New gene delivery carrier

Abstract

To allow transplanted cells to have fusion capability and to express cell fusion capability at a desired time at a transplantation site, irrespective of concomitant use of an immunosuppressant, in a situation where enhancing fusion capability of transplanted cells stops their proliferation during their production, while enhancing their proliferation prevents fusion at the transplantation site, the aim being to achieve long-term engraftment solving a problem in which the cells do not engraft, are immunologically rejected, and disappear.SOLUTION: A cell is provided that contains, as a foreign gene, an intercellular fusion gene, the intercellular fusion gene being expressed in the presence of an extracellular factor.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[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. [Background technology]

[0002] Since regenerative medicine products face the challenge of cell transplants being lost due to immune rejection, various methods to improve cell survival are being investigated. The problem of loss due to immune rejection has also arisen in the case of muscle cell transplants. The last paragraph of the left column on page 9 of Non-Patent Document 1 describes that a drawback of cell delivery by injection is that it can result in a harsh immune environment due to either immune rejection or inflammatory cytokines, which can cause 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 being considered as a relatively rapid form of "adult gene recombination" in muscle. Non-patent document 3 describes the administration of myoblasts to Duchenne muscular dystrophy (DMD) mice, and from the last paragraph in the left column to the first paragraph in the right column on page 178, it states that the challenge of MMT (Myoblast Transfer Therapy) in humans is to avoid immune rejection.

[0003] Furthermore, in myoblast transplantation, the standard approach is to use immunosuppressants in combination to address immune rejection, but engraftment remains an issue. Non-Patent Document 4 describes the results of a blinded trial of myoblast transplantation in human Duchenne muscular dystrophy (DMD) patients without the use of immunosuppressants, but 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. On page 557, left column, first paragraph of Non-Patent Document 5, it is stated that it is still unclear whether some form of immunosuppression is 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) decreases if immunosuppressants are not used. Furthermore, the last line of the third paragraph in the left column on page 836 of Non-Patent Document 6 states that a very low cell survival rate is observed.

[0004] Patent Document 1 describes expressing a nucleic acid encoding the fusion gene Myomaker in non-muscle cells such as fibroblasts that lack the ability to fuse, 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. 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. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent Application Publication No. 2019 / 0358294 [Non-patent literature]

[0006] [Non-Patent Document 1] Taimoor H. Qazi, et al., Cell therapy to improve regeneration of skeletal muscle injuries. Journal of Cachexia, Sarcopenia and Muscle. 2019;(3):501-516 [Non-patent document 2] Georges von Degenfeld, et al., Myoblast-mediated gene transfer for therapeutic angiogenesis and arteriogenesis, British Journal of Pharmacology (2003) 140, 620-626 [Non-patent document 3] TA Partlidge, et al., Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature. 1989; (337): 176-179 [Non-patent document 4] JP 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. [Non-patent document 5] J. HUARD, et al., HUMAN MYOBLAST TRANSPLANTATION: PRELIMINARY RESULTS OF 4 CASES. Muscle Nerve 1992;(15):550?56 [Non-patent document 6] KARLIJN J. WILSCHUT, et al., Concise Review: Stem Cell Therapy for Muscular Dystrophies. STEM CELLS TRANSLATIONAL MEDICINE. 2012; (1): 833?842. Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[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 an intercellular fusion gene as an exogenous gene, wherein the intercellular fusion gene is expressed in the presence of an extracellular factor. [2] The cell according to [1], wherein the timing of the initiation 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 according to [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 according to [3], wherein the cell-cell fusion gene is a muscle-related gene. [5] The cells according to [4], wherein the muscle-related genes are 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 cells according to any one of [1] to [5], which are muscle cells or myoid cells. [7] The cell according to [6], wherein the cell is a human muscle cell or a human myoid cell. [8] The cell according to any one of [1] to [7], wherein the cell further expresses a gene encoding a drug. [9] The cell according to any one of [1] to [8], wherein the cell vector system in which the cell-cell fusion gene is expressed in the presence of an extracellular factor is Cre-Lox, Flp-FRT, Vika-Vox, ERT, ERT2, Destabilizing Domain (DD), TetON, or TetOFF.

[10] A cell medicine containing the cells according to [1] to [9] and extracellular factors, which has the added property of improving cell engraftment after cell transplantation, wherein the extracellular factors can be arbitrarily exposed to the cells so that the expression of the cell-cell fusion gene does not affect the proliferation ability required for the production of 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 the expression of the cell-cell fusion gene does not affect the proliferation ability required for the production of cell-based medicines. [Effects of the Invention]

[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. [Brief explanation of the drawings]

[0011] [Figure 1A] Figure 1A shows the plasmid vector (Vector 1; SEQ ID NO: 1) introduced into the cells of the present invention. In Vector 1, skeletal muscle-related genes (MyoD and Myogenin) for cell-cell fusion are designed to be inverted. The two loxP sites are on the same DNA strand but in opposite directions, so the genes between the loxP sites are inverted by Cre. [Figure 1B] Figure 1B shows the plasmid vector (Vector 2; SEQ ID NO: 2) introduced into the cells of the present invention. Vector 2 serves to switch the reverse orientation of Vector 1 to the forward orientation. This switch functions by exposure to trimethoprim (TMP). [Figure 1C] Figure 1C shows a plasmid vector (Vector 3; SEQ ID NO: 3) introduced into a cell of the present invention. Vector 3 synthesizes mRNA using the vector as a template and introduces the mRNA into the cell. Vector 3 also plays a role in integrating Vector 1 and Vector 2 into the nucleic acid of the cell. [Figure 2A] Figure 2A shows a fusion detection system using the Flp-FRT system. [Figure 2B] FIG. 2B shows the results of an in vitro fusion test of the cell vector. [Figure 3A] FIG. 3A shows the results of an in vivo fusion test of cell vectors (photograph). [Figure 3B] FIG. 3B is a graph quantifying the results of the test in FIG. 3A. [Figure 4A] FIG. 4A shows the results of a cell-vector Matrigel plug assay (photograph). [Figure 4B] FIG. 4B is a graph quantifying the results of the assay in FIG. 4A. [Figure 4C] FIG. 4C shows the site of gel administration in mice. [Figure 5A] FIG. 5A shows the results of the efficacy test of the cell vector (days 21 and 28). [Figure 5B] FIG. 5B is a graph showing the results (blood flow) of the efficacy test quantified. [Figure 5C] FIG. 5C is a table showing the numerical results of the efficacy test (number of positive cells) and a corresponding graph. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. The present invention relates to cells that contain 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 to 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 a period preparing for DNA replication (G1 phase), a period replicating DNA (S phase), a period preparing for cell division (G2 phase), a period of cell division (M phase), and a resting phase (G0 phase), making it difficult to align the cell division period and other conditions for all cells in the cell population. If a gene capable of fusion is introduced into cells as an exogenous gene without controlling its expression, the expression of this gene has a strong influence, causing the introduced transplant cells to fuse with each other before transplantation into the body and subsequently to stop proliferating.To avoid fusion between transplant cells after gene introduction and before transplantation as much as possible, and to proliferate as many transplant cells as possible during production and enable efficient fusion with somatic cells in the body, we controlled the timing of the onset of expression of the cell-cell fusion gene in the presence of extracellular factors so as not to affect the proliferation ability of the transplant cells.

[0016] In the present invention, the cell-cell fusion genes are 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, and examples include 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 fertilization-related genes refer to genes involved in the fusion of sperm and eggs, such as CD9, Izumo1, Juno, Spaca6, TMEM95, Sof1, Fimp, and Dcst1 / Dsct2. The virus-related genes involved in membrane fusion 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 a 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). In another embodiment, the cell-cell fusion gene is Myomaker. Myomaker is a membrane protein that is highly expressed during the differentiation process of myoblasts, 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 transplanted cells are not 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 they are administered, and the transplanted cells may be either 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 are, 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 be fused with somatic cells such as muscle cells or myoid cells in the body, but examples include relatively available somatic cells such as fibroblasts derived from skin or hair, red blood cells, and stem cells derived from bone marrow and adipose tissue. In the present invention, the transplant cells are preferably cells derived from humans. In the present invention, the cells to be fused with the transplanted cells are not particularly limited as long as they can be fused with the transplanted 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 may contain and express a drug gene without being encoded in the cell vector. In the present invention, a pharmaceutical gene may be single-stranded or double-stranded. Examples of single-stranded pharmaceutical genes include mRNA and miRNA. Examples of double-stranded pharmaceutical 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 pharmaceutical gene is not particularly limited. For example, drug genes include genes involved in the cell cycle (CDK family, Cyclin family, p16, p21, p27, E2F, and their mutants, etc.), genes related to chromosome structure (genes encoding telomerase, TERT, ZSCAN, SV40 Large T antigen, HPV E6 / E7, Ras, Rb, recombinase, integrase, nuclease, helicase, ligase, replicase, B-cell lymphoma 2, CRISPR Cas, and their mutants, etc.), reprogramming-related genes (Oct3 / 4, c-Myc, Klf4, Sox2, NANOG, ASCL1, PITX3, NURR1, LMX1A, and their mutants, etc.), and skeletal muscle-related genes (CD56, Pax3, Pax7, Myogenin, Myf5, MyoD, Myomaker, Myomixer, Myosin Heavy Chain, Desmin, Dystrophin, Myothin, Laminin, etc.). A / C, CAV, CAPN, SGCG, TRIM32, TCAP, FKRP, EMD, PABP, DMPK, ZNF9, FCMD, POMENTI, Collagen, SEPN1, RYRI, MTM, TNNT, NEB, TPM, ACTN, GNE, DYSF, CRYAB, ACTA and their variants), 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 variants), genes related to transcription factors (Runt domain including RUNX and its variants, helix-turn-helix, helix-loop-helix, zinc finger, leucine zipper, β-sheet motif and their variants), genes related to enzymes (glucose-6-phosphatase, t-PA, collagenase, alglucosidase, urate oxidase, alkaline phosphatase, glycosaminoglycan degrading enzyme, β-glucuronidase, glutamic acid carboxypeptidase, sphingomyelin phosphodiesterase, α-L-isothiazolinone ... These include genes related to membrane proteins (monotopic, including neprilysin, as well as polytopic and their variants), genes related to chimeric antigen receptors (CAR and its variants), antibody genes (genes encoding mouse antibodies, chimeric antibodies, humanized antibodies, human antibodies, and antibodies without substem indicating their origin), blood coagulation-related factors, serum proteins, hormones, vaccines, interferons, erythropoietins, cytokines, toxins, and fusion proteins. In the present invention, for example, by introducing the above-mentioned genes into a vector in the transplanted cells (i.e., a cell vector system) or by introducing the genes directly into the transplanted cells without introducing them into a vector, the lifespan of the transplanted cells into which the genes have been introduced can be extended or differentiation into the desired cells can be promoted.

[0021] In the present invention, genes can be introduced into cells by any known method, including biochemical methods such as lipofection and polybrene, physical methods such as electroporation, or a combination of these. In the present invention, when lipofection is used, 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 a density of 1 to 2 x 10 4 cells / cm 2 If the cells are human myoblasts, more preferably 2 x 10 4 cells / cm 2 It is sown in. For example, when using lipofection 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 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 a density of 1 to 2 x 10 3 cells / cm 2 If the cells are human myoblasts, more preferably 2 x 10 3 cells / cm 2 It is sown in. For example, when a viral vector is introduced into cells using the polybrene method, the viral vector is mixed with a polybrene solution and added to the cells to be introduced with a gene at a multiplicity of infection (MOI) of 0.1 to 100. Preferably, from the viewpoint of achieving a high final introduction efficiency and survival rate, the viral vector is added to the cells to be introduced with a gene at an MOI of 1 to 10. In the present invention, when electroporation is used, the cells into which the gene is introduced are prepared at about 1,000 to 1,000,000 cells / mL and electroporated. When the cells into which the gene is introduced are human somatic cells, the cells are more preferably prepared at about 1 to 10x10 4 The cells are adjusted to 10x10 cells / mL and electroporated, and if the cells are human myoblasts, more preferably 10x10 4 Cells / mL are adjusted and electroporated. For example, when using electroporation to introduce nucleic acid molecules such as plasmid DNA or mRNA into cells, 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 survival rate, 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 in which the cell-cell fusion gene is expressed in the presence of an extracellular factor is not particularly limited, as long as it is possible to adjust the timing of the initiation of expression of the cell-cell fusion gene so as not to affect the proliferation ability of the transplanted cells, and examples thereof include Cre-Lox, Flp-FRT, Vika-Vox, ERT, ERT2, Destabilizing Domain (DD), TetON, and TetOFF. In one embodiment of the present invention, the vector encoding the cell-cell fusion gene is integrated into the genome of the fused cells. In the present invention, the extracellular factor is not particularly limited as long as it can regulate the timing of initiation of cell-cell fusion gene expression in the cell vector system, and examples thereof include trimethoprim (TMP) and tamoxifen. In one embodiment, the extracellular factor is not particularly limited as long as it can migrate to the administration site 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 controlled by adjusting the timing and site of TMP administration using the Cre-Lox or Flp-FRT cell vector system. In another embodiment, the timing of exposure of the cell vector in the transplanted cells to TMP can be controlled by administering the transplanted cells and TMP at different sites. In the present invention, the timing of termination of expression of the cell-cell fusion gene 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, and is characterized by comprising the above-mentioned cells of the present invention and extracellular factors, and being able to expose the extracellular factors to the transplanted cells 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, transplant cells can be administered to a subject requiring muscle cells or myoid cells at a rate of, for example, 10,000 to 100,000,000 cells / kg body weight. In the pharmaceutical of the present invention, when the extracellular factor is TMP, for example, 1 to 200 mg / kg body weight can be administered to a subject requiring muscle cells or myoid cells. 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 composition 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 a mammal, and a human subject is preferred.

[0024] In the present invention, the transplanted cells and the extracellular factors may be administered simultaneously or separately. In the present invention, the timing of exposure of the transplanted cells to the extracellular factors can be controlled by administering the transplanted cells and the extracellular factors at different sites. In one embodiment, the transplanted 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 in a vector, and expression of the cell-cell fusion gene is initiated only when transplant cells containing such a vector are exposed to an extracellular factor. Therefore, it takes a certain amount of time for the expression of the cell-cell fusion gene to be initiated by exposure of the transplant cells to the extracellular factor, and fusion between the transplanted cells does not initiate immediately after exposure and before transplantation, so the transplanted cells and the extracellular factor may be administered simultaneously. When the transplanted cells and the extracellular factor are administered separately, the order of administration is not limited. Since transplanted cells are usually eliminated by the host's immune response approximately 24 hours after transplantation, if the transplanted cells are administered first and then the extracellular factor is administered, the extracellular factor should be administered at the same or a different site within at least 24 hours, e.g., within a few hours, e.g., 2 to 3 hours, after the administration of the transplanted cells. Furthermore, if the extracellular factor is administered first and then the transplanted cells are administered, 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 should be administered within at least 10 hours, e.g., within 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour, after the administration of the transplanted cells. Furthermore, when TMP is administered orally, its half-life is just under 7 hours, so TMP should be 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 increase or decrease in 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).

[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 such a way that expression of the cell-cell fusion gene does not affect the proliferation ability required for the production of a cell medicine.

[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 administering the cells of the present invention and an extracellular factor into the muscle of a tissue of a subject in need of improved blood flow, and is characterized in that the cells of the present invention are 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 a cell-based medicine. In the present invention, the pharmacological genes that can be used are 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, improvement in blood flow can be determined 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 cell-based pharmaceuticals due to the expression of the cell-cell fusion gene. [Example]

[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. Preparation of cell vectors Using the Cre-Lox cell vector system (Vector Builder) and the φC31 integrase system (Vector Builder), MyoD (SEQ ID NO: 4) and Myogenin (SEQ ID NO: 5) are used as cell-cell fusion genes, and trimethoprim (TMP) is used as an extracellular factor to regulate the initiation of cell-cell fusion gene expression. When the two loxP sites are on the same DNA strand (Vector 1) but positioned in opposite directions, the gene between the loxP sites is inverted by Cre in Vector 2. The φC31 integrase system is a combination of mRNA produced from vector 3 and 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 to 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, and the vectors were added to the cells at a concentration of 1 to 20 pg / cell. Hereinafter, the primary muscle cells into which the three plasmids have been introduced are referred to as the cell vector of the present invention.

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

[0031] Example 3. Cell-vector in vivo fusion assay Using the Flp-FRT system (Vector Builder), we constructed a system in which luminescence is observed only when the cell vector and mouse somatic cells fuse. This is the same system as in Example 2, except that Flp-expressing transgenic mice are used instead of C2C12 (Flp), and fusion results in luminescence of Luciferin. The cell vector of the present invention was fused with mouse somatic cells in vivo. The cell vector was administered to the left and right thighs of the mice, and TMP was administered to the same sites the following day. In the group transplanted with only the cell vector (cell vector transplant group), luminescence attenuated (Figure 3A, left column). However, in the group administered both the cell vector and TMP, the luminescence attenuation was suppressed (Figure 3A, center column). This indicates that exposure to TMP initiated expression of the cell-cell fusion gene, resulting in fusion with mouse somatic cells. Furthermore, in the group administered with only the vehicle (phosphate-buffered saline) (vehicle group), no luminescence was observed (Figure 3A, right column). A graph of this quantification is also shown (Figure 3B). The device used was the IVIS Imaging System (Revvity).

[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 Biosciences, Inc.). Seven days later, the gel was removed and evaluated (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 significantly higher vascular area than the bFGF (Fibroblast Spray) (Kaken Pharmaceutical Co., Ltd.) group and the human mesenchymal stem cell (MSC) group (Figure 4A, B). Matrigel alone was used as the medium.

[0033] Example 5. Drug efficacy test of cell vectors A hindlimb ischemia model was created in mice by isolating the artery and vein in one thigh. The test substance was then administered intramuscularly to the ischemic limb. To initiate cell-cell fusion gene expression from the human cell vector, TMP was administered intraperitoneally 2-3 hours before the administration of the test substance on the same day. As a result, the human cell vector group ("test product") showed excellent improvement in blood flow (right column of Figure 5A, Figure 5B), and the number of blood vessels was significantly higher than in the other groups (right column of Figure 5A, Figure 5C).

Claims

1. 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.

2. 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.

3. 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.

4. The cell of claim 3 , wherein the cell-cell fusion gene is a muscle-related gene.

5. 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.

6. The cell of claim 1 , wherein the cell is a muscle cell or a myoid cell.

7. The cell of claim 6 , wherein the cell is a human muscle cell or a human myoid cell.

8. The cell of claim 1 , wherein the cell further expresses a gene encoding a drug.

9. The cell described in claim 1, wherein the cell vector system in which the cell-cell fusion gene is expressed in the presence of an extracellular factor is Cre-Lox, Flp-FRT, Vika-Vox, ERT, ERT2, Destabilizing Domain (DD), TetON, or TetOFF.

10. 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.

11. 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.

Images