using TransIT-293 Transfection Reagent (Mirus Bio). The amount of viral vector used for transfection was 1 x 10 ４ ~1 x 10 ６ For each 293T cell, 1 to 4 μg of each pCAG-FctHe80 p / lΔMG was used, and helper plasmids were added in the following proportions: pcN (0.125 to 0.5 μg), pCXN2-P (0.125 to 0.05 μg), pcL (0.125 to 0.5 μg), pcM (0.05 to 0.125 μg), and pcG (0.05 to 0.125 μg).

[0078] The viral vector and helper plasmid were introduced into 293T cells, which were then cultured at 37°C. Three days after introduction, the cells were passaged and further co-cultured with Vero cells constitutively expressing the M and G genes (Vero-MG cells). The Vero-MG cells were collected as recombinant virus-infected Vero cells.

[0079] Recovery of M and G gene-deficient recombinant viruses The recombinant virus-infected Vero-MG cells prepared above were washed twice with 20 mM HEPES buffer (Thermo Fisher Scientific, pH 7.5) and then resuspended in 20 mM HEPES (pH 7.5) / 250 mM MgCl ２ A 1% FCS buffer solution was added, and the mixture was left to stand at 37° C. for 90 minutes. After standing, the added buffer solution was collected, and the mixture was centrifuged at 3,500 rpm for 5 minutes at 4° C. The supernatant obtained was used as a viral vector solution and stored at −80° C.

[0080] Introduction of Bornavirus vector into mesenchymal stem cells Human bone marrow-derived mesenchymal stem cells (MSCs) (PromoCell #C-12974) were seeded onto a 6-well plate coated with RetroNectin™ (Takara Bio Inc.) and cultured in Cellartis MSC Xeno-Free Culture Medium (Takara Bio Inc.). After removing the culture supernatant from the mesenchymal stem cells, the viral vector solution prepared above was added to the mesenchymal stem cells at an MOI of 0.25 or 0.5 and allowed to stand at 37°C for 90 minutes. After standing, the cells were washed twice with a sufficient amount of Cellartis MSC Xeno-Free Culture Medium and again continued to be cultured at 37°C.

[0081] Transplantation of Bornavirus vector-introduced MSCs: After culturing in a 10-cm dish, infected MSCs were detached using Accumax (Nacalai Tesque), centrifuged at 500 x g for 5 minutes, and then transferred to 10 μL of Cellartis MSC Xeno-Free Culture Medium at a concentration of 1.0 x 10 ６ The suspension was adjusted to contain 1000 MSCs. The occipital region of 6-week-old wild-type SD rats (Japan SLC) or F344 / NJcl-rnu / rnu immunodeficient rats (Japan CLEA) was incised under isoflurane inhalation anesthesia, and 10 μL of the above MSC suspension was administered between the occipital bone and the first cervical vertebra for intrathecal transplantation. The lumbar region of 4-week-old ALS model mice, B6SJL-Tg(SOD1*G93A)1Gur / J (The Jackson Laboratory), was incised under isoflurane inhalation anesthesia, and 10 μL of the above MSC suspension was administered intrathecally via lumbar puncture.

[0082] Test Example 1 Each viral vector recovered by the above method was inoculated into MSCs at an MOI of 1.0. Four days after inoculation, 5.0 × 10 cells were placed on a 12-well plate coated with RetroNectin (Takara Bio Inc.). ４Each vector-introduced MSC was seeded. Every four days after seeding, the cells were detached using Accumax (Nacalai Tesque), suspended in Cellartis MSC Xeno-Free Culture Medium, and the viable cell count was measured using a TC20 Automated Cell Counter (Bio-Rad). All groups were tested at n = 3. The viral vectors used for inoculation were pCAG-FctHe80 p / lΔMG Empty-scFv (Empty-scFv), p / lΔMG NTF-scFv (BDNF-scFv, FGF2-scFv, HGF-scFv, IGF1-scFv, VEGFA-scFv), p / lΔMG Empty-GFP (Empty-GFP), and p / lΔMG NTF-GFP (BDNF-GFP, FGF2-GFP, HGF-GFP, IGF1-GFP, VEGFA-GFP). MSCs not transfected with the vector (Mock) served as a control.

[0083] The results are shown in Figure 1. The Empty-scFv, Empty-GFP, and Mock groups in Figures 1A to 1E show the results for the same sample. Compared to the control, MSC proliferation was significantly higher in the BDNF (Figure 1A), FGF2 (Figure 1B), HGF (Figure 1C), IGF1 (Figure 1D), and VEGFA (Figure 1E) expression vectors, regardless of whether the gene carried in the vector was scFv or GFP.

[0084] Test Example 2: MSCs transfected with pCAG-FctHe80 p / lΔMG BDNF-scFv (BDNF group), FGF2-scFv (FGF2 group), HGF-scFv (HGF group), or IGF1-scFv (IGF1 group) were transplanted intrathecally into wild-type SD rats (Japan SLC). A control group (Control) was administered with MSCs transfected with pCAG-FctHe80 p / lΔMG scFv, which does not carry a growth factor gene, and the amount of scFv in serum was compared and quantified. All groups were tested with n=3. Whole blood was collected from the tail vein every two days after administration, centrifuged at 3,000 x g for 5 minutes, and serum was collected. Using the collected serum and the Nano-Glo HiBiT Lytic Detection System (Promega), the relative amount of scFv was measured by chemiluminescence from the HiBiT tag linked to the scFv terminus. 10 μL of collected serum was dispensed into a 96-well plate, and 40 μL of Nano-Glo HiBiT Lytic Reagent, prepared by mixing the included LgBit Protein and Nano-Glo HiBiT Lytic Substrate with Nano-Glo HiBiT Lytic Buffer, was added to each well, and the luminescence was measured using a GloMax Discover Microplate Reader (Promega).

[0085] The results of quantifying the amount of scFv using the above method are shown in Figure 2. In the BDNF, FGF2, HGF, and IGF1 groups expressing growth factor genes, the relative amount of scFv in serum was significantly increased from 8 to 14 days after administration compared to the control. These results suggest that the expression of growth factors increases the proliferation and engraftment efficiency of MSCs compared to the control, and increases the amount of scFv expressed in vivo.

[0086] In addition, 14 days after administration, cerebral cortex tissue, cerebellum tissue, and spinal fluid were collected and the amount of scFv in the tissues was relatively quantified. The collected tissues were dissolved in RIPA buffer (50 mM Tris-HCl pH 7.7, 150 mM NaCl, 1% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS), and the protein amount of each sample was measured by colorimetric detection using the extracted fractions and Pierce BCA Protein Assay Kits (Thermo Fisher Scientific). Similarly, the amount of scFv in 1 mg of protein extracted from each tissue was relatively quantified using the HiBiT chemiluminescence method described above.

[0087] The results are shown in Figure 3. Compared with the group that did not express growth factor genes (scFv), the groups that received MSCs that expressed BDNF and HGF (scFv + BDNF and scFv + HGF) showed significantly higher amounts of scFv in the tissues. These results suggest that the expression of growth factors such as BDNF and HGF increases the amount of scFv in each tissue and maintains this level for a long period of time.

[0088] Test Example 3: MSCs transduced with pCAG-FctHe80 p / lΔMG BDNF-scFv (BDNF group) or HGF-scFv (HGF group) were intrathecally transplanted into immunodeficient F344 / NJcl-rnu / rnu rats (CLEA Japan). Additionally, a control group (Control) administered with MSCs transduced with pCAG-FctHe80 p / lΔMG scFv not carrying a growth factor gene and a control group (Mock) administered with MSCs not transduced with a vector were used to comparatively quantify the amount of scFv in serum. All groups were tested with n=3. Whole blood was collected from the tail vein every 6 days after administration, centrifuged at 3000 x g for 5 minutes, and serum was collected. Using the collected serum and the Nano-Glo HiBiT Lytic Detection System (Promega), the relative amount of scFv was measured by chemiluminescence from the HiBiT tag linked to the scFv terminus. 10 μL of collected serum was dispensed into a 96-well plate, and 40 μL of Nano-Glo HiBiT Lytic Reagent, prepared by mixing the included LgBit Protein and Nano-Glo HiBiT Lytic Substrate with Nano-Glo HiBiT Lytic Buffer, was added to each well, and the luminescence was measured using a GloMax Discover Microplate Reader (Promega).

[0089] The results of quantifying the amount of scFv using the above method are shown in Figure 4. In the BDNF and HGF groups, the relative serum scFv amount was significantly increased compared to the control, and no decrease in scFv amount was observed even after 40 days of administration. These results suggest that the expression of BDNF and HGF increases the proliferation and engraftment efficiency of MSCs compared to the control, increases the amount of scFv expression in vivo, and maintains the scFv amount for a long period of time.

[0090] Test Example 4: MSCs transfected with pCAG-FctHe80 p / lΔMG HGF-scFv were transplanted intrathecally into G93 mutant SOD1 model mice B6SJL-Tg(SOD1*G93A)1Gur / J (The Jackson Laboratory) (REVec-MSC ALS group). Control groups included a group of ALS model mice administered the same amount of culture medium alone as above (Non-treat ALS), and a group of wild-type C57BL / 6JJmsSlc mice (Japan SLC) administered the same amount of culture medium alone (Non-treat WT), and individual weight changes were compared. All groups were tested with n = 6. The results are shown in Figure 5. The weight of each individual was measured every two weeks after administration. As a result, the group administered with vector-introduced MSCs that simultaneously express HGF and scFv (REVec-MSC ALS) showed a significant improvement in weight loss compared to the non-treated group (Non-treat ALS group).

[0091] In addition, to quantify the amount of phosphorylated neurofilament (pNF-H) in serum, a brain inflammation marker, whole blood was collected from the tail vein at 18 weeks after administration, and after centrifugation at 3000 x g for 5 minutes, serum was collected. The amount of pNF-H in serum was quantified by ELISA using the collected serum and Phosphorylated Neurofilament NF-H ELISA Kit Version 2 (EnCor Biotechnology). The collected serum was mixed with the dilution buffer provided with the kit and dispensed into a 96-well plate, then allowed to stand at room temperature for 2 hours. After standing, each well was washed with TBS-T, and an HRP-conjugated pNF-H monoclonal antibody was administered. After standing for 2 hours, the reaction mixture was diluted with the tetramethylbenzidine reaction solution and 2N H ２ SO ４ The solutions were mixed and the absorbance at 450 nm was measured using a GloMax Discover Microplate Reader (Promega).

[0092] The results of quantifying serum pNF-H levels in each group using the above method are shown in Figure 6. Compared to the control Non-treat / ALS group and the group administered MSCs without vector (MSC / ALS), the group administered MSCs with HGF-scFv vector (REVec-MSC / ALS) showed a significant decrease in serum pNF-H levels at 18 weeks after administration. These results suggest that gene cell therapy, which involves transplanting MSCs transduced with growth factor (HGF) and scFv genes using a Borna virus vector, significantly suppresses brain inflammation and exhibits greater efficacy in ALS than conventional cell therapy using MSC transplantation.

Claims

1. A borna virus vector carrying a nucleic acid encoding a growth factor.

2. The bornavirus vector of claim 1, further comprising a gene of interest.

3. A bornavirus vector according to claim 1 or 2, wherein the growth factor is an NTF (neurotrophic factor).

4. The bornavirus vector of claim 1 or 2, wherein the growth factor is selected from the group consisting of BDNF, CXCR4, FGF2, GDNF, HGF, IGF1, Neurogenin1, Notch1 domain, PDGFA, PDGFB, TGFB1, and VEGFA.

5. A bornavirus vector according to claim 2, wherein the gene of interest is a gene encoding an antibody or an antibody fragment.

6. The antibody fragment is Fab, Fab', F(ab') ２ 6. The bornavirus vector of claim 5, which is an Fv, scFv, scFab, dsFv, ds-scFv, diabody, triabody, tetrabody, or minibody.

7. A bornavirus vector according to claim 5 or 6, wherein the antibody or antibody fragment is a humanized antibody or antibody fragment.

8. A bornavirus vector according to claim 5 or 6, wherein the antibody or antibody fragment has binding activity to a mutant SOD1 protein.

9. A Borna virus vector according to claim 1 or 2, comprising: (a) a cDNA of a recombinant viral RNA having at least the N gene, X gene, P gene, and L gene in the Borna disease virus genome in the same order as in the Borna disease virus genome, with the nucleic acid inserted into the untranslated region linked downstream of the translated region of the P gene; (b) DNA encoding a ribozyme; and (c) a promoter sequence, with (b) located upstream and downstream of (a), and (a) and (b) located downstream of (c).

10. A Borna virus vector according to claim 9, wherein (a) is a cDNA of a recombinant viral RNA having at least the N gene, X gene, P gene, and L gene in the Borna disease virus genome in the same order as in the Borna disease virus genome, and having a sequence in which the nucleic acid and the gene of interest are inserted into the untranslated region linked downstream of the translated region of the P gene.

11. A recombinant virus comprising RNA encoded by the bornavirus vector of claim 1 or 2.

12. A cell into which the nucleic acid has been introduced by infection with the recombinant virus of claim 11.

13. The cell according to claim 12, wherein the cell is an oligodendrocyte precursor cell, a glial neural precursor cell, a dopaminergic neural precursor cell, a microglia, a neural precursor cell, a neural stem cell, a mesenchymal stem cell, a Muse cell, an iPS cell, or an ES cell.

14. A pharmaceutical composition comprising the cells of claim 13.

15. The pharmaceutical composition according to claim 14, for use in the treatment and / or prevention of amyotrophic lateral sclerosis.

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