Novel gene transfer carrier

Abstract

Enhancing the fusion ability of transplant cells tends to arrest their proliferation in the production thereof, whereas promoting the proliferation of transplant cells tends to hinder their fusion at a transplantation site. Under such circumstances, the purpose of the present invention is to enable long-term engraftment of transplant cells regardless of concomitant use of immunosuppressants by endowing transplant cells with fusion ability and allowing the transplant cells to exert the fusion ability at a transplantation site at a desired time point, thereby overcoming the problem of graft failure and immune rejection resulting in loss of cells. The present invention provides a cell containing a cell-cell fusion gene as a foreign gene, wherein the cell-cell fusion gene is allowed to be expressed in the presence of an extracellular factor.

Description

Novel gene transporter

[0001] The present invention relates to a vector prepared from fusion-competent cells for the purpose of sustained drug release, and to a method for promoting stable expression of a foreign gene through long-term engraftment via fusion.

[0002] Because regenerative medicine products face the challenge of cell loss due to immune rejection after cell transplantation, various methods to improve cell survival have been explored. The problem of cell loss due to immune rejection has also occurred in muscle cell transplants. The final paragraph in the left column on page 9 of Non-Patent Document 1 describes a drawback of cell delivery by injection, which can lead to a harsh immune environment due to either immune rejection or inflammatory cytokines, leading to cell death. The abstract of Non-Patent Document 2 states that myoblast-mediated gene transfer is a useful tool for investigating promising new approaches to therapeutic angiogenesis, enabling strong and long-term expression and considered a relatively rapid form of "adult gene modification" in muscle. Non-Patent Document 3 describes the administration of myoblasts to Duchenne muscular dystrophy (DMD) mice, and from the final paragraph in the left column to the first paragraph in the right column on page 178, it states that the challenge of myoblast transfer therapy (MMT) in humans is to avoid immune rejection.

[0003] Furthermore, while the standard approach for myoblast transplantation is to address immune rejection with the use of immunosuppressants, engraftment remains a challenge. Non-Patent Document 4 describes the results of a blinded study of myoblast transplantation in human Duchenne muscular dystrophy (DMD) patients without the use of immunosuppressants. However, the final line of the abstract of Non-Patent Document 4 concludes that myoblast transplantation and gene therapy for DMD cannot be performed without immunosuppression. Non-Patent Document 5, page 557, left column, first paragraph, states that it is still unclear whether some forms of immunosuppression are necessary, even for immunocompatible donors and recipients. Table 1 of Non-Patent Document 6 examines intramuscular transplantation of myoblasts in human Duchenne muscular dystrophy (DMD) patients, and shows that the percentage of dystrophin-positive fibers (muscle fibers) is reduced without the use of immunosuppressants. Furthermore, the final line of the third paragraph, left column, on page 836 of Non-Patent Document 6, states that a very low cell survival rate was observed.

[0004] Patent Document 1 describes expressing a nucleic acid encoding the fusion gene Myomaker in non-muscle cells, such as fibroblasts, that lack fusion ability, and fusing them with muscle cells, such as myoblasts, but does not confirm actual engraftment after transplantation. Patent Document 1 also does not describe expressing the fusion gene Myomaker in muscle cells.

[0034] Patent Document 2 lists, in some embodiments, characteristics of myomerger polypeptides, such as increased membrane permeability (compared to, for example, wild-type myomerger polypeptides or to the absence of myomerger polypeptides), increased ability to activate fusion, and increased expression levels during myogenesis in vivo. However, Patent Document 2 does not mention that the myomerger polypeptides do not affect the proliferation ability required for the production of cell medicines, or that they improve engraftment after cell transplantation. Claim 1 of Patent Document 3 describes a method for producing skeletal muscle cells from pluripotent stem cells, but in reality, it merely describes a method of introducing a vector encoding MyoD or Myf5 into human iPS cells, expressing the vector, and inducing differentiation into skeletal muscle cells. Therefore, regardless of whether or not immunosuppressants are used concomitantly, there has been a need to confer fusion ability to transplanted cells and to enable them to exert this cell fusion ability at a desired time, thereby avoiding the cells failing to engraft and being rejected by the immune system, resulting in their disappearance, and enabling them to engraft.

[0005] U.S. Patent Application Publication No. 2019 / 0358294 U.S. Patent Application Publication No. 2021 / 0253655 International Publication No. 2020 / 090836

[0006] Taimoor H. Qazi, et al., Cell therapy to improve regeneration of skeletal muscle injuries. Journal of Cachexia, Sarcopenia and Muscle. 2019;(3):501-516Georges von Degenfeld, et al., Myoblast-mediated gene transfer for therapeutic angiogenesis and arteriogenesis, British Journal of Pharmacology (2003) 140, 620-626T. A. Partlidge, et al., Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature. 1989; (337): 176-179J. P. Tremblay, et al., RESULTS OF A TRIPLE BLIND CLINICAL STUDY OF MYOBLAST TRANSPLANTATIONS WITHOUT IMMUNOSUPPRESSIVE TREATMENT IN YOUNG BOYS WITH DUCHENNE MUSCULAR DYSTROPHY. Cell Transplantation. 1933; (2): 99-112.J. HUARD, et al., HUMAN MYOBLAST TRANSPLANTATION: PRELIMINARY RESULTS OF 4 CASES. Muscle Nerve 1992;(15):550-56KARLIJN J. WILSCHUT, et al., Concise Review: Stem Cell Therapy for Muscular Dystrophies. STEM CELLS TRANSLATIONAL MEDICINE. 2012; (1): 833-842.Hongmei Luo et al., Functional Non-coding RNA During Embryonic Myogenesis and Postnatal Muscle Development and Disease, Frontiers in Cell and Developmental Biology, January 2021, Volume 9, Article 628339, p1-15Xiaoming Yang et al., Changes of Gene Expression Patterns of Muscle Pathophysiology-Related Transcription Factors During Denervated Muscle Atrophy, Frontiers in Physiology, June 2022, Volume 13, Article 923190, p1-16 and Supplementary Tables.Tsuyoshi Morita et al., Actin-related protein 5 functions as a novel modulator of MyoD and MyoG in skeletal muscle and in rhabdomyosarcoma, eLife 2022;11:e77746 (https: / / doi.org / 10.7554 / eLife.77746)Rachel S. Donocoff et al., Optimization of tamoxifen-induced Cre activity and its effect on immune cell populations, Scientific Reports (2020) 10:15244 (https: / / doi.org / 10.1038 / s41598-020-72179-0)Peter S. Zammit, et al., Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis, Seminars in Cell & Developmental Biology (2017)Michael V. Taylor, et al., Mef2 and the skeletal muscle differentiation program, Seminars in Cell & Developmental Biology 72 (2017) 33-44Naoki Ito, et al., Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells, SCIENTIFIC REPORTS, 7:8097 (2017); DOI:10.1038 / s41598-017-08232-2Bide Chen, et al., The regulatory role of Myomaker and Myomixer-Myomerger-Minion in muscle development and regeneration, Cellular and Molecular Life Sciences (2020) 77:1551-1569Francisco Hermandez-Torres et al., Pitx2 in Embryonic and Adult Myogenesis, Frontiers in Cell and Developmental Biology, 01 May 2017, Volume 5, Article 46Jinhong Meng et al., Contribution of Human Muscle-Derived Cells to Skeletal Muscle Regeneration in Dystrophic Host Mice, PLoS ONE, March 2011, Volume 6, Issue 3, e17454.

[0007] In a situation where promoting the fusion ability of transplanted cells stops their proliferation during production, and promoting the proliferation of transplanted cells prevents fusion of the transplanted cells at the transplant site, the present invention aims to provide transplanted cells with fusion ability and enable them to exert their cell fusion ability at the transplant site at a desired time, regardless of whether an immunosuppressant is used in combination, thereby addressing the problem of cells not taking root and being rejected by the immune system and disappearing, thereby achieving long-term engraftment.

[0008] The present inventors recognized the problem that promoting the fusion ability of transplanted cells stops the proliferation of the transplanted cells during production, and promoting the proliferation of transplanted cells prevents the transplanted cells from fusing at the transplant site. The present inventors discovered that by regulating the timing of the onset of expression of cell-cell fusion genes, transplanted cells can be efficiently proliferated ex vivo without affecting their proliferation ability, and can be fused with muscle cells in vivo, thereby improving engraftment after cell transplantation. Based on this finding, the present inventors conducted further research and completed the present invention.

[0009] That is, the present invention relates to the following: [1] A cell comprising a cell-cell fusion gene as an exogenous gene, wherein the cell-cell fusion gene is expressed in the presence of an extracellular factor. [2] The cell described in [1], wherein the timing of the onset of expression of the cell-cell fusion gene can be arbitrarily adjusted so as not to affect the proliferation ability of the cell. [3] The cell described in [1] or [2], wherein the cell-cell fusion gene is a muscle-related gene group, a fertilization-related gene group, or a virus-related gene group related to membrane fusion. [4] The cell described in [3], wherein the cell-cell fusion gene is a muscle-related gene. [5] The cell according to [4], wherein the muscle-related gene is Myomaker, Myomixer, M-cadherin, CD56, Pax3, Pax7, MyoD, Myf5, Myogenin, ACTA, Myosin Heavy Chain, Desmin, Wnt, GSK3 inhibitor, Six1 / 4, P21, Ezh2, Notch1, PitX1, PitX2, MEF2A, MEF2B, MEF2C, IGF, TGF inhibitor, Sirt1, HDAC, Sms1, Sms2, or MyD88. [6] The cell according to [1] to [5], wherein the cell is a muscle cell or a myoid cell. [7] The cell according to [6], wherein the cell is a human muscle cell or a human myoid cell. [8] The cell according to [1] to [7], wherein the cell further expresses a gene encoding a drug. [9] The cells according to any one of [1] to [8], wherein the cell vector system or gene expression control system in which the cell-cell fusion gene is expressed in the presence of an extracellular factor is Cre-Lox, Flp-FRT, Vika-Vox, Cre-ERT, Cre-ERT2, ERT, ERT2, Destabilizing Domain (DD), TetON, or TetOFF.

[10] A cell medicine having an added property of improving engraftment after cell transplantation, comprising the cells according to any one of [1] to [9] and an extracellular factor, wherein the pharmaceutical composition can be arbitrarily exposed to the extracellular factor so that the expression of the cell-cell fusion gene does not affect the proliferation ability required for manufacturing the cell medicine.

[11] A method for promoting cell-cell fusion, comprising exposing the cells to be fused to the cells and extracellular factors described in [1] to [9], wherein the extracellular factors are exposed to the cells described in [1] to [9] so that expression of the cell-cell fusion gene does not affect the proliferation ability required for the production of a cell-based pharmaceutical.

[0010] By using the cells of the present invention, regardless of whether or not an immunosuppressant is used in combination, it is possible to efficiently proliferate transplanted cells ex vivo and fuse them with muscle cells in vivo without affecting the proliferation ability of the transplanted cells, thereby improving engraftment after cell transplantation.

[0011] Figure 1A shows a plasmid vector (Vector 1; SEQ ID NO: 1) introduced into cells of the present invention. In Vector 1, skeletal muscle-related genes (MyoD and Myogenin) for cell-cell fusion are designed in opposite orientations. The two loxP sites are located on the same DNA strand but in opposite orientations, allowing Cre to invert the gene between the loxP sites. Figure 1B shows a plasmid vector (Vector 2; SEQ ID NO: 2) introduced into cells of the present invention. Vector 2 switches the reverse orientation of Vector 1 to forward orientation. This switch functions upon exposure to trimethoprim (TMP). Figure 1C shows a plasmid vector (Vector 3; SEQ ID NO: 3) introduced into cells of the present invention. Vector 3 synthesizes mRNA using the vector as a template and introduces the mRNA into the cell. Vector 3 also integrates Vectors 1 and 2 into the cellular nucleic acid. Figure 2A shows a fusion detection system using the Flp-FRT system. Figure 2B shows the results of an in vitro fusion test of cell vectors. Figure 3A shows the results of an in vivo fusion test of a cell vector (photograph). Figure 3B is a graph quantifying the results of the test in Figure 3A. Figure 4A shows the results of a cell vector Matrigel plug assay (photograph). Figure 4B is a graph quantifying the results of the assay in Figure 4A. Figure 4C shows the site of gel administration in mice. Figure 5A shows the results of a drug efficacy test of a cell vector (days 21 and 28). Figure 5B is a graph quantifying the results of the drug efficacy test (blood flow). Figure 5C is a table quantifying the results of the drug efficacy test (number of positive cells) and a corresponding graph.

[0012] The present invention relates to a cell that contains a cell-cell fusion gene as an exogenous gene, in which the cell-cell fusion gene is expressed in the presence of an extracellular factor, and a method for promoting cell-cell fusion using the same.

[0013] The cells of the present invention (hereinafter also referred to as transplant cells) contain a vector encoding a cell-cell fusion gene. In one embodiment, the cell-cell fusion gene is expressed in the presence of extracellular factors so as not to affect the proliferation ability of the transplanted cells. In another embodiment, the cell-cell fusion gene is expressed only in the presence of extracellular factors so as not to affect the proliferation ability of the transplanted cells.

[0014] By adjusting the timing of expression of the cell-cell fusion gene encoded by the vector, the cells of the present invention can efficiently proliferate transplanted cells ex vivo without affecting the cell proliferation ability of the transplanted cells, and can promote fusion with muscle cells or myoid cells in the body at the intended time after transplantation, thereby improving engraftment.

[0015] In the present invention, "not affecting the proliferation potential" or "not affecting the proliferation potential" of transplanted cells means that the transplanted cells can proliferate efficiently without affecting mitosis and cytokinesis during the M phase. A cell population of transplanted cells contains a mixture of cells in the G1 phase (preparing for DNA replication), the S phase (replicating DNA), the G2 phase (preparing for cell division), the M phase (cell division), and the quiescent phase (GO), making it difficult to align the cell division phase and other conditions for all cells in the cell population. If a fusion-competent gene is introduced into cells as an exogenous gene without controlling its expression, the expression of this gene can have a strong effect, causing the introduced transplanted cells to fuse with each other before transplantation into a living body and subsequently to cease proliferation. By controlling the onset of cell-cell fusion gene expression in the presence of extracellular factors, we have achieved a method of avoiding fusion between transplanted cells after gene introduction and before transplantation as much as possible, allowing as many transplanted cells as possible to proliferate during production and efficiently fuse with somatic cells in the living body, without affecting the proliferation potential of the transplanted cells.

[0016] In the present invention, the cell-cell fusion genes refer to muscle-related genes, fertilization-related genes, or virus-related genes involved in membrane fusion. The muscle-related genes refer to genes involved in muscle cell differentiation and fusion, such as Myomaker, Myomixer, M-cadherin, CD56, Pax3, Pax7, MyoD, Myf5, Myogenin, ACTA, Myosin Heavy Chain, Desmin, Wnt, GSK3 inhibitor, Six1 / 4, P21, Ezh2, Notch1, PitX1, PitX2, MEF2A, MEF2B, MEF2C, IGF, TGF inhibitor, Sirt1, HDAC, Sms1, Sms2, and MyD88. The term "muscle-related gene" is generally known to those skilled in the art, as described in, for example, Non-Patent Documents 7 to 9, and includes coding RNAs related to non-coding RNAs, but is not limited to non-coding RNAs. Fertilization-related genes refer to genes involved in sperm-egg fusion, such as CD9, Izumo1, Juno, Spaca6, TMEM95, Sof1, Fimp, and Dcst1 / Dsct2. Virus-related membrane fusion genes refer to genes involved in virus-cell fusion, such as Hemagglutinin, Syncytin-1, Syncytin-2, Hap2 / Gcs1, Arginine, Snare, Snap-25 / SNAP-29, MAP-Tau, p-Tau, Tmem16f, Tubulin, Gp41, Gp120, CD4, Ccr5, and Cxcr4. In one embodiment, the cell-cell fusion gene is the skeletal muscle-related gene MyoD and / or Myogenin. For example, MyoD is NM_002478.5 (MYOD1) (SEQ ID NO: 4), and Myogenin is NM_002479.6 (MYOG) (SEQ ID NO: 5). Furthermore, as shown in, for example, Non-Patent Documents 11 to 16 listed below, various genes other than MyoD (SEQ ID NO: 4) and Myogenin (SEQ ID NO: 5) are "muscle-related genes that are cell-cell fusion genes" and are capable of muscle differentiation and cell-cell fusion.The first and second paragraphs in the left column of the introduction of Non-Patent Document 11 describe that MyoD, Myf5, and Myogenin, which share a common "bHLH" domain, share a common muscle differentiation function. Figure 2 in Non-Patent Document 12 describes that MEF2A, MEF2B, and MEF2C cooperate with transcription factors containing bHLH domains to promote muscle differentiation (page 39, left column, third paragraph). The abstract of Non-Patent Document 13 reports that Pax3, Pax7, MyoD, PitX1, and MEF2B are genes that promote muscle cell function acquisition. The right column of the introduction on page 1551 of Non-Patent Document 14 describes that Myomaker and Myomixer both directly mediate cell-cell fusion. The second paragraph in the left column on page 5 of Non-Patent Document 15 describes PitX1 and PitX2 of the PitX gene family, suggesting that PitX2 is important for regulating skeletal muscle formation. The last paragraph of the left column on page 6 to the first paragraph of the left column on page 7 and the third paragraph of the left column on page 12 of Non-Patent Document 16 describe the promotion of myotube formation (the formation of a fusion of multiple muscle cells resulting from skeletal muscle differentiation) by separating and culturing CD56-positive and -negative cells. In another embodiment, the cell-cell fusion gene is Myomaker. Myomaker is a membrane protein that is highly expressed during myoblast differentiation, and its deletion is known to significantly impair myoblast fusion. For example, the nucleotide sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2 described in Patent Document 1 can be used as Myomaker.

[0017] In the present invention, the transplant cells are not particularly limited as long as they can fuse with somatic cells, such as muscle cells or myoid cells, in the living body of the subject to which the transplant cells are administered. The transplant cells may be muscle cells or somatic cells other than muscle cells. In the present invention, muscle cells include, for example, myoblasts, skeletal muscle cells, cardiac muscle cells, smooth muscle cells, and myoid cells. In the present invention, myoid cells include, for example, muscle cells derived from pluripotent stem cells and muscle cells derived from somatic stem cells such as mesenchymal stem cells. In the present invention, the cells other than muscle cells are not particularly limited as long as they can fuse with somatic cells, such as muscle cells or myoid cells, in the living body. Examples of such cells include relatively available somatic cells such as fibroblasts derived from skin or hair, erythrocytes, and stem cells derived from bone marrow and adipose tissue. In the present invention, the transplant cells are preferably human-derived cells. In the present invention, the cells fused with the transplant cells are not particularly limited as long as they can fuse with the transplant cells, but somatic cells such as muscle cells or myoid cells are preferred.

[0018] In the present invention, in addition to the cell vector encoding the cell-cell fusion gene, the transplanted cells may further encode a gene encoding a drug (hereinafter also referred to as a drug gene) in the same or a different cell vector, or the transplanted cells may contain and express a drug gene without being encoded by the cell vector. In the present invention, the drug gene may be single-stranded or double-stranded. Examples of single-stranded drug genes include mRNA and miRNA. Examples of double-stranded drug genes include viral vectors, plasmid DNA, and siRNA.

[0019] In one embodiment, the drug gene is encoded by a drug-releasing cell vector. In the present invention, expression of the cell-cell fusion gene is initiated by exposure to an extracellular factor. However, by encoding a drug gene in the same vector as the vector encoding the cell-cell fusion gene or in a different vector, expression of the drug gene can also be initiated upon exposure to an extracellular factor. For example, expression of the cell-cell fusion gene can be initiated upon exposure to an extracellular factor, and then or prior to that, expression of the drug gene can also be initiated, resulting in a drug-releasing cell vector.

[0020] In the present invention, the drug gene is not particularly limited. For example, the drug gene may be a gene involved in the cell cycle (such as the CDK family, the Cyclin family, p16, p21, p27, E2F, and mutants thereof), a gene involved in the structure of chromosomes (such as telomerase, TERT, ZSCAN, SV40 Large T antigen, HPV E6 / E7, Ras, Rb, recombinase, integrase, nuclease, helicase, ligase, replicase, B-cell lymphoma 2, CRISPR), or ... cell cycle (such as CDK family, Cyclin family, p16, p21, p27, E2F, mutants thereof, and mutants thereof), a gene involved in the cell cycle (such as CDK family, Cyclin family, p16, p21, p27, E2F, mutants thereof, and mutants thereof), a gene involved in the cell cycle (such as CDK family, Cyclin family, p16, p21, p27, E2F, mutants thereof), a gene involved in the cell cycle (such as CDK family, Cyclin family, p16, p21, p27, E2F, mutants thereof), a gene involved in the cell cycle (such as CDK family, Cyclin family, p16, p21, p27, E Genes encoding Cas and their mutants, etc.), reprogramming-related genes (Oct3 / 4, c-Myc, Klf4, Sox2, NANOG, ASCL1, PITX3, NURR1, LMX1A and their mutants, etc.), skeletal muscle-related genes (CD56, Pax3, Pax7, Myogenin, Myf5, MyoD, Myomaker, Myomixer, Myosin Heavy Chain, Desmin, Dystrophin, Myotilin, Laminin) A / C, CAV, CAPN, SGCG, TRIM32, TCAP, FKRP, EMD, PABP, DMPK, ZNF9, FCMD, POMGnT1, Collagen, SEPN1, RYRI, MTM, TNNT, NEB, TPM, ACTN, GNE, DYSF, CRYAB, ACTA and their mutants, etc.), genes related to growth factors (VEGF, IGF, FGF, HGF, EGF, TGF, NGF, BDNF, GDNF, BMP, PDGF, EPO, TPO, G-CSF, GM-CSF and their families and their mutants, etc.), genes related to transcription factors (Runt domain including RUNX and its mutants, helix-turn-helix, helix-loop-helix, zinc finger, leucine-nucleotide genes related to PAR, β-sheet motif and their variants, etc.), enzymes (glucose-6-phosphatase, t-PA, collagenase, alglucosidase, urate oxidase, alkaline phosphatase, glycosaminoglycan degrading enzyme, β-glucuronidase, glutamic acid carboxypeptidase, sphingomyelin phosphodiesterase, α-L-iduronidase, iduronate sulfatase, N-acetylgalactosamine-6-sulfatase,Genes related to membrane proteins (cadherin, EGF receptor, HGF receptor, EpCAM, HER2, FGF receptor, NGF receptor, TGF receptor, calcium channel, sodium channel, potassium channel, junctophilin, ryanodine receptor, GPCR, monotopic including neprilysin, polytopic and their mutants, etc.), genes related to chimeric antigen receptors (CAR and its mutants, etc.), antibody genes (mouse antibody, chimeric antibody, Examples of drug genes include genes encoding antibodies, humanized antibodies, human antibodies, and antibodies without a substem indicating their origin), blood coagulation-related factors (factors I to XIII), serum proteins (albumin, immunoglobulins, transferrin, antitrypsin, haptoglobin, fibrinogen, thrombin, antithrombin, thrombomodulin), hormones (T3, T4, insulin, estrogen, progesterone, testosterone, serotonin, cortisol, adrenaline), vaccines, interferons, erythropoietin, cytokines (TNF, IFN, CTLA, JAK, Syk, S1P, BLyS, CXCL), toxins, and fusion proteins. Furthermore, genes other than cell-cell fusion genes and genes other than muscle-related genes can also be used as drug genes. In the present invention, for example, by introducing the above-mentioned genes into a vector in transplant cells (i.e., a cell vector system) or directly into the transplant cells without introducing them into a vector, it is possible to prolong the lifespan of the transplant cells into which the genes have been introduced or to promote their differentiation into desired cells.

[0021] In the present invention, any known method can be used to introduce a gene into cells. Examples of such methods include biochemical methods such as lipofection and polybrene, physical methods such as electroporation, or a combination of these. When lipofection is used in the present invention, the cells into which the gene is introduced are approximately 1,000 to 100,000 cells / cm. ２ When the cells to be transfected are human somatic cells, the cells are more preferably seeded at 1 to 2 x 10 ４ cells / cm ２ and if the cells are human myoblasts, more preferably 2 x 10 ４ cells / cm ２ For example, when using the lipofection method to introduce nucleic acid molecules such as plasmid DNA or mRNA (e.g., mRNA synthesized from Vector 3) into cells, the plasmid DNA or mRNA is mixed with a lipofection reagent such as Lipofectamine (Thermo Fisher Scientific), ViaFect (Promega), or PEI MAX (Polysciences) at a concentration of 1 to 100 pg / cell and added to the cells to be introduced. Preferably, from the viewpoint of achieving high final introduction efficiency and viability, the plasmid DNA or mRNA (e.g., mRNA synthesized from Vector 3) is added to the cells to be introduced at a concentration of 1 to 20 pg / cell. In the present invention, when the polybrene method is used, the cells to be introduced are cultured at a density of approximately 1,000 to 100,000 cells / cm. ２ When the cells to be transfected are human somatic cells, the cells are more preferably seeded at 1 to 2 x 10 ３ cells / cm ２ and if the cells are human myoblasts, more preferably 2 x 10 ３ cells / cm ２For example, when a viral vector is introduced into cells using the polybrene method, the viral vector is mixed with a polybrene solution to give a multiplicity of infection (MOI) of 0.1 to 100, and then added to the cells into which the gene is to be introduced. Preferably, from the viewpoint of achieving high final introduction efficiency and survival rate, the viral vector is added to the cells into which the gene is to be introduced at an MOI of 1 to 10. When the electroporation method is used in the present invention, the cells into which the gene is to be introduced are prepared at about 1,000 to 1,000,000 cells / mL and electroporated. When the cells into which the gene is to be introduced are human somatic cells, the cells are more preferably prepared at a density of 1 to 10 x 10 ４ cells / mL and electroporated, and if the cells are human myoblasts, more preferably 10 x 10 ４ The solution is adjusted to a concentration of 1 to 20 pg / cell and electroporated. For example, when nucleic acid molecules such as plasmid DNA or mRNA are introduced into cells using electroporation, the plasmid DNA or mRNA is mixed with an electroporation reagent such as NEON Nxt Resuspension buffer (Thermo Fisher Scientific) at a concentration of 1 to 100 pg / cell and added to the cells to be introduced. Preferably, from the viewpoint of achieving high final introduction efficiency and viability, the plasmid DNA or mRNA (e.g., mRNA synthesized from Vector 3) is added to the cells to be introduced at a concentration of 1 to 20 pg / cell.

[0022] In the present invention, the cell vector system or gene expression control system in which the cell-cell fusion gene is expressed in the presence of extracellular factors is not particularly limited, as long as it can regulate the timing of the onset of cell-cell fusion gene expression so as not to affect the proliferation ability of the transplanted cells. For example, Cre-Lox, Flp-FRT, Vika-Vox, Cre-ERT, Cre-ERT2, ERT, ERT2, Destabilizing Domain (DD), TetON, or TetOFF can be used. In one embodiment of the present invention, the vector encoding the cell-cell fusion gene is integrated into the genome of the fused cell. The gene recombination systems Cre-ERT and Cre-ERT2 are described, for example, in Non-Patent Document 10. Other examples include the following vectors: (1) A vector using Pax7, PitX1, MEF2B, and MyoD, with the cell vector system being TetON (Vector ID: VB230925-1617tfv, VectorBuilder). (2) A vector using MyoD and Myomaker (also known as MYMK) and using the TetON cell vector system (Vector ID: VB230426-1754vqg, VectorBuilder). (3) A vector using MyoD and NCAM1 (also known as CD56) and using the TetON cell vector system (Vector ID: VB230426-1760wdu, VectorBuilder). (4) A vector using MyoD as a muscle-related gene and using ERT2 as the cell vector system (Vector ID: VB221223-1034msj, VectorBuilder). (5) A vector using MyoD and Myogenin as muscle-related genes and using Cre-Lox as the cell vector system (Vector ID: VB220304-1379fmf, VectorBuilder). (6) A vector (Vector ID: VB210606-1094uwz, VectorBuilder) using MyoD and Myomaker (also known as MYMK) as muscle-related genes and a Cre-Lox cell vector system.In the present invention, the extracellular factor is not particularly limited as long as it can regulate the initiation timing of cell-cell fusion gene expression in the cell vector system. For example, trimethoprim (TMP) or tamoxifen can be used. In one embodiment, the extracellular factor is not particularly limited as long as it can migrate to the site of administration of transplanted cells and initiate expression of the cell-cell fusion gene when exposed to the transplanted cells. In one embodiment, the timing of cell-cell fusion gene expression can be regulated by using a Cre-Lox or Flp-FRT cell vector system and adjusting the time and site of TMP administration. In another embodiment, the timing of exposure of the cell vector in the transplanted cells to TMP can be regulated by administering the transplanted cells and TMP at different sites. In the present invention, the timing of termination of cell-cell fusion gene expression is not particularly limited.

[0023] The pharmaceutical of the present invention is a cell pharmaceutical with the added property of improving cell engraftment after cell transplantation. It contains the cells of the present invention and an extracellular factor, and is characterized in that the transplanted cells can be exposed to the extracellular factor so that expression of the cell-cell fusion gene does not affect the proliferation ability required for the production of the cell pharmaceutical. In the pharmaceutical of the present invention, the transplanted cells can be administered to a subject in need of muscle cells or myoid cells, for example, at a dose of 10,000 to 100,000,000 cells / kg body weight. When the extracellular factor of the pharmaceutical of the present invention is TMP, for example, the dose can be administered to a subject in need of muscle cells or myoid cells at a dose of 1 to 200 mg / kg body weight. When the extracellular factor is other than TMP, those skilled in the art can appropriately determine the dosage, using 1 to 200 mg / kg body weight as a guide. The pharmaceutical of the present invention can be administered to a subject in need of muscle cells or myoid cells, for example, a subject with lower limb ischemia. In the present invention, the subject is a human or mammal, with human subjects being preferred.

[0024] In the present invention, transplant cells and extracellular factors may be administered simultaneously or separately. By administering the transplant cells and extracellular factors at different sites, the timing of exposure of the transplant cells to the extracellular factors can be controlled. In one embodiment, the transplant cells are administered intramuscularly, subcutaneously, intravascularly, or directly to the transplantation site, and the extracellular factors are administered intramuscularly, subcutaneously, intravascularly, or directly to the transplantation site, intraperitoneally, or orally. Unlike Patent Document 1, in which the expression of the cell-cell fusion gene Myomaker is not regulated, in the present invention, cell-cell fusion genes such as MyoD and Myogenin are encoded by a vector, and expression of the cell-cell fusion gene is initiated only upon exposure of transplant cells containing such a vector to an extracellular factor. Therefore, it takes some time for the expression of the cell-cell fusion gene to be initiated upon exposure of the transplanted cells to an extracellular factor, and fusion between the transplanted cells does not initiate immediately after exposure and before transplantation. Therefore, the transplanted cells and extracellular factors may be administered simultaneously. When the transplanted cells and extracellular factors are administered separately, the order of administration is not limited. Since transplanted cells are usually eliminated by the host's immune response within approximately 24 hours after transplantation, when administering transplanted cells first and then administering an extracellular factor, the extracellular factor should be administered at the same or a different site within at least 24 hours, for example, within a few hours, such as 2 to 3 hours, after administration of the transplanted cells. Furthermore, when administering an extracellular factor first and then administering transplanted cells, the extracellular factor should be administered within the serum half-life of the extracellular factor. When TMP is used intravenously as the extracellular factor, its serum half-life is approximately 10 hours, so TMP is administered within at least 10 hours, for example, within 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour, after administration of the transplanted cells. Furthermore, when TMP is administered orally, its half-life is just under 7 hours, so TMP is administered within at least 7 hours, for example, within 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour, after administration of the transplanted cells.In one aspect, the engraftment after cell transplantation can be determined by measuring the luminescence intensity of luciferase expressed under the Flp-FRT system using an imaging device (IVIS Imaging System), thereby determining whether the engraftment increases or decreases.

[0025] In one aspect, the present invention relates to a method for promoting cell-cell fusion in vivo or in vitro. The method of the present invention comprises exposing the cells of the present invention described above and extracellular factors to cells to be fused (somatic cells such as muscle cells or myoid cells), and is characterized in that the cells of the present invention are exposed to the extracellular factors in a manner that does not affect the proliferation ability required for the production of cell-based pharmaceuticals due to the expression of a cell-cell fusion gene.

[0026] In another aspect, the present invention relates to a method for improving blood flow in a subject by fusing transplanted cells with the subject and continuously releasing a drug. The method of the present invention comprises intramuscularly administering the cells of the present invention and extracellular factors to tissue of a subject requiring improved blood flow, and is characterized in that the cells of the present invention are exposed to the extracellular factors so that expression of the cell-cell fusion gene does not affect the proliferation ability required for the production of cell-based medicines. In the present invention, the drug genes can be hFGF2 [NM-001361665.2] (SEQ ID NO: 6) and hHGF [NM_000601.6] (SEQ ID NO: 7), which are known angiogenic factors. In the present invention, improved blood flow can be assessed by measuring blood flow volume and the number of CD31-positive cells (number of blood vessels).

[0027] In yet another aspect, the present invention relates to a method for promoting cell-cell fusion in vitro. The method of the present invention comprises exposing the cells of the present invention described above and extracellular factors to cells to be fused (somatic cells such as muscle cells or myoid cells) in vitro, and is characterized in that the cells of the present invention are exposed to the extracellular factors in a manner that does not affect the proliferation ability required for the production of a cell-based pharmaceutical product due to the expression of the cell-cell fusion gene.

[0028] The present invention will be described in more detail below based on examples, but it goes without saying that the present invention is not limited to these examples.

[0029] Example 1. Construction of Cellular Vectors. The Cre-Lox cell vector system (Vector Builder) and the φC31 integrase system (Vector Builder) were used. The intercellular fusion genes MyoD (SEQ ID NO: 4) and Myogenin (SEQ ID NO: 5) were used as intercellular fusion genes, and trimethoprim (TMP) was used as an extracellular factor to regulate the initiation of intercellular fusion gene expression. When the two loxP sites are located on the same DNA strand (Vector 1) but in opposite orientations, the gene between the loxP sites is inverted by Cre in Vector 2. The φC31 integrase system combines mRNA produced by Vector 3 with Vector 1 or 2. In the presence of φC31 integrase, integration into the genome begins at the "attB" sequence of Vectors 1 and 2. The constructed Vectors 1-3 are shown in Figures 1A, 1B, and 1C, respectively. These vectors were introduced into primary muscle cells. The vectors were introduced using the lipofection method, with the vectors added to the cells at a concentration of 1-20 pg / cell. Hereinafter, the primary muscle cells into which the three plasmids have been introduced will be referred to as the cell vector of the present invention.

[0030] Example 2. In vitro fusion assay of cell vectors. Using the Flp-FRT system (Vector Builder), we constructed a system in which luciferin luminescence was observed only upon fusion between a human cell vector and mouse cells (Figure 2A). Specifically, a human cell vector infected with FRT lentivirus (FRT) and an immortalized mouse myoblast cell line (C2C12(Flp)) infected with Flp lentivirus (C2C12(Flp)) were co-cultured under TMP exposure. After cell lysis, luciferin was added and a luciferase assay was performed. Luciferin luminescence was confirmed, suggesting heterologous fusion (Figure 2B).

[0031] Example 3. In Vivo Fusion Test of Cell Vectors Using the Flp-FRT system (Vector Builder), a system was constructed in which luminescence was observed only upon fusion of the cell vector with mouse somatic cells. The system was the same as in Example 2, except that Flp-expressing transgenic mice were used instead of C2C12(Flp). Luciferin luminescence was observed upon fusion. The cell vector of the present invention was used to fusion with mouse somatic cells in vivo. The cell vector was administered to the left and right thighs of mice, followed the next day by TMP administration to the same site. Luminescence attenuation was observed in the group receiving only the cell vector (cell vector-transplanted group) (Figure 3A, left panel). However, the luminescence attenuation was suppressed in the group receiving both the cell vector and TMP (Figure 3A, center panel). This indicates that exposure to TMP initiated expression of the cell-cell fusion gene, resulting in fusion with mouse somatic cells. Furthermore, no luminescence was observed in the group receiving only vehicle (phosphate-buffered saline) (vehicle group) (Figure 3A, right panel). The numerical results are shown in a graph (Figure 3B). The IVIS Imaging System (Revvity) was used.

[0032] Example 4. Cell Vector Matrigel Plug Assay Matrigel (Corning) was mixed with the cell vector of the present invention (human cell product group) and administered to NOD / SCID mice (Charles River, Inc.), and the gel was removed and evaluated 7 days later (Figure 4A). The administration site was subcutaneous on the back of the mouse (Figure 4B, right). The cell vector of the present invention (human cell product group) induced a significantly higher blood vessel area than the bFGF (Fiblast Spray) (Kaken Pharmaceutical Co., Ltd.) group and the human mesenchymal stem cell (MSC) group (Figures 4A and 4B). Matrigel alone was used as the vehicle.

[0033] Example 5. Efficacy Test of Cell Vectors A hind limb ischemia model was created by removing the artery and vein from one side of a mouse's thigh. The test substance was then administered intramuscularly to the ischemic limb. Furthermore, to initiate cell-cell fusion gene expression from the human cell vector, TMP was administered intraperitoneally 2-3 hours prior to the administration of the test substance on the same day. The results showed that the human cell vector group ("test product") exhibited superior improvement in blood flow (Figure 5A, right column; Figure 5B), and the number of blood vessels was significantly greater than that of the other groups (Figure 5A, right column; Figure 5C).

Claims

A cell comprising an intercellular fusion gene as an exogenous gene, wherein the intercellular fusion gene is expressed in the presence of an extracellular factor. 　 The cell according to claim 1, wherein the timing of initiation of expression of the cell-cell fusion gene can be arbitrarily adjusted so as not to affect the proliferation ability of the cell. 　 The cell according to claim 1, wherein the cell-cell fusion gene is a muscle-related gene group, a fertilization-related gene group, or a virus-related gene group involved in membrane fusion. 　 The cell of claim 3 , wherein the cell-cell fusion gene is a muscle-related gene. 　 The cell of claim 4, wherein the muscle-related gene is Myomaker, Myomixer, M-cadherin, CD56, Pax3, Pax7, MyoD, Myf5, Myogenin, ACTA, Myosin Heavy Chain, Desmin, Wnt, GSK3 inhibitor, Six1 / 4, P21, Ezh2, Notch1, PitX1, PitX2, MEF2A, MEF2B, MEF2C, IGF, TGF inhibitor, Sirt1, HDAC, Sms1, Sms2, or MyD88. 　 The cell of claim 1 , wherein the cell is a muscle cell or a myoid cell. 　 The cell of claim 6 , wherein the cell is a human muscle cell or a human myoid cell. 　 The cell of claim 1 , wherein the cell further expresses a gene encoding a drug. 　 The cell described in claim 1, wherein the cell vector system or gene expression control system in which the cell-cell fusion gene is expressed in the presence of an extracellular factor is Cre-Lox, Flp-FRT, Vika-Vox, Cre-ERT, Cre-ERT2, ERT, ERT2, Destabilizing Domain (DD), TetON, or TetOFF. 　 A cell drug comprising the cells of claim 1 and extracellular factors, which has the added property of improving cell engraftment after transplantation, and wherein the extracellular factors can be arbitrarily exposed to the cells so that expression of the cell-cell fusion gene does not affect the proliferation ability required for the production of the cell drug. 　 A method for promoting cell-cell fusion, comprising exposing the cells to be fused to the cells described in claim 1 and an extracellular factor, wherein the extracellular factor is exposed to the cells described in claim 1 so that expression of the cell-cell fusion gene does not affect the proliferation ability required for the production of cell medicines.

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