Method for improving the efficiency of inducing pluripotent stem cells

Abstract

The present invention provides methods for improving the efficiency of inducing pluripotent stem cells, as well as vectors and compositions for use therein. In a pluripotent stem cell induction process that includes the introduction of a vector sequentially containing the KLF gene, the OCT gene, and the SOX gene, the efficiency of pluripotent stem cell induction was significantly improved by further introducing a vector containing the KLF gene but not the OCT gene and the SOX gene. The methods of the present invention have excellent characteristics because they allow for efficient induction of pluripotent stem cells under temperature conditions that closely resemble physiological conditions, as well as rapid removal of the vector after pluripotent stem cell induction. The present invention enables more efficient induction of pluripotent stem cells.

Description

Technical Field

[0001] The present invention relates to a method for improving the induction efficiency of pluripotent stem cells. Furthermore, the present invention relates to a gene delivery vector and a gene delivery composition for use in the induction of pluripotent stem cells, as well as uses thereof. Specifically, the present invention relates to a technique for improving the efficiency of inducing pluripotent stem cells, wherein the induction of pluripotent stem cells comprises the step of introducing a vector containing, in sequence, a KLF gene, an OCT gene, and a SOX gene. Background Art

[0002] Since the induction of pluripotent stem cells by somatic cells (induced pluripotent stem cells (iPS) cells; also known as "artificial pluripotent stem cells" or "induced pluripotent stem cells") was reported (non-patent literature 1), iPS cells have been produced by introducing reprogramming factors into a variety of mammalian cells, including human and mouse cells (non-patent literatures 1 to 9). Many of them use retroviruses to introduce reprogramming factors. However, since retroviruses have the risk of causing tumors due to integration into the host genome, their use is limited (non-patent literature 3). In order to solve this problem, people have tried to use adenovirus to induce iPS cells, but as long as DNA-type vectors are used, it is impossible to completely eliminate the worry about integration into the genome. In addition, the iPS cell induction efficiency of these vectors is extremely low (non-patent literatures 10 to 13).

[0003] To address these issues, the present inventors previously developed a system for inducing iPS cells using RNA-based virus Sendai virus vectors (Patent Documents 1 and 2). The efficiency of iPS cell induction using Sendai virus vectors was significantly higher than previous cases using other vectors. Since Sendai viruses do not have a DNA phase in their life cycle, and therefore there is no concern about their integration into the host genome, they are extremely superior in terms of safety. Moreover, the vector can be easily removed after iPS cell induction. However, the technology of the present invention for further improving the efficiency of iPS cell induction is still unknown.

[0004] Prior art literature

[0005] [Patent Document]

[0006] [Patent Document 1] International Publication WO 2010 / 008054

[0007] [Patent Document 2] International Publication WO 2012 / 029770

[0008] [Non-patent literature]

[0009] [Non-patent document 1] Takahashi, K. and Yamanaka, S. (2006) Cell 126, 663-676

[0010] [Non-patent document 2] Maherali, N. et al., (2007) Cell Stem Cell 1, 55-70

[0011] [Non-patent document 3] Okita, K. et al., (2007) Nature 448, 313-317

[0012] [Non-patent document 4] Wernig, M. et al., (2007) Nature 448, 318-324

[0013] [Non-patent document 5] Takahashi, K. et al., (2007) Cell 131, 861-872

[0014] [Non-patent document 6] Yu, J. et al., (2007) Science 318, 1917-1920

[0015] [Non-patent document 7] Lowry, WE et al., (2008) Proc. Natl. Acad. Sci. USA 105, 2883-2888

[0016] [Non-patent document 8] Park, IH et al., (2008) Nature 451, 141-146

[0017] [Non-patent document 9] Masaki, H. et al., (2008) Stem Cell Res. 1, 105-115

[0018] [Non-patent document 10] Stadtfeld, M. et al., (2008) Science 322, 945-949

[0019] [Non-patent document 11] Okita, K. et al., (2008) Science 322, 949-953

[0020] [Non-patent document 12] Yu, J. et al., (2009) Science 324, 797-801

[0021] [Non-patent document 13] Zhou, W. et al., (2009) Stem Cells 27, 2667-2674

[0022] Summary of the Invention

[0023] Problems to be solved by the present invention

[0024] The present invention relates to a method for improving the induction efficiency of pluripotent stem cells. Furthermore, an object of the present invention is to provide a gene delivery vector and a gene delivery composition for use in inducing pluripotent stem cells, as well as uses thereof. Specifically, an object of the present invention is to provide a method for improving the efficiency of inducing pluripotent stem cells, and a gene delivery vector and composition used therein, wherein the induction of pluripotent stem cells comprises the step of introducing a vector that sequentially contains the KLF gene, the OCT gene, and the SOX gene in a single vector.

[0025] Means of solving the problem

[0026] The present inventors have sought a method for further improving the efficiency of inducing pluripotent stem cells in the induction of pluripotent stem cells, wherein the induction of the pluripotent stem cells includes the step of introducing a vector, wherein the vector sequentially contains the KLF gene, the OCT gene, and the SOX gene (i.e., reprogramming factors) in one vector. Specifically, when a temperature-sensitive vector is used as a vector for inducing pluripotent stem cells, although pluripotent stem cells are induced with a relatively high efficiency when cultured at a low temperature (36°C) after the vector is introduced, the induction efficiency of the pluripotent stem cells may decrease significantly if they are cultured at a standard cell culture temperature of 37°C. Although temperature-sensitive vectors are superior in that they can be quickly removed after the induction of pluripotent stem cells, the problem is that in order to effectively induce such pluripotent stem cells, it is necessary to culture at a low temperature after the vector is introduced.

[0027] The present inventors have conducted intensive research on methods for inducing pluripotent stem cells with high efficiency, using a temperature-sensitive Sendai virus vector and avoiding low-temperature culture after vector introduction. As a result, they discovered that, when introducing a vector containing the KLF, OCT, and SOX genes in sequence within a single vector, the efficiency of pluripotent stem cell induction can be significantly increased without low-temperature culture by further introducing a vector containing the KLF gene but not containing the OCT and SOX genes. In particular, by further combining these vectors with a vector containing the KLF gene but not containing the OCT and SOX genes, iPS cells can be induced with significantly higher efficiency without low-temperature culture, compared to when a vector containing the KLF, OCT, and SOX genes in sequence within a single vector is used alone in combination with a vector expressing the MYC gene.

[0028] This allows for efficient induction of pluripotent stem cells without low-temperature culture, and the establishment of a system for rapid vector removal after induction of pluripotent stem cells.

[0029] That is, the present invention relates to methods for improving the efficiency of inducing pluripotent stem cells, as well as gene delivery vectors and compositions used therein. More specifically, it relates to the invention described in each claim, wherein the induction of pluripotent stem cells includes the step of introducing a vector that sequentially contains the KLF gene, the OCT gene, and the SOX gene in a single vector. Inventions consisting of any combination of two or more inventions described in multiple claims that reference the same claim are also inventions intended to be protected by this application. More specifically, the present invention relates to the following:

[0030] [1] A method for improving the efficiency of pluripotent stem cell induction in a method for inducing pluripotent stem cells without low-temperature culture, the method comprising introducing a temperature-sensitive Sendai virus vector comprising, in sequence, the KLF gene, the OCT gene, and the SOX gene in one vector, wherein the method comprises further introducing a Sendai virus vector comprising the KLF gene but not the OCT gene and the SOX gene;

[0031] [2] The method of [1], wherein the culture is carried out at about 37°C;

[0032] [3] The method of [1] or [2], wherein the temperature-sensitive Sendai virus vector comprising the KLF gene, the OCT gene, and the SOX gene in sequence in one vector is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, D433A, R434A, K437A, and L511F mutations in the P protein, and N1197S and K1795E mutations in the L protein;

[0033] [4] The method of any one of [1] to [3], wherein the Sendai virus vector comprising the KLF gene but not the OCT gene and the SOX gene is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein;

[0034] [5] The method of any one of [1] to [4], further comprising introducing a temperature-sensitive Sendai virus vector into which the MYC gene is inserted;

[0035] [6] The method of [5], wherein the temperature-sensitive Sendai virus vector inserted with the MYC gene is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, D433A, R434A, K437A, and L511F mutations in the P protein, and L1361C, L1558I, N1197S, and K1795E mutations in the L protein;

[0036] [7] Use of a Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene in the manufacture of a medicament for improving the efficiency of pluripotent stem cell induction in a method for inducing pluripotent stem cells without low-temperature culture, wherein the method for inducing pluripotent stem cells comprises introducing a temperature-sensitive Sendai virus vector containing the KLF gene, the OCT gene, and the SOX gene in sequence in one vector;

[0037] [8] The use of [7], wherein the method of inducing pluripotent stem cells is a method involving culturing at about 37°C;

[0038] [9] [7] or [8], wherein the temperature-sensitive Sendai virus vector comprising the KLF gene, the OCT gene and the SOX gene in sequence in one vector is an F gene-deleted Sendai virus vector comprising G69E, T116A and A183S mutations in the M protein, A262T, G264R and K461G mutations in the HN protein, D433A, R434A, K437A and L511F mutations in the P protein, and N1197S and K1795E mutations in the L protein;

[0039]

[10] The use of any one of [7] to [9], wherein the Sendai virus vector comprising the KLF gene but not the OCT gene and the SOX gene is an F gene-deficient Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein;

[0040]

[11] A composition for inducing pluripotent stem cells, wherein the composition comprises:

[0041] (a) a temperature-sensitive Sendai virus vector comprising the KLF gene, the OCT gene, and the SOX gene in sequence in one vector, and

[0042] (b) Sendai virus vector containing the KLF gene but not the OCT gene and SOX gene;

[0043]

[12] A composition for inducing pluripotent stem cells, wherein the composition comprises:

[0044] (a) a temperature-sensitive Sendai virus vector comprising the KLF gene, the OCT gene, and the SOX gene in sequence in one vector, which is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, D433A, R434A, K437A, and L511F mutations in the P protein, and N1197S and K1795E mutations in the L protein;

[0045] (b) a Sendai virus vector comprising the KLF gene but not the OCT gene and the SOX gene, which is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, an L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein;

[0046]

[13] The composition of

[11] or

[12] , further comprising a temperature-sensitive Sendai virus vector into which the MYC gene is inserted;

[0047]

[14]

[13] The composition of claim 13, wherein the temperature-sensitive Sendai virus vector inserted with the MYC gene is an F gene-deficient Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, D433A, R434A, K437A, and L511F mutations in the P protein, and L1361C, L1558I, N1197S, and K1795E mutations in the L protein;

[0048]

[15] The composition of any one of

[11] to

[14] , which is a composition for inducing pluripotent stem cells by culturing in the absence of low-temperature culture;

[0049]

[16] A kit for inducing pluripotent stem cells, wherein the kit comprises:

[0050] (a) a temperature-sensitive Sendai virus vector comprising the KLF gene, the OCT gene, and the SOX gene in sequence in one vector, and

[0051] (b) Sendai virus vector containing the KLF gene but not the OCT gene and SOX gene;

[0052]

[17] A kit for inducing pluripotent stem cells, wherein the kit comprises:

[0053] (a) a temperature-sensitive Sendai virus vector comprising the KLF gene, the OCT gene, and the SOX gene in sequence in one vector, which is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, D433A, R434A, K437A, and L511F mutations in the P protein, and N1197S and K1795E mutations in the L protein;

[0054] (b) a Sendai virus vector comprising the KLF gene but not the OCT gene and the SOX gene, which is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, an L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein;

[0055]

[18] The kit of

[16] or

[17] , further comprising a temperature-sensitive Sendai virus vector into which the MYC gene is inserted;

[0056]

[19]

[18] The kit of

[18] , wherein the temperature-sensitive Sendai virus vector inserted with the MYC gene is an F gene-deficient Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, D433A, R434A, K437A, and L511F mutations in the P protein, and L1361C, L1558I, N1197S, and K1795E mutations in the L protein;

[0057]

[20] The kit according to any one of

[16] to

[19] , which is a kit for inducing pluripotent stem cells by culturing them without low-temperature culture;

[0058] This application is intended to cover any of the inventions described herein and any combination thereof. Within these inventions, this application is also intended to cover inventions that exclude any of the inventions described herein or any combination thereof. Furthermore, certain specific embodiments of the invention described in this application disclose not only those embodiments but also inventions that are obtained by excluding those embodiments from the superordinate inventions disclosed in this application that include those embodiments.

[0059] Effects of the Invention

[0060] The present invention utilizes a vector containing the KLF gene, the OCT gene, and the SOX gene in sequence, which significantly improves the efficiency of pluripotent stem cell induction. In particular, it is useful to be able to induce pluripotent stem cells with high efficiency without low-temperature culture, and the use of a temperature-sensitive vector allows for rapid removal of the vector after pluripotent stem cell induction. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The figure shows the induction efficiency of alkaline phosphatase-positive iPS-like cells. The figure shows alkaline phosphatase staining images of vector-infected human neonatal foreskin-derived fibroblasts (BJ) on day 28.

[0063] Figure 2 The induction efficiency of alkaline phosphatase-positive iPS-like cells is shown. All conditions are the same as Figure 3 The same ones in .

[0064] Figure 3 Alkaline phosphatase-stained colonies are shown. The number of alkaline phosphatase-positive colonies that appeared under conditions 1 and 2 was significantly higher.

[0065] Figure 4 The induction efficiency of alkaline phosphatase-positive iPS-like cells is shown. The induction efficiency of pluripotent stem cells from BJ cells is shown. All conditions are the same as Figure 3 The same ones in .

[0066] Figure 5 The figure shows the induction efficiency of alkaline phosphatase-positive iPS-like cells. The figure shows the alkaline phosphatase staining results of human adult skin-derived fibroblasts (HDF) on day 28 after infection with the vector.

[0067] Figure 6 The induction efficiency of alkaline phosphatase-positive iPS-like cells is shown. The induction efficiency of pluripotent stem cells from human adult skin-derived fibroblasts (HDF) is shown. All conditions are the same as Figure 5 The same ones in .

[0068] Figure 7 The figure shows the induction efficiency of alkaline phosphatase-positive iPS-like cells. The figure shows the results of alkaline phosphatase staining of human fetal lung cell-derived fibroblasts (MRC-5) on day 28 after infection with the vector.

[0069] Figure 8 Figure 2 shows the induction efficiency of alkaline phosphatase-positive iPS-like cells. Figure 2 shows the induction efficiency of alkaline phosphatase-positive colonies. All conditions were the same as Figure 7 The same ones in .

[0070] Figure 9The in vivo pluripotency of induced pluripotent stem cells was demonstrated.

[0071] Figure 10 Karyotyping of induced pluripotent stem cells was performed.

[0072] Figure 11 Shown is the induction of iPS cells from mouse fibroblasts (alkaline phosphatase staining results).

[0073] Figure 12 Confirmation of vector removal by anti-Sendai virus antibody staining is shown.

[0074] Figure 13 Confirmation of marker gene expression by immunostaining is shown.

[0075] Figure 14 The results of evaluation of mouse iPS cells (embryoid body formation) are shown.

[0076] Figure 15 The results of evaluation of mouse iPS cells (in vitro differentiation into endoderm) are shown.

[0077] Figure 16 The results of evaluation of mouse iPS cells (in vitro differentiation into mesoderm) are shown.

[0078] Figure 17 The results of evaluation of mouse iPS cells (in vitro differentiation into ectoderm) are shown.

[0079] Figure 18 The results of evaluation of mouse iPS cells (in vitro differentiation into ectoderm) are shown.

[0080] Figure 19 Shown is the induction of iPS cells from human monocytes.

[0081] Figure 20 Shown is the induction of iPS cells from human monocytes (alkaline phosphatase staining images).

[0082] Figure 21 The induction of iPS cells from human monocytes (iPS cell induction efficiency) is shown.

[0083] Figure 22 Shown is the induction of iPS cells from human peripheral blood mononuclear cells (appearance of cells from donor 1).

[0084] Figure 23 Shown is the induction of iPS cells from human peripheral blood mononuclear cells (appearance of cells from donor 2).

[0085] Figure 24Shown is the induction of iPS cells from human peripheral blood mononuclear cells (alkaline phosphatase staining).

[0086] Figure 25 Shown is the induction (induction efficiency) of iPS cells from human peripheral blood mononuclear cells.

[0087] Figure 26 Shown is the induction of iPS cells from human monocytes (appearance of cells from donor 3).

[0088] Figure 27 Shown is the induction of iPS cells from human monocytes (appearance of cells from donor 4).

[0089] Figure 28 Shown is the induction of iPS cells from human monocytes (alkaline phosphatase staining).

[0090] Figure 29 Shown is the induction of iPS cells from human monocytes (induction efficiency).

[0091] Modes for Carrying Out the Invention

[0092] The following will describe in detail the mode for carrying out the present invention.

[0093] The present invention relates to a method for improving the efficiency of pluripotent stem cell induction, comprising the step of introducing a vector containing the KLF gene but not the OCT gene and the SOX gene during the induction of pluripotent stem cells, wherein the induction of pluripotent stem cells comprises the step of introducing a vector containing the KLF gene, the OCT gene, and the SOX gene in sequence within a single vector. More specifically, the method of the present invention improves the efficiency of pluripotent stem cell induction in a method of inducing pluripotent stem cells by introducing a temperature-sensitive viral vector containing the KLF gene, the OCT gene, and the SOX gene in sequence within a single vector, and further comprising the step of introducing a viral vector containing the KLF gene but not the OCT gene and the SOX gene.

[0094] After vector introduction, culturing at a low temperature (e.g., at 36.5°C, and more specifically, at 36°C or lower) is unnecessary. That is, the method of the present invention is superior using a temperature-sensitive variant vector because pluripotent stem cells can be induced with high efficiency without low-temperature culturing (e.g., culturing at less than 36.5°C), and the vector can be rapidly removed after induction of the pluripotent stem cells. For example, the present invention relates to a method for improving the efficiency of pluripotent stem cell induction in a method for inducing pluripotent stem cells without low-temperature culturing by introducing a temperature-sensitive Sendai virus vector that sequentially includes the KLF gene, the OCT gene, and the SOX gene in a single vector, the method comprising further introducing a Sendai virus vector that contains the KLF gene but does not include the OCT gene and the SOX gene. The cell culture temperature can be appropriately selected; for example, it can be approximately 37°C, particularly 36.5°C to 37.5°C, preferably 36.6°C to 37.4°C, and more preferably 36.7°C to 37.3°C. In the present invention, the induction of pluripotent stem cells includes the generation of multipotent stem cells, and the improvement of the efficiency of pluripotent stem cell induction includes the improvement of the efficiency of the generation of multipotent stem cells.

[0095] Although the introduction of the MYC gene may not be necessary for the induction of pluripotent stem cells, the efficiency of pluripotent stem cell induction can be improved by introducing the MYC gene. For example, the MYC gene can be introduced by using a temperature-sensitive viral vector, preferably a temperature-sensitive Sendai virus vector.

[0096] The present invention provides a method for reprogramming cells, comprising introducing or contacting differentiated cells, such as somatic cells, with a vector containing, in sequence, the KLF gene, the OCT gene, and the SOX gene, and a vector containing the KLF gene but not the OCT gene and the SOX gene. Furthermore, the present invention provides a method for introducing the aforementioned reprogramming factor genes during cell reprogramming, comprising introducing these reprogramming factor genes using a vector containing, in sequence, the KLF gene, the OCT gene, and the SOX gene, and a vector containing the KLF gene but not the OCT gene and the SOX gene, as well as compositions comprising the vectors used in those methods.

[0097] In the present invention, "pluripotent stem cells" refer to stem cells prepared from the inner cell mass of an animal embryo at the blastocyst stage, or cells with a phenotype similar to these cells. Specifically, the induced pluripotent stem cells of the present invention are cells that express alkaline phosphatase (an indicator of ES-like cells). Here, "ES-like cells" refers to pluripotent stem cells with properties and / or morphology similar to ES cells. Furthermore, when pluripotent stem cells are cultured, they preferably form flat colonies containing cells with a higher nuclear volume than cytoplasm ratio. They may be cultured with feeder cells as appropriate. Furthermore, unlike cultured cells such as MEFs, which cease to proliferate within a few weeks, pluripotent stem cells can be passaged for extended periods of time. This can be confirmed by maintaining their proliferative capacity after, for example, passage every three days for at least 15 times, preferably at least 20 times, at least 25 times, at least 30 times, at least 35 times, or at least 40 times. Furthermore, pluripotent stem cells preferably express endogenous OCT3 / 4 or Nanog, or more preferably, they express both OCT3 / 4 and Nanog. In addition, pluripotent stem cells preferably express TERT and show telomerase activity (activity of synthesizing telomeric repeat sequences). In addition, pluripotent stem cells preferably have the ability to differentiate into three germ layers (endoderm, mesoderm and ectoderm) (this can be confirmed, for example, during teratoma formation and / or embryoid body formation). More preferably, pluripotent stem cells produce germline chimeras when transplanted into blastocysts. Pluripotent stem cells capable of germline transmission are referred to as germline-competent pluripotent stem cells. The confirmation of these phenotypes can be carried out by known methods (WO 2007 / 69666; Ichisaka T. et al., Nature 448 (7151): 313-7, 2007).

[0098] In addition, in the present invention, "(differentiated)" means that the differentiation stage of the cell has progressed compared to before, and may refer to, for example, more differentiated than pluripotent stem cells, and includes a state in which the ability to differentiate into multiple cell lineages (such as somatic stem cells) is still retained, as well as a terminally differentiated state. Differentiated cells are cells derived from pluripotent stem cells (except pluripotent stem cells). Differentiated cells can be, for example, cells that do not have the ability to differentiate into three germ layers (endoderm, mesoderm, and ectoderm). Such cells will not have the ability to form three germ layers unless reprogrammed. In addition, differentiated cells can be, for example, cells that are unable to generate cells other than the germ layer type to which they belong. Differentiated cells can be somatic cells, and for example, they can be cells other than germ cells.

[0099] In the present invention, reprogramming in the present invention refers to: making the differentiated state of a certain cell become a state compared to an undifferentiated state, including, for example, dedifferentiation of differentiated cells, such as inducing cells with differentiated pluripotency, such as pluripotent stem cells, from cells that do not have differentiated pluripotency. In addition, dedifferentiation in the present invention refers to the conversion of specific cells into a more immature (e.g., undifferentiated) state. Dedifferentiation can also refer to the return of specific cells to their initial state or intermediate state of differentiation. In addition, dedifferentiation can also refer to the change from a cell that cannot generate cells other than the germ layer type to which it belongs to a cell to a cell that can differentiate into other germ layers. Dedifferentiation also includes, for example, the acquisition of three germ layer differentiation ability by cells that do not have the three germ layer differentiation ability. In addition, dedifferentiation includes the generation of pluripotent stem cells.

[0100] In the present invention, somatic cells include, for example, cells other than pluripotent stem cells and germ cells. Somatic cells include, for example, cells other than pluripotent stem cells in cells constituting multicellular organisms, and cultured cells thereof. Somatic cells include, for example, somatic stem cells and terminally differentiated cells.

[0101] Low-temperature culture in the present invention refers to culture at a temperature below 36.5°C. Low-temperature culture refers to culture at a temperature of preferably below 36.4°C, more preferably 36.3°C, 36.2°C, 36.1°C, 36°C, 35.9°C, 35.8°C, 35.7°C, 35.6°C, 35.5°C, 35.4°C, 35.3°C, 35.2°C or 35.1°C, and even more preferably below 35°C. The lower limit is, for example, 30°C, preferably 31°C, more preferably 32°C, 33°C or 34°C. In addition, about 37°C in the present invention refers specifically to 36.5°C to 37.5°C, preferably 36.6°C to 37.4°C, and more preferably 36.7°C to 37.3°C.

[0102] Temperature sensitivity in the present invention means that the activity is significantly reduced at a standard temperature for cell culture (e.g., 37°C to 38°C) compared to a low temperature (e.g., 30°C to 36°C). More preferably, it means that the activity is significantly reduced at 37°C compared to 36°C. For example, in the case of an expression vector, the expression level of a temperature-sensitive vector at a standard temperature for cell culture (e.g., 37°C to 38°C) is significantly lower than the expression level at a low temperature (e.g., 30°C to 36°C). For example, TS 7 (Y942H / L1361C / L1558I mutations in L protein), TS 12 (D433A / R434A / K437A mutations in P protein), TS 13 (D433A / R434A / K437A mutations in P protein and L1558I mutation in L protein), TS 14 (D433A / R434A / K437A mutations in P protein and L1361C in L protein), and TS 15 (D433A / R434A / K437A mutations in P protein and L1361C / L1558I in L protein) mutations in Sendai virus are temperature-sensitive mutations.

[0103] The vector in the present invention is preferably a viral vector. In the present invention, a viral vector is a vector having a genomic nucleic acid of viral origin and capable of expressing a transgene by incorporating a transgene into the nucleic acid. Since the Sendai virus vector is a non-chromosomal integrative viral vector and is expressed in the cytoplasm, there is no risk that the introduced gene will become integrated into the host's chromosome (nuclear chromosome). Therefore, the vector is safe and can be removed after reprogramming is completed. In the present invention, the Sendai virus vector includes infectious virus particles, as well as a composition of the viral core, viral genome and viral proteins, and a complex comprising non-infectious virus particles, etc., which are complexes with the ability to be introduced into cells and express the loaded gene. For example, in Sendai virus, the ribonucleoprotein (core part of the virus) composed of the Sendai virus genome and the Sendai virus proteins (NP, P and L proteins) bound thereto can express a transgene in the cell after being introduced into the cell (WO00 / 70055). Transfection reagents, etc. can be used to appropriately carry out the introduction of cells. Such ribonucleoproteins (RNPs) are also included in the Sendai virus vector of the present invention.

[0104] Sendai virus is a virus of the order Mononegavirales, belonging to the family Paramyxoviridae (which includes the genera Paramyxovirus, Morbillivirus, Mumpsvirus, and Pneumovirus). Its genome consists of a single-stranded negative-strand RNA (the antisense strand of the sense strand encoding viral proteins). This minus-strand RNA is also known as negative-strand RNA.

[0105] In addition to paramyxoviruses (Paramyxoviridae viruses), the order Univirales also includes viruses belonging to families such as Rhabdoviridae (including genera such as Vesiculovirus, Lyssavirus and Ephemerovirus) and Filoviridae (Virus, vol. 57, No. 1: pp 29-36, 2007; Annu. Rev. Genet. 32, 123-162, 1998; Fieldsvirology fourth edition, Philadelphia, Lippincott-Raven, 1305-1340, 2001; Microbiol. Immunol. 43, 613-624, 1999; Field Virology, Thirdedition pp. 1205-1241, 1996). Examples of other Paramyxoviridae viruses besides Sendai virus include Newcastle disease virus, mumps virus, measles virus, respiratory syncytial virus (RS virus), rinderpest virus, distemper virus, simian parainfluenza virus (SV5), human parainfluenza virus types I, II, and III, influenza virus belonging to the Orthomyxoviridae family, vesicular stomatitis virus belonging to the Rhabdoviridae family, and rabies virus. Other examples include Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), seal distemper virus (PDV), feline distemper virus (CDV), dolphin morbillivirus (DMV), peste des petits ruminants virus (PDPR), measles virus (MV), rinderpest morbillivirus (RPV), Hendra virus (Hendra), Nipah virus (Nipah), human parainfluenza virus-2 (HPIV-2), simian parainfluenza virus 5 (SV5), human parainfluenza virus-4a (HPIV-4a), human parainfluenza virus-4b (HPIV-4b), mumps virus (Mumps), and Newcastle disease virus (NDV). More preferably, examples include viruses selected from the group consisting of Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), phoinosepticemia virus (PDV), feline distemper virus (CDV), dolphin measles virus (DMV), peste des petits ruminants virus (PDPR), measles virus (MV), rinderpest morbillivirus (RPV), Hendra virus (Hendra), and Nipah virus (Nipah).

[0106] Examples of the accession numbers of the nucleotide sequences of Sendai virus in the database are as follows: NP genes are M29343, M30202, M30203, M30204, M51331, M55565, M69046, and X17218; P genes are M30202, M30203, M30204, M55565, M69046, X00583, X17007, and X17008; M genes are D11446, K02742, M30202, M30203, M30204, M69046, U31956, X00584 , and X53056; the F genes are D00152, D11446, D17334, D17335, M30202, M30203, M30204, M69046, X00152, and X02131; the HN genes are D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, and X56131; the L genes are D00053, M30202, M30203, M30204, M69040, X00587, and X58886. Examples of viral genes encoded by other viruses include: CDV, AF014953 for the NP gene (also known as the N gene); DMV, X75961; HPIV-1, D01070; HPIV-2, M55320; HPIV-3, D10025; Mapuera, X85128; Mumps, D86172; MV, K01711; NDV, AF064091; PDPR, X74443; PDV, X75717; RPV, X68311; SeV, X00087; SV5, M81442; and Tupaia, AF079780. For the P gene, there are CDV, X51869; DMV, Z47758; HPIV-1, M74081; HPIV-3, X04721; HPIV-4a, M55975; HPIV-4b, M55976; Mumps, D86173; MV, M89920; NDV, M20302; PDV, X75960; RPV, X68311; SeV, M30202; SV5, AF052755; and Tupaia, AF079780; for the C gene, there are CDV, AF014953; DMV, Z47758; HPIV-1.M74081; HPIV-3, D00047; MV, ABO16162; RPV, X68311; SeV, AB005796; and Tupaia, AF079780; for the M gene, CDV, M12669; DMV Z30087; HPIV-1, S38067; HPIV-2, M62734; HPIV-3, D00130; HPIV-4a, D10241; HPIV-4b, D10242; Mumps, D86171; MV, AB012948; NDV, AF089819; PDPR, Z47977; PDV, X75717; RPV, M34018; SeV, U31956; and SV5, M32248; for the F gene, CDV, M21849; DMV, AJ224704; HPN-1, M22347; H PIV-2, M60182; HPIV-3.X05303, HPIV-4a, D49821; HPIV-4b, D49822; Mumps, D86169; MV, AB003178; NDV, AF048763; PDPR, Z37017; PDV, AJ224706; RPV, M21514; SeV, D17334; and SV5, AB021962; and for HN (H or G gene), CDV, AF112189; DMV, AJ224705; HPIV-1, U709498; HPIV-2.D000865; HPIV-3, AB012132; HPIV-4A, M34033; HPIV-4B, AB006954; Mumps, X99040; MV, K01711; NDV, AF204872; PDPR, Z81358; PDV, Z36979; RPV, AF132934; SeV, U06433; and SV-5, S76876. However, multiple strains are known for each of the genes listed above, and due to strain differences, genes composed of sequences different from those listed above may exist. Sendai viruses carrying viral genes derived from any of these genes can be used as vectors of the present invention. For example, the Sendai virus vectors of the present invention comprise a nucleotide sequence that is 90% or greater, preferably 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater, identical to the coding sequence of any of the above viral genes. Furthermore, the Sendai virus vector of the present invention comprises, for example, a nucleotide sequence encoding an amino acid sequence having 90% or greater, preferably 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity to the amino acid sequence encoded by the coding sequence of any of the above-mentioned viral genes. Furthermore, the Sendai virus vector of the present invention comprises, for example, a nucleotide sequence encoding an amino acid sequence having 10 or fewer, preferably 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid substitution, insertion, deletion, and / or addition in the amino acid sequence encoded by the coding sequence of any of the above-mentioned viral genes.

[0107] The sequences referenced by database accession numbers for nucleotide sequences and amino acid sequences described in this specification may, for example, be sequences as of the filing date or priority date of this application, and may be identified as such, preferably as of the filing date of this application. Sequences at various time points can be identified by reference to the revision history of the database.

[0108] The Sendai virus vector used in the present invention may be a derivative, including viruses with modified viral genes, and chemically modified viruses, etc., so that the ability of the virus to introduce genes is not impaired.

[0109] In addition, Sendai virus can be derived from natural strains, wild-type strains, mutant strains, laboratory-passaged strains, and artificially constructed strains. An example is the Z strain (Medical Journal of Osaka University Vol.6, No.1, March 1955p1-15). That is, these viruses can be viral vectors with structures similar to those isolated from viruses in nature, or viruses artificially modified by genetic recombination, as long as the desired reprogramming can be induced. For example, they may have mutations or deletions in any gene of the wild-type virus. In addition, incomplete viruses such as DI particles (J.Virol.68:8413-8417,1994) can also be used. For example, viruses with mutations or deletions in at least one gene encoding a viral envelope protein or capsid protein can be preferably used. Such viruses are, for example, viral vectors that can replicate genomes in infected cells but cannot form infectious viral particles. Since there is no concern about spreading the infection to the surrounding area, such transfection-defective viral vectors are highly safe. For example, a viral vector can be used that does not contain at least one gene encoding an envelope protein (such as F and / or HN or spike protein, or a combination thereof) (WO00 / 70055 and WO00 / 70070; Li, H.-O. et al., J. Virol. 74(14):6564-6569 (2000)). If the genomic RNA encodes proteins necessary for genome replication (e.g., NP, P, and L proteins), the genome can replicate in infected cells. In order to produce a defective virus, for example, the defective gene product or a protein capable of complementing it is trans-supplied to the virus-producing cell (WO00 / 70055 and WO00 / 70070; Li, H.-O. et al., J. Virol. 74(14):6564-6569 (2000)). In addition, methods are also known for collecting viral vectors in the form of non-infectious viral particles (VLPs) without complete complementation of defective viral proteins (WO 00 / 70070). In addition, when viral vectors are collected in the form of RNPs (e.g., RNPs containing N, L, and P proteins and genomic RNA), vectors can be produced without complete complementation of envelope proteins.

[0110] In addition, it is also preferred to use a vector carrying a mutant viral protein gene. The present invention particularly provides a method for delivering genes in the reprogramming of Sendai virus vectors having mutations and / or deletions in viral genes and a method for producing reprogrammed cells, and compositions and kits thereof. For example, multiple mutations are known in envelope proteins and capsid proteins, including attenuated mutations and temperature-sensitive mutations. Sendai viruses with these mutant protein genes can advantageously be used in the present invention. In the present invention, it is ideal to use a vector with reduced cytotoxicity. Cytotoxicity can be measured, for example, by quantitatively releasing lactate dehydrogenase (LDH) from cells. For example, a vector with significantly reduced cytotoxicity compared to the wild type can be used. Regarding the degree of reduction in cytotoxicity, for example, a vector can be used: when infected with human-derived Hela cells (ATCC CCL-2) or monkey-derived CV-1 cells (ATCC CCL 70) at an MOI (multiplicity of infection) of 3, the level of LDH released in the culture medium obtained after 3 days of culture is significantly reduced compared to the wild type, for example, by 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 50% or more. In addition, mutations that reduce cytotoxicity also include temperature-sensitive mutations. Temperature-sensitive mutations refer to mutations that significantly reduce activity at the normal temperature of the viral host (e.g., 37°C to 38°C) compared to low temperatures (30°C to 36°C, e.g., 30°C to 32°C). Proteins with such temperature-sensitive mutations are useful because viruses can be prepared at permissive temperatures (low temperatures). Viral vectors having temperature-sensitive mutations useful in the present invention exhibit a growth rate or gene expression level that is, for example, 1 / 2 or less, preferably 1 / 3 or less, more preferably 1 / 5 or less, more preferably 1 / 10 or less, and more preferably 1 / 20 or less when infected at 37°C compared to when infected in cultured cells at 32°C.

[0111] The Sendai virus vector used in the present invention can be wild type, as long as it does not suppress reprogramming, can make reprogramming factor expression, and can induce or support the induction of reprogramming by the reprogramming factor, and it is preferably at least one, more preferably at least 2,3,4,5 or more viral genes have deletion or mutation. Deletion and mutation can be arbitrarily combined and imported into each gene. Here, mutation can be dysfunctional mutation or temperature-sensitive mutation, and preferably such mutation, it is compared with wild type or the virus without the mutation, at least 37 DEG C by virus particle formation ability (or in the case of non-infectious virus, non-infectious virus-like particle (NTVLP) formation ability (Inoueviral et al., J.Virol.77:3238-3246,2003)), proliferation rate and / or the expression level of any carried gene is reduced to preferably 1 / 2 or less, more preferably 1 / 3 or less, more preferably 1 / 5 or less, more preferably 1 / 10 or less, and more preferably 1 / 20 or less. The use of such modified viral vectors may be particularly useful for inducing pluripotent stem cells. For example, the Sendai virus vectors preferably used herein have at least two deleted or mutated viral genes. Such viruses include those lacking at least two viral genes, those with mutations in at least two viral genes, and those with mutations in at least one viral gene and at least one viral gene deleted. The at least two mutated or deleted viral genes are preferably genes encoding proteins that constitute the envelope. For example, vectors with a deletion of the F gene and further deletion of the M and / or HN genes, or further mutations in the M and / or HN genes (e.g., NTVLP formation-inhibiting mutations and / or temperature-sensitive mutations) are preferably used in the present invention. In addition, vectors with a deletion of the F gene and deletion of the M or HN gene, while the remaining M or HN gene has a mutation (e.g., NTVLP formation-inhibiting mutations and / or temperature-sensitive mutations) are also preferably used in the present invention. The vectors used in the present invention more preferably have at least three deleted or mutated viral genes (preferably at least three genes encoding proteins that constitute the envelope, F, HN, and M). Such viral vectors include vectors with at least three gene deletions, vectors with mutations in at least three genes, vectors with mutations in at least one gene and at least two gene deletions, and vectors with mutations in at least two genes and at least one gene deletion. As examples of more preferred embodiments, vectors lacking the F gene and also lacking the M and HN genes, or having mutations in the M and HN genes (e.g., mutations that inhibit NTVLP formation and / or temperature sensitivity) are preferably used in the present invention. Furthermore, vectors lacking the F gene and also lacking the M or HN gene, with mutations in the remaining M or HN gene (e.g., mutations that inhibit NTVLP formation and / or temperature sensitivity) are also preferred in the present invention. Such mutant viruses can be prepared according to known methods.

[0112] For example, preferred mutations in the Sendai virus M gene include amino acid substitutions at any of positions 69 (G69), 116 (T116), and 183 (A183) in the M protein (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246). Viruses having a genome encoding a mutant M protein, in which the amino acids at any one of the three positions, preferably a combination of any two positions, and more preferably all three positions, in the Sendai virus M protein are substituted with other amino acids, are preferably used in the present invention.

[0113] Preferred amino acid mutations are substitutions with other amino acids having different side chain chemical properties, for example, substitutions with amino acids having a score of 3 or less, preferably 2 or less, more preferably 1 or less, and even more preferably 0, according to the BLOSUM62 matrix (Henikoff, S. and Henikoff, JG (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919). Specifically, G69, T116, and A183 of the Sendai virus M protein can be substituted with Glu (E), Ala (A), and Ser (S), respectively. Furthermore, mutations homologous to mutations in the M protein of the temperature-sensitive P253-505 measles virus strain (Morikawa, Y. et al., Kitasato Arch. Exp. Med. 1991:64;15-30) can also be utilized. Mutations can be introduced using, for example, oligonucleotides using known mutagenesis methods.

[0114] Preferred examples of HN gene mutations include amino acid substitutions at any of positions 262 (A262), 264 (G264), and 461 (K461) of the Sendai virus HN protein (Inoue, M. et al., J. Virol. 2003, 77:3238-3246). Viruses having genomes encoding mutant HN proteins in which the amino acids at any one of these three positions, preferably a combination of any two positions, and more preferably all three positions, are substituted with other amino acids are preferably used in the present invention. As described above, preferred amino acid substitutions are substitutions with other amino acids having different side chain chemical properties. As a preferred example, A262, G264, and K461 of the Sendai virus HN protein are substituted with Thr (T), Arg (R), and Gly (G), respectively. Furthermore, for example, using the temperature-sensitive vaccine strain Urabe AM9 of mumps virus as a reference, amino acids 464 and 468 of the HN protein can be mutated (Wright, KE et al., Virus Res. 2000: 67; 49-57).

[0115] Furthermore, Sendai virus may have mutations in the P and / or L genes. Specific examples of such mutations include mutations of Glu at position 86 (E86) of the SeV P protein and substitution of Leu at position 511 (L511) of the SeV P protein with other amino acids. As described above, preferred amino acid substitutions are substitutions with amino acids having chemically different side chains. Specific examples include substitutions of the amino acid at position 86 with Lys and substitutions of the amino acid at position 511 with Phe. Furthermore, examples in the L protein include substitutions of Asn at position 1197 (N1197) and / or Lys at position 1795 (K1795) of the SeV L protein with other amino acids. As described above, preferred amino acid substitutions are substitutions with amino acids having chemically different side chains. Specific examples include substitutions of the amino acid at position 1197 with Ser and substitutions of the amino acid at position 1795 with Glu. Mutations in the P and L genes can significantly enhance persistent infectivity, inhibition of secondary particle release, and inhibition of cytotoxicity. Furthermore, these effects can be dramatically enhanced when combined with mutations and / or deletions in the envelope protein gene. Examples of L gene substitutions include substitutions of Tyr at position 1214 (Y1214) and / or Met at position 1602 (M1602) in the SeVL protein with other amino acids. As mentioned above, preferred amino acid substitutions are those with chemically different side chains. Specific examples include substitutions of the amino acid at position 1214 with Phe and the amino acid at position 1602 with Leu. Any combination of these mutations is acceptable.

[0116] For example, Sendai virus vectors in which at least G at position 69, T at position 116, and A at position 183 of the SeV M protein, at least A at position 262, G at position 264, and K at position 461 of the SeV HN protein, at least L at position 511 of the SeV P protein, and at least N at position 1197 and K at position 1795 of the SeV L protein are substituted with other amino acids, and the F gene is defective or deleted; and Sendai virus vectors in which the F gene is defective or deleted and has the same or lower cytotoxicity as those described above and / or has the same or higher inhibition of NTVLP formation at 37°C as those described above are particularly preferably used for expressing nuclear reprogramming factors in the present invention. Specific substitution examples include G69E, T116A, and A183S substitutions of the M protein, A262T, G264R, and K461G substitutions of the HN protein, L511F substitution of the P protein, and N1197S and K1795E substitutions of the L protein.

[0117] Mutations in the L protein also include substitutions of amino acids at any of positions selected from among position 942 (Y942), position 1361 (L1361), and position 1558 (L1558) of the SeV L protein with other amino acids. As described above, amino acid substitutions are preferably with other amino acids having different side chain chemical properties. Specific examples include substitutions of the amino acid at position 942 with His, the amino acid at position 1361 with Cys, and the amino acid at position 1558 with Ile. L proteins with substitutions at least at position 942 or 1558 are particularly preferred. For example, mutant L proteins in which position 1361 is substituted with other amino acids in addition to position 1558 are preferred. Furthermore, mutant L proteins in which position 1558 and / or 1361 are substituted with other amino acids in addition to position 942 are also preferred. These mutations can improve the temperature sensitivity of the L protein.

[0118] Examples of P protein mutations include substitutions of amino acids at any of positions selected from among position 433 (D433), position 434 (R434), and position 437 (K437) of the SeV P protein with other amino acids. As described above, amino acid substitutions are preferably with amino acids having different side chain chemical properties. Specific examples include substitutions of the amino acid at position 433 with Ala(A), the amino acid at position 434 with Ala(A), and the amino acid at position 437 with Ala(A). P proteins with substitutions at all three of these positions are particularly preferred. These mutations can improve the temperature sensitivity of the P protein.

[0119] The present invention is preferably used with a Sendai virus vector encoding a mutant P protein in which at least three amino acids, D at position 433, R at position 434, and K at position 437, are substituted with other amino acids, and a mutant L protein in which at least L at position 1558 of the SeV L protein is substituted (preferably a mutant L protein in which at least L at position 1361 is also substituted with other amino acids); and a Sendai virus vector encoding a mutant F gene having the same or lower cytotoxicity and / or the same or higher temperature sensitivity as those described above. In addition to the above mutations, each viral protein may also have mutations in other amino acids (e.g., 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid). Since the vector containing the mutations shown above shows high temperature sensitivity, after reprogramming is completed, the vector can be easily removed by culturing the cells at a normal temperature (e.g., about 37°C, particularly 36.5°C to 37.5°C, preferably 36.6°C to 37.4°C, and more preferably 36.7°C to 37.3°C). For vector removal, culturing can also be performed at a slightly higher temperature (e.g., 37.5-39°C, preferably 38-39°C or 38.5-39°C).

[0120] More specifically, Sendai virus vectors having, for example, the following mutations can be preferably used (International Publication No. WO2010 / 008054):

[0121] TS 7:L(Y942H / L1361C / L1558I);

[0122] TS 12:P(D433A / R434A / K437A);

[0123] TS 13:P(D433A / R434A / K437A),L(L1558I);

[0124] TS 14: P(D433A / R434A / K437A), L(L1361C); and

[0125] TS 15:P(D433A / R434A / K437A),L(L1361C / L1558I).

[0126] Specific vectors include, for example, a Sendai virus vector (e.g., strain Z) in which the M protein has G69E, T116A, and A183S mutations, the HN protein has A262T, G264R, and K461G mutations, the P protein has L511F mutation, and the L protein has N1197S and K1795E mutations, resulting in a deletion of the F gene. Furthermore, a vector derived by introducing a TS7, TS12, TS13, TS14, or TS15 mutation into this vector is more preferred. Specifically, examples include SeV18+ / TSΔF (WO2010 / 008054 and WO2003 / 025570) and SeV(PM) / TSΔF, as well as vectors derived by further introducing the aforementioned TS7, TS12, TS13, TS14, or TS15 mutations into these vectors, but are not limited thereto.

[0127] "TSΔF" means carrying G69E, T116A and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, as well as missing F gene.

[0128] The temperature-sensitive Sendai virus vector containing the KLF gene, OCT gene, and SOX gene in sequence in a single vector is an F gene-deleted Sendai virus vector, specifically carrying G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, an L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein. Preferably, it further has the aforementioned TS7, TS12, TS13, TS14, or TS15 mutations. More preferably, it is an F gene-deleted Sendai virus vector carrying G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, an L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, and further carrying a TS12 mutation. In Sendai virus containing KLF, OCT and SOX genes, KLF, OCT and SOX genes are preferably integrated in this order immediately after the P gene of Sendai virus, ie, immediately downstream of the P gene (just on the 5' side of the negative-strand RNA genome).

[0129] There is no limitation thereto, and specifically, SeV(PM)KOS / TS7ΔF, SeV(PM)KOS / TS12ΔF and the like are preferred vectors, and SeV(PM)KOS / TS12ΔF is more preferred.

[0130] At this time, it is preferably combined with a Sendai virus vector expressing the KLF gene (a vector carrying the KLF gene but not comprising the OCT gene and the SOX gene). The Sendai virus vector encoding the KLF gene (i.e., carrying the KLF gene but not comprising the OCT gene and the SOX gene) may or may not be temperature-sensitive, and preferably the vector has a mutation that inhibits NTVLP formation at 37°C. NTVLP formation can be measured according to the reference Inoue et al., J. Virol. 77: 3238-3246, 2003. A preferred vector is, for example, a Sendai virus vector carrying G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutations in the P protein, and N1197S and K1795E mutations in the L protein with an F gene deletion. The Sendai virus vector carrying the KLF gene but not comprising the OCT gene and the SOX gene can express the loaded gene (KLF gene) at 37°C. For this vector, a vector that has higher efficiency in expressing the loaded gene at 37°C than a vector containing the KLF gene, OCT gene, and SOX gene in sequence in one vector is used. In particular, a vector with lower temperature sensitivity (high resistance at 37°C and relatively high expression level of the loaded gene) than the temperature-sensitive Sendai virus vector containing the KLF gene, OCT gene, and SOX gene in sequence in one vector is preferred. The KLF gene can be inserted, for example, upstream of the N gene of the Sendai virus vector (to the 3' side of the N gene in the genome), and specifically, SeV18+KLF4 / TSΔF is a preferred vector.

[0131] In this case, it is preferred to combine it with a temperature-sensitive viral vector encoding the MYC gene. The temperature-sensitive viral vector encoding the MYC gene is, for example, a temperature-sensitive Sendai virus vector encoding the MYC gene. More specifically, it is a Sendai virus vector carrying the G69E, T116A, and A183S mutations in the M protein, the A262T, G264R, and K461G mutations in the HN protein, the L511F mutation in the P protein, and the N1197S and K1795E mutations in the L protein. It is also preferred that it further has the TS7, TS12, TS13, TS14, or TS15 mutations described above. More preferably, it is a Sendai virus vector carrying the G69E, T116A, and A183S mutations in the M protein, the A262T, G264R, and K461G mutations in the HN protein, the L511F mutation in the P protein, and the N1197S and K1795E mutations in the L protein, and further carrying the TS15 mutation in the F gene deletion. The MYC gene is integrated, for example, immediately after the HN gene, that is, immediately downstream of the HN gene (just 5' to the negative-strand RNA genome), for example, between the HN gene and the L gene. More specifically, it can be SeV(HNL)c-rMYC / TS12ΔF, SeV(HNL)c-rMYC / TS13ΔF, or SeV(HNL)c-rMYC / TSΔ15. Favorable results can be obtained by combining, specifically, SeV(PM)KOS / TS12ΔF, SeV(HNL)c-rMYC / TS15ΔF, and SeV18+KLF4 / TSΔF.

[0132] The cytotoxicity of the vector can be measured, for example, by quantifying lactate dehydrogenase (LDH) released from the cells. Specifically, for example, HeLa (ATCC CCL-2) or monkey CV-1 (ATCC CCL 70) is infected with an MOI of 3, and the amount of LDH released in the culture medium after 3 days of culture is measured. The lower the amount of LDH released, the lower the cytotoxicity. In addition, temperature sensitivity can be measured by measuring the rate of viral proliferation or the expression level of the gene carried at the normal temperature of the viral host (for example, 37°C to 38°C). If the virus amplification rate and / or the expression level of the gene carried are reduced compared to those without mutations, the mutation is judged to be a temperature-sensitive mutation, and the lower the proliferation rate and / or expression level, the higher the temperature sensitivity of the judgment.

[0133] In addition, when using enveloped viruses, viruses can be used that contain proteins in their envelope that are different from the envelope proteins originally carried by the virus. For example, when making a virus, by causing the virus-producing cells to express the desired exogenous envelope protein, a virus containing the exogenous envelope protein can be made. Such proteins are not particularly limited, and proteins such as the desired adhesion factors, ligands, receptors, etc. that confer the ability to infect mammalian cells can be used. Specific examples include the G protein (VSV-G) of vesicular stomatitis virus (VSV). The VSV-G protein can be derived from any VSV strain, for example, the VSV-G protein derived from the Indiana serotype strain (J. Virology 39: 519-528 (1981)) can be used, but is not limited thereto. The Sendai virus vector used in the present invention can contain any combination of envelope proteins derived from other viruses.

[0134] Reconstruction of recombinant Sendai viruses carrying nuclear reprogramming factors can be performed using known methods. Specifically, Sendai viruses can typically be produced by the following steps: (a) transcribing cDNA encoding Sendai virus genomic RNA (negative strand) or its complementary strand (positive strand) in cells expressing the viral proteins (NP, P, and L) necessary for virion formation, and (b) recovering the culture supernatant containing the generated virus. The viral proteins necessary for particle formation can be expressed from the transcribed viral genomic RNA, or they can be supplied in trans from a source other than the genomic RNA. For example, expression plasmids encoding the NP, P, and L proteins can be introduced into cells for supply. If the viral genes necessary for particle formation are missing from the genomic RNA, these viral genes can be expressed separately in the virus-producing cells to complement particle formation. To express the viral proteins or RNA genome within the cells, a vector encoding the protein or genomic RNA is introduced into the host cells, linked to a vector downstream of an appropriate promoter capable of functioning in the host cell. The transcribed genomic RNA replicates in the presence of the viral proteins and forms infectious virions. When producing a defective virus with a defective gene (such as an envelope protein gene), the defective protein, other viral proteins that can complement those proteins or their functions, etc. can be expressed in virus-producing cells.

[0135] For example, Sendai virus can be produced by the following known methods (WO97 / 16539; WO97 / 16538; WO00 / 70055; WO00 / 70070; WO01 / 18223; WO03 / 025570; WO2005 / 071092; WO2006 / 137517; WO2007 / 083644; WO2008 / 007581; Hasan, MK et al., J. Gen. Virol. 78:2813-2820, 1997; Kato, A. et al., 1997, EMBO J. 16:578-587; and Yu, D. et al., 1997, Genes Cells 2:457-466; Durbin, AP et al., 1997, Virology 235:323-332; Whelan, SP et al., 1995, Proc. Natl. Acad. Sci. USA 92: 8388-8392; Schnell. MJ et al., 1994, EMBO J. 13: 4195-4203; Radecke, F. et al., 1995, EMBO J.14:5773-5784; Lawson, ND et al., Proc.Natl.Acad.Sci.USA92:4477-4481; Garcin, D. et al., 1995, EMBO J.14:6087-6094; Kato, A. et al., 1996, Genes Cells 1:569-579; Baron, MD and Barrett, T., 1997, J. Virol. 71:1265-1271; Bridgen, A. and Elliott, RM, 1996, Proc. Natl. Acad. Sci. USA 93:15400-15404; Tokusumi, T. et al., Virus Res. 2002:86;33-38, Li, H.-O. et al., J. Virol. 2000:74;6564-6569). For other RNA virus propagation methods and methods for producing recombinant viruses, see "Uirusu-gaku Jikken-gaku Kakuron (Detailed Virology Experiments)" 2nd revised edition (National Institute of Infectious Diseases Students Institute edition, Maruzen, 1982).

[0136] In the Sendai virus described above, the KLF gene, OCT gene, and SOX gene were integrated sequentially. In another Sendai virus, the KLF gene was integrated (the OCT gene and SOX gene were not integrated into this vector). Generally, these foreign genes can be inserted immediately before (3' side on the genome) or immediately after (5' side on the genome) any viral gene (NP, P, M, F, HN, or L).

[0137] Regarding Sendai viruses containing the KLF, OCT, and SOX genes, it is preferred that the KLF, OCT, and SOX genes be integrated sequentially immediately after the Sendai virus P gene, that is, immediately downstream of the P gene (immediately 5' on the negative-strand RNA genome). For example, a transcription start signal (S sequence), a transcription termination signal (E sequence), and spacer sequences (intervening sequences (I sequences)) may be included between the P gene and the KLF gene; however, no other transcription units (e.g., transcription units encoding protein-coding genes) are included. Because the Sendai virus carries negative-strand RNA as its genome, contrary to the usual practice, the 3' end corresponds to the upstream end of the genome, and the 5' end corresponds to the downstream end. When the KLF, OCT, and SOX genes are located in this order in the Sendai virus genome, the KLF gene is located closest to the 3' end, while the SOX gene is located closest to the 5' end. Because negative-strand RNA encodes genes in the antisense orientation, the KLF, OCT, and SOX genes are encoded as the antisense strand on the genome, rather than the protein-coding strand (sense strand). When the Sendai virus genome enters cells, it uses it as a template to produce antigenomic RNA. The antigenomic RNA is the positive strand, while the KLF, OCT, and SOX genes are carried as the protein-coding strand (sense strand). Within the antigenomic RNA, the KLF, OCT, and SOX genes are positioned so that, of the three genes, the KLF gene is located closest to the 5' end, while the SOX gene is located closest to the 3' end.

[0138] These three genes are preferably adjacent to each other (i.e., no other genes exist between them). Each of these three genes can be suitably sandwiched between the Sendai virus S (start) sequence and the E (stop) sequence. The S sequence is a signal sequence that initiates transcription, while the E sequence terminates transcription. The region between the S and E sequences constitutes a single transcription unit. If appropriate, a sequence that acts as a spacer (intervening sequence) can be inserted between the E sequence of a specific gene and the S sequence of the next gene. For example, a Sendai virus vector containing a nucleic acid having the following composition can be advantageously used: S-KLF gene-EIS-OCT gene-EIS-SOX gene-E (S, I, and E refer to the S sequence, I (intervening) sequence, and E sequence, respectively). The P gene and KLF gene on the Sendai virus genome will be connected in the following manner: P gene-EIS-KLF gene-, and so on.

[0139] When the OCT gene, SOX gene, and KLF gene are located in this order, for example, a Sendai virus vector containing a nucleic acid having a genome structure of S-OCT gene-EIS-SOX gene-EIS-KLF gene-E (S, I, and E represent the S sequence, I (intervening) sequence, and E sequence, respectively) can be advantageously used. The P gene and SOX gene on the Sendai virus genome are linked in the following manner: P gene-EIS-SOX gene-, and so on.

[0140] The desired S sequence of Sendai virus can be used as the S sequence, but the 3'-UCCCWVUUWC-5' (W = A or U; V = A, C, or G) sequence (SEQ ID NO: 1) is preferably used. In particular, 3'-UCCCAGUUUC-5' (SEQ ID NO: 2), 3'-UCCCACUUAC-5' (SEQ ID NO: 3), and 3'-UCCCACUUUC-5' (SEQ ID NO: 4) are preferred. When these sequences are expressed as DNA sequences encoding the positive strand, they are 5'-AGGGTCAAAG-3' (SEQ ID NO: 5), 5'-AGGGTGAATG-3' (SEQ ID NO: 6), and 5'-AGGGTGAAAG-3' (SEQ ID NO: 7), respectively. The E sequence of the Sendai virus vector is preferably, for example, 3'-AUUCUUUUU-5' (5'-TAAGAAAAA-3' for DNA encoding the positive strand). The I sequence can be, for example, any three bases, and specifically, 3'-GAA-5' (5'-CTT-3' in positive-strand DNA) can be used.

[0141] The genome of wild-type Sendai virus comprises, in sequence, the nucleocapsid (N) gene, phosphorus (P) gene, matrix (M) gene, fusion (F) gene, hemagglutinin-neuraminidase (HN) gene and large (L) gene, as well as a short 5'-followed sequence (trailer) region after the 3' short leader region. In Sendai virus vectors, viral genes can also be configured in this order. The preparation of recombinant vectors and various mutant vectors comparable to wild-type viruses is known. It has also been shown that gene introduction can be achieved using a single RNP without its envelope (WO00 / 70055). Therefore, Sendai virus RNP can be used as a viral vector for reprogramming.

[0142] If Sendai virus carries NP gene, P gene and L gene, it is enough to function as a vector. They can replicate the genome and express the exogenous genes (KLF, OCT, SOX, etc.) carried in cells. In Sendai virus that carries NP gene, P gene and L gene as viral genes, the group of KLF, OCT and SOX genes is inserted, for example, between the P gene and the L gene. In the vector containing M gene, the group of KLF, OCT and SOX genes is inserted, for example, between the P gene and the M gene (WO97 / 16539). When the F gene is included in the Sendai virus vector with M gene deletion, the group of KLF, OCT and SOX genes is inserted between the P gene and the F gene (WO00 / 70070). For Sendai virus vectors with deletion of M and F genes, the position of insertion is between the P gene and the HN gene; and for Sendai virus vectors with deletion of F, M and HN genes, the position of insertion is between the P gene and the L gene (WO2003 / 025570, WO 2006 / 137517). Vectors with deleted viral genes are preferred because they are very safe. In the present invention, vectors with at least the F gene deleted can be preferably used.

[0143] Furthermore, for a vector containing the KLF gene but not the OCT gene and the SOX gene, the KLF gene is inserted, for example, upstream of the NP gene (between the 3' leader sequence and the NP gene).

[0144] The above-mentioned Sendai virus vector containing KLF, OCT and SOX genes and the Sendai virus vector containing KLF gene but not containing OCT gene and SOX gene can be suitably used for gene delivery in reprogramming, but they are more preferably used for reprogramming that also involves MYC gene introduction. The vector expressing Glis1 gene (Maekawa et al., Nature, 474:225-229, 2011) can also be combined to replace MYC gene. MYC gene or Glis1 gene can be inserted into the above-mentioned Sendai virus containing KLF, OCT and SOX genes or the Sendai virus vector containing KLF gene but not containing OCT gene and SOX gene, but can also be used after being inserted into other vectors. When MYC gene or Glis1 gene is inserted into other vectors, desired vectors such as plasmids, viral vectors and non-viral vectors (such as liposomes) can be used. Examples of viral vectors include adenoviral vectors and retroviral vectors, but are not limited thereto. More preferably, MYC gene or Glis1 gene is inserted into Sendai virus vector. In the present invention, the Sendai virus vector containing the KLF, OCT, and SOX genes and the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene are preferably used in combination with another Sendai virus vector into which the MYC gene or the Glis1 gene is inserted. The Sendai virus vector can be used as a starting Sendai virus vector to insert the MYC gene or the Glis1 gene.

[0145] When MYC gene or Glis1 gene are inserted into Sendai virus genome, the gene insertion position in the vector can select desired site. For example, MYC gene or Glis1 gene are preferably located at the rear (5 ' side) of negative strand RNA genome, for example, closer to 5 ' end than the center of negative strand RNA virus genome (position is closer to 5 ' end than the gene at the center). In other words, in a plurality of protein coding sequences located on genome, it is preferably located at a position (seeing embodiments) closer to 3 ' end than 5 ' end. MYC gene or Glis1 gene can be located at, for example, the most 5 ' side (i.e., the first position from 5 ' end), or the second or third position from 5 ' end. MYC gene or Glis1 gene can be located at the second position from 5 ' end of genome, specifically, when L gene is located at the most 5 ' side of genome, followed by HN gene, it can be located between HN gene and L gene (between HN-L). Specifically, it is preferred to insert the MYC gene or Glis1 gene immediately upstream (3' side, for example, between the HN gene and the L gene) or immediately downstream (5' side, for example, between the L gene and the 5'-following sequence) of the L gene in the Sendai virus genome. Most preferred are Sendai virus vectors in which the MYC gene has been inserted between the HN gene and the L gene. The Sendai virus vector may be, for example, an F gene-deleted Sendai virus vector (e.g., strain Z) in which the M protein has G69E, T116A, and A183S mutations, the HN protein has A262T, G264R, and K461G mutations, the P protein has an L511F mutation, and the L protein has N1197S and K1795E mutations; and a vector produced by further introducing a TS7, TS12, TS13, TS14, or TS1 mutation into this vector is more preferred.

[0146] The MYC gene, including not only wild-type c-MYC but also T58A mutants, N-MYC and L-MYC, can induce pluripotent stem cells (WO2007 / 69666; Blelloch R. et al., Cell Stem Cell, 1: 245-247, 2007). Thus, since family genes can be selected and used in various ways, MYC family genes can be appropriately selected to induce reprogramming. In addition, continuous A or T nucleotide sequences in the MYC gene can be replaced by appropriately introducing silent mutations that do not change the encoded amino acid sequence.

[0147] For example, it has been found that the amount of wild-type c-MYC expressed by RNA viral vectors such as Sendai virus vectors is small. However, by introducing one or more, preferably two or more, three or more, or four or more, or all five mutations selected from a378g, t1122c, t1125c, a1191g, and a1194g into wild-type c-MYC, the gene can be stably and highly expressed from the vector. In the present invention, for example, a modified c-MYC gene (referred to as "c-rMYC") shown in SEQ ID NO: 8 (SEQ ID NO: 9 is an amino acid sequence) can be appropriately used. Specifically, examples include SeV(HNL)c-rMYC / TSΔF, SeV(L)c-rMYC / TSΔF, SeV(HNL)c-rMYC / TS15ΔF (WO2010 / 008054; Fusaki et al., Proc Jpn Acad Ser B Phys Biol Sci. Vol. 85, p348-362 (2009)), and SeV(HNL)c-rMYC / TS15ΔF is particularly preferred, but is not limited thereto.

[0148] When appropriate, genes for cell reprogramming can be further carried in the above-mentioned Sendai virus vectors carrying KLF, OCT, and SOX genes, Sendai virus vectors containing KLF genes but not OCT and SOX genes, and / or Sendai virus vectors containing MYC genes. In addition, other vectors carrying genes for cell reprogramming can also be combined with the above-mentioned Sendai virus vectors. The genes to be carried can be desired genes involved in the induction of various stem cells such as induced pluripotent stem cells from differentiated cells. For example, genes that increase reprogramming efficiency can be carried.

[0149] The present invention provides the Sendai virus vector of the present invention for the purpose of introducing a gene in cell reprogramming, and these vectors are used for expressing reprogramming factors in cells to induce the reprogramming of these cells. In addition, the present invention provides agents (transfer agents, gene transfer agents) containing the Sendai virus vector of the present invention for introducing a gene in cell reprogramming, and agents for expressing reprogramming factors in cells containing these vectors. In addition, the present invention relates to agents for expressing reprogramming factors in cells to induce the reprogramming of the cells containing the Sendai virus vector of the present invention. In addition, when implementing the reprogramming of cells, the vector of the present invention can also be used to express desired genes in these cells. The Sendai virus vector of the present invention can be used for cell reprogramming according to the present invention. The induction of reprogramming can specifically be the induction of pluripotent stem cells. The present invention can be used for medical and non-medical uses, and can be used in medical and non-medical embodiments. For example, the present invention can be used for treatment, surgery, and / or diagnostic purposes, or non-treatment, non-surgery, and / or non-diagnostic purposes.

[0150] In the present invention, nuclear reprogramming factors refer to genes or their products that can be used alone or in combination with multiple factors to induce the differentiated state of a certain cell to a relatively undifferentiated state, including, for example, genes or their products for inducing dedifferentiation of differentiated cells. In the present invention, nuclear reprogramming factors (a nuclear reprogramming factor) include: factors necessary for nuclear reprogramming and auxiliary factors (auxiliary factors) that improve the efficiency of nuclear reprogramming. In the present invention, the vector can be made to carry the desired gene for nuclear reprogramming. For example, genes for the manufacture of pluripotent stem cells can be further loaded. Specifically, as nuclear reprogramming factors for inducing pluripotent stem cells, for example, genes (ES cell-specific genes, etc.) that are expressed in ES cells, early embryos, etc., but are not expressed or expressed in a variety of somatic cells that are differentiated are reduced. Such genes are preferably genes encoding transcription factors, nuclear proteins, etc. Methods for identifying nuclear reprogramming genes are known (WO 2005 / 80598), and indeed, genes identified using this method have been shown to be useful for reprogramming into pluripotent stem cells (WO 2007 / 69666).

[0151] Examples of such genes include DPPA5 (developmental pluripotency-associated 5, ES cell-specific gene 1 (ESG1); accession numbers NM_001025290, NM_025274, XM_236761), F-box protein 15 (Fbx15, NM_152676, NM_015798), Nanog (NM_024865, AB093574), ECAT1 (ES cell-associated transcript 1; AB211062, AB211060), ERAS (ES cell-expressed Ras; NM_181532, NM_181548), DNMT3L (DNA (cytosine-5-)- Methyltransferase 3-like; NM_013369, NM_019448), ECAT8 (AB211063, AB211061), GDF3 (growth differentiation factor 3; NM_020634, NM_008108), SOX15 (SRY (sex determining region Y)-box 15; NM_006942, NM_009235), DPPA4 (developmental pluripotency associated 4; NM_018189, NM_028610), DPPA2 (NM_138815, NM_028615), FTHL17 (transferrin, heavy polypeptide-like 17; NM_031894, NM_031261), SAL L4 (sal-like 4; NM_020436, NM_175303), OCT3 / 4 (also known as POU5F1; NM_002701, NM_203289, NM_013633, NM_001009178), SOX2 (NM_003106, NM_011443, XM_574919), Rex-1 (ZFP42 (zinc finger protein 42 homolog); NM_174900, NM_009556), Utf1 (undifferentiated embryonic cell transcription factor 1; NM_003577, NM_009482), TCL1A (T-cell leukemia / lymphoma 1A; NM_021966), , NM_009337), DPPA3 (also known as Stella, NM_199286, NM_139218, XM_216263), KLF4 (Kruppel-like factor 4; NM_004235, NM_010637), catenin β1 (cadherin-related protein β1; NM_001904, NM_007614; containing S33Y mutant), c-MYC (NM_002467, NM_010849; containing T58A mutant), STAT3 (signal transducer and activator of transcription 3; NM_139276, NM_213659), GRB2 (growth factor receptor binding protein 2;NM_002086, NM_008163), Glis1 gene (Maekawa et al., Nature, 474: 225-229, 2011; NM_147193, NM_147221) and genes of other members of the family to which these genes belong. When these genes are introduced into cells, pluripotent stem cells can be induced (WO2007 / 69666). Therefore, Sendai virus vectors comprising KLF, OCT and SOX genes, Sendai virus vectors containing KLF genes but not containing OCT genes and SOX genes, and / or Sendai virus vectors containing MYC genes can be further loaded with genes of the above genes that have not yet been loaded into the vector. Alternatively, other vectors carrying these genes can be used in combination with these Sendai virus vectors. These genes can be respectively grouped into different vectors, or multiple genes can be grouped together into a single vector. In addition, each gene can be incorporated into a single vector, or different types of vectors (including chromosomal integration viral vectors and / or non-viral vectors) can be used in combination with the Sendai viral vector of the present invention comprising the KLF gene, OCT gene, and SOX gene, or the Sendai viral vector containing the KLF gene but not the OCT gene and the SOX gene. In addition, each viral vector can be packaged separately and used in combination when used. Alternatively, multiple viral vectors carrying different genes can be pre-combined into a kit or mixed into a composition. In addition, in the reprogramming of cells, especially the production of pluripotent stem cells, it is also preferred to use one or more non-integrating viral vectors containing any combination (or all) of these genes, as well as a kit or composition containing the vector. In the case of a composition, the vector can be suitably combined with a pharmaceutically acceptable carrier and / or vehicle, for example, mixed in sterile water, pH buffer, physiological saline, culture medium, etc. In these systems, part or most of the nuclear reprogramming gene can also be replaced with its expression product as a protein. More specifically, the compositions and kits of the present invention may also contain other vectors (chromosomally integrative viral vectors and / or non-viral vectors) expressing reprogramming factors and / or compounds, proteins, etc. that induce reprogramming, as long as they include the above-mentioned Sendai virus vector containing the KLF gene, the OCT gene, and the SOX gene, or a Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene. Preferably, all the necessary factors for reprogramming are expressed by the Sendai virus vector;However, only a part may be expressed by the Sendai virus vector while the other part may be supplied by other vectors and / or compounds (such as proteins or low molecular weight compounds). In addition, the method for producing reprogrammed cells of the present invention is not limited to a method in which all gene introductions are carried out using Sendai virus vectors. More specifically, the method of the present invention only requires the use of the above-mentioned Sendai virus vectors containing at least the KLF gene, the OCT gene, and the SOX gene, and the Sendai virus vectors containing the KLF gene but not the OCT gene and the SOX gene, and also includes the combined use of other vectors (chromosomal integration virus vectors and / or non-viral vectors) expressing reprogramming factors and / or compounds that induce reprogramming. Preferably, they are used in combination with the above-mentioned Sendai virus vectors containing the MYC gene or the Glis1 gene. ;

[0152] The present invention also provides the following:

[0153] [1] A method for improving the efficiency of inducing pluripotent stem cells (including improving the efficiency of producing pluripotent stem cells) in a method for inducing pluripotent stem cells by introducing a vector containing the KLF gene, the OCT gene, and the SOX gene in sequence in one vector (including a method for producing pluripotent stem cells), wherein the method comprises the step of further introducing a vector containing the KLF gene but not containing the OCT gene and the SOX gene;

[0154] [2] The method of [1], wherein the vector comprising the KLF gene, the OCT gene and the SOX gene in sequence is a temperature-sensitive viral vector;

[0155] [3] The method of [2], wherein the temperature-sensitive viral vector is a temperature-sensitive Sendai virus vector;

[0156] [4][3] The method of claim 4, wherein the temperature-sensitive Sendai virus vector is an F gene-deleted Sendai virus vector comprising mutations selected from the group consisting of TS 7 (Y942H, L1361C, and L1558I mutations in L protein), TS 12 (D433A, R434A, and K437A mutations in P protein), TS 13 (D433A, R434A, and K437A mutations in P protein and L1558I mutation in L protein), TS 14 (D433A, R434A, and K437A mutations in P protein and L1361C in L protein), and TS 15 (D433A, R434A, and K437A mutations in P protein and L1361C and L1558I mutations in L protein).

[0157] [5] The method of [4], wherein the temperature-sensitive Sendai virus vector is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, and further comprising TS 12 mutations (D433A, R434A, and K437A mutations in the P protein);

[0158] [6] The method of any one of [3] to [5], wherein the KLF gene, the OCT gene, and the SOX gene are integrated in this order immediately after the P gene of Sendai virus (on the 5′ side of the P gene of the negative-strand RNA genome);

[0159] [7] The method of [3], wherein the temperature-sensitive Sendai virus vector is selected from the group consisting of SeV(PM)KOS / TS7ΔF and SeV(PM)KOS / TS12ΔF;

[0160] [8] The method of any one of [1] to [7], wherein the vector containing the KLF gene but not the OCT gene and the SOX gene is a Sendai virus vector;

[0161] [9][8], wherein the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene is a Sendai virus vector having a mutation that inhibits NTVLP formation at 37°C;

[0162]

[10] The method of [8], wherein the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene is an F gene-deficient Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein;

[0163]

[11] The method of any one of [8] to

[10] , wherein the KLF gene is integrated upstream of the N gene (on the 3' side of the N gene in the negative-strand RNA genome);

[0164]

[12] [8], wherein the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene is SeV18+KLF4 / TSΔF;

[0165]

[13] The method according to any one of [1] to

[12] , further comprising the step of introducing a vector into which the MYC gene is inserted;

[0166]

[14]

[13] , wherein the vector into which the MYC gene is inserted is a Sendai virus vector;

[0167]

[15]

[14] , wherein the Sendai virus vector inserted with the MYC gene is a temperature-sensitive Sendai virus vector;

[0168]

[16] The method of

[15] , wherein the temperature-sensitive Sendai virus vector inserted with the MYC gene is an F gene-deleted Sendai virus vector comprising a mutation selected from the group consisting of TS 7, TS 12, TS 13, TS 14, and TS 15;

[0169]

[17] The method of

[16] , wherein the temperature-sensitive Sendai virus vector inserted with the MYC gene is an F gene-deleted Sendai virus vector comprising G69E, T116A and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, and further comprising TS15 mutations (D433A, R434A and K437A mutations in the P protein, and L1361C and L1558I mutations in the L protein);

[0170]

[18] The method of any one of

[14] to

[17] , wherein the MYC gene is integrated immediately before the L gene (on the 3' side of the negative-strand RNA genome);

[0171]

[19] The method of

[14] , wherein the temperature-sensitive Sendai virus vector into which the MYC gene is inserted is SeV(HNL)c-rMYC / TS15ΔF;

[0172]

[20] The method of any one of

[13] to

[19] , wherein the MYC gene is c-rMyc;

[0173]

[21] The method of any one of [1] to

[20] , which does not involve culturing at low temperature; and

[0174]

[22] The method of

[21] , wherein the culturing is performed at about 37°C.

[0175] The present invention also provides the following:

[0176] [1] Use of a vector comprising the KLF gene but not the OCT gene and the SOX gene in the manufacture of a medicament for improving the efficiency of inducing pluripotent stem cells (including improving the efficiency of generating pluripotent stem cells) in a method for inducing pluripotent stem cells by introducing a vector comprising the KLF gene, the OCT gene and the SOX gene in sequence in one vector (including a method for generating pluripotent stem cells);

[0177] [2] The use of [1], wherein the vector sequentially comprising the KLF gene, the OCT gene and the SOX gene is a temperature-sensitive viral vector;

[0178] [3] The use of [2], wherein the temperature-sensitive viral vector is a temperature-sensitive Sendai virus vector;

[0179] [4] The use of [3], wherein the temperature-sensitive Sendai virus vector is an F gene-deleted Sendai virus vector comprising a mutation selected from the group consisting of TS 7, TS 12, TS 13, TS 14, and TS 15;

[0180] [5][4] The use of temperature-sensitive Sendai virus vector is an F gene-deleted Sendai virus vector comprising G69E, T116A and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, D433A, R434A, K437A, and L511F mutations in the P protein, and N1197S and K1795E mutations in the L protein, and further comprising TS 12 mutation (D433A, R434A and K437A mutations in the P protein);

[0181] [6] The use of any one of [3] to [5], wherein the KLF gene, OCT gene and SOX gene are integrated in this order immediately after the P gene of Sendai virus (on the 5' side of the P gene of the negative-strand RNA genome);

[0182] [7] The use of [3], wherein the temperature-sensitive Sendai virus vector is selected from the group consisting of SeV(PM)KOS / TS7ΔF and SeV(PM)KOS / TS12ΔF;

[0183] [8] The use of any one of [1] to [7], wherein the vector containing the KLF gene but not the OCT gene and the SOX gene is a Sendai virus vector;

[0184] [9][8], wherein the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene is a Sendai virus vector having a mutation that inhibits NTVLP formation at 37°C;

[0185]

[10] [8] The use of, wherein the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene is an F gene-deficient Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein;

[0186]

[11] The use of any one of [8] to

[10] , wherein the KLF gene is integrated upstream of the N gene (on the 3' side of the N gene in the negative-strand RNA genome);

[0187]

[12] [8], wherein the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene is SeV18+KLF4 / TSΔF;

[0188]

[13] The use of any one of [1] to

[12] , further comprising the step of introducing a vector into which the MYC gene is inserted;

[0189]

[14] The use of

[13] , wherein the vector having the MYC gene inserted therein is a Sendai virus vector;

[0190]

[15]

[14] The use of, wherein the Sendai virus vector having the MYC gene inserted therein is a temperature-sensitive Sendai virus vector;

[0191]

[16]

[15] The use of the temperature-sensitive Sendai virus vector inserted with the MYC gene is an F gene-deleted Sendai virus vector comprising a mutation selected from the group consisting of TS 7, TS 12, TS 13, TS 14 and TS 15;

[0192]

[17]

[16] The use of a temperature-sensitive Sendai virus vector inserted with a MYC gene is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, and further comprising TS15 mutations (D433A, R434A, and K437A mutations in the P protein, and L1361C and L1558I mutations in the L protein);

[0193]

[18] The use of any one of

[14] to

[17] , wherein the MYC gene is integrated immediately before the L gene (on the 3' side of the negative-strand RNA genome);

[0194]

[19] The use of

[14] , wherein the temperature-sensitive Sendai virus vector having the MYC gene inserted therein is SeV(HNL)c-rMYC / TS15ΔF;

[0195]

[20] The use of any one of

[13] to

[19] , wherein the MYC gene is c-rMyc;

[0196]

[21] The use of any one of [1] to

[20] , which does not involve culturing at low temperature during induction and / or production; and

[0197]

[22] The use of

[21] , wherein the culturing is performed at about 37°C.

[0198] The present invention also provides the following:

[0199] [1] A composition and kit for inducing pluripotent stem cells (including generating pluripotent stem cells), comprising (a) a vector comprising a KLF gene, an OCT gene, and a SOX gene in sequence in one vector and (b) a vector comprising a KLF gene but not comprising an OCT gene and a SOX gene;

[0200] [2] The composition and kit of [1], wherein the vector sequentially comprising the KLF gene, the OCT gene and the SOX gene is a temperature-sensitive viral vector;

[0201] [3] The composition and kit of [1], wherein the temperature-sensitive viral vector is a temperature-sensitive Sendai virus vector;

[0202] [4] The composition and kit of [3], wherein the temperature-sensitive Sendai virus vector is an F gene-deleted Sendai virus vector comprising a mutation selected from the group consisting of TS 7, TS 12, TS 13, TS 14, and TS 15;

[0203] [5][4] The composition and kit of [5], wherein the temperature-sensitive Sendai virus vector is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, and further comprising TS 12 mutations (D433A, R434A, and K437A mutations in the P protein);

[0204] [6] The composition and kit of any one of [3] to [5], wherein the KLF gene, OCT gene and SOX gene are integrated in this order immediately after the P gene of Sendai virus (on the 5′ side of the P gene of the negative-strand RNA genome);

[0205] [7] The composition and kit of [3], wherein the temperature-sensitive Sendai virus vector is selected from the group consisting of SeV(PM)KOS / TS7ΔF and SeV(PM)KOS / TS12ΔF;

[0206] [8] The composition and kit of any one of [1] to [7], wherein the vector containing the KLF gene but not the OCT gene and the SOX gene is a Sendai virus vector;

[0207] [9][8] The composition and kit of [8], wherein the Sendai virus vector comprising the KLF gene but not the OCT gene and the SOX gene is a Sendai virus vector having a mutation that inhibits NTVLP formation at 37°C;

[0208]

[10] [8] The composition and kit, wherein the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene is an F gene-deficient Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein;

[0209]

[11] The composition and kit of any one of [8] to

[10] , wherein the KLF gene is integrated upstream of the N gene (on the 3' side of the N gene in the negative-strand RNA genome);

[0210]

[12] [8] The composition and kit, wherein the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene is SeV18+KLF4 / TSΔF;

[0211]

[13] The composition and kit of any one of [1] to

[12] , further comprising a vector into which the MYC gene is inserted;

[0212]

[14] The composition and kit of

[13] , wherein the vector having the MYC gene inserted therein is a Sendai virus vector;

[0213]

[15] The composition and kit of

[14] , wherein the Sendai virus vector having the MYC gene inserted therein is a temperature-sensitive Sendai virus vector;

[0214]

[16] The composition and kit of

[15] , wherein the temperature-sensitive Sendai virus vector inserted with the MYC gene is an F gene-deleted Sendai virus vector comprising a mutation selected from the group consisting of TS 7, TS 12, TS 13, TS 14 and TS15;

[0215]

[17]

[16] The composition and kit of, wherein the temperature-sensitive Sendai virus vector inserted with the MYC gene is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, and further comprising TS 15 mutations (D433A, R434A, and K437A mutations in the P protein, and L1361C and L1558I mutations in the L protein);

[0216]

[18] The composition and kit of any one of

[14] to

[17] , wherein the MYC gene is integrated immediately before the L gene (on the 3' side of the negative-strand RNA genome);

[0217]

[19] The composition and kit of

[14] , wherein the temperature-sensitive Sendai virus vector having the MYC gene inserted therein is SeV(HNL)c-rMYC / TS15ΔF;

[0218]

[20] The composition and kit of any one of

[13] to

[19] , wherein the MYC gene is c-rMyc;

[0219]

[21] The composition and kit of any one of [1] to

[20] , which does not involve culturing at low temperature during induction and / or production; and

[0220]

[22] The composition and kit of

[21] , wherein the culture is performed at about 37°C.

[0221] The present invention also provides the following:

[0222] [1] A composition and kit for improving the efficiency of inducing pluripotent stem cells (including improving the efficiency of generating pluripotent stem cells), comprising (a) a vector comprising the KLF gene, the OCT gene, and the SOX gene in sequence in one vector and (b) a vector comprising the KLF gene but not the OCT gene and the SOX gene;

[0223] [2] The composition and kit of [1], wherein the vector sequentially comprising the KLF gene, the OCT gene and the SOX gene is a temperature-sensitive viral vector;

[0224] [3] The composition and kit of [1], wherein the temperature-sensitive viral vector is a temperature-sensitive Sendai virus vector;

[0225] [4] The composition and kit of [3], wherein the temperature-sensitive Sendai virus vector is an F gene-deleted Sendai virus vector comprising a mutation selected from the group consisting of TS 7, TS 12, TS 13, TS 14, and TS 15;

[0226] [5][4] The composition and kit of [5], wherein the temperature-sensitive Sendai virus vector is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, and further comprising TS 12 mutations (D433A, R434A, and K437A mutations in the P protein);

[0227] [6] The composition and kit of any one of [3] to [5], wherein the KLF gene, OCT gene and SOX gene are integrated in this order immediately after the P gene of Sendai virus (on the 5′ side of the P gene of the negative-strand RNA genome);

[0228] [7] The composition and kit of [3], wherein the temperature-sensitive Sendai virus vector is selected from the group consisting of SeV(PM)KOS / TS7ΔF and SeV(PM)KOS / TS12ΔF;

[0229] [8] The composition and kit of any one of [1] to [7], wherein the vector containing the KLF gene but not the OCT gene and the SOX gene is a Sendai virus vector;

[0230] [9][8] The composition and kit of [8], wherein the Sendai virus vector comprising the KLF gene but not the OCT gene and the SOX gene is a Sendai virus vector having a mutation that inhibits NTVLP formation at 37°C;

[0231]

[10] [8] The composition and kit, wherein the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene is an F gene-deficient Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein;

[0232]

[11] The composition and kit of any one of [8] to

[10] , wherein the KLF gene is integrated upstream of the N gene (on the 3' side of the N gene in the negative-strand RNA genome);

[0233]

[12] [8] The composition and kit, wherein the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene is SeV18+KLF4 / TSΔF;

[0234]

[13] The composition and kit of any one of [1] to

[12] , further comprising using a vector into which the MYC gene is inserted;

[0235]

[14] The composition and kit of

[13] , wherein the vector having the MYC gene inserted therein is a Sendai virus vector;

[0236]

[15] The composition and kit of

[14] , wherein the Sendai virus vector having the MYC gene inserted therein is a temperature-sensitive Sendai virus vector;

[0237]

[16] The composition and kit of

[15] , wherein the temperature-sensitive Sendai virus vector inserted with the MYC gene is an F gene-deleted Sendai virus vector comprising a mutation selected from the group consisting of TS 7, TS 12, TS 13, TS 14 and TS15;

[0238]

[17]

[16] The composition and kit of, wherein the temperature-sensitive Sendai virus vector inserted with the MYC gene is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, and further comprising TS 15 mutations (D433A, R434A, and K437A mutations in the P protein, and L1361C and L1558I mutations in the L protein);

[0239]

[18] The composition and kit of any one of

[14] to

[17] , wherein the MYC gene is integrated immediately before the L gene (on the 3' side of the negative-strand RNA genome);

[0240]

[19] The composition and kit of

[14] , wherein the temperature-sensitive Sendai virus vector having the MYC gene inserted therein is SeV(HNL)c-rMYC / TS15ΔF;

[0241]

[20] The composition and kit of any one of

[13] to

[19] , wherein the MYC gene is c-rMyc;

[0242]

[21] The composition and kit of any one of [1] to

[20] , which does not involve culturing at low temperature during induction and / or production; and

[0243]

[22] The composition and kit of

[21] , wherein the culture is performed at about 37°C.

[0244] In the present invention described herein, a preferred combination is a vector in which the KLF, OCT, and SOX genes are inserted in this order into a single Sendai virus vector (preferably SeV(PM)KOS / TS12ΔF) and a Sendai virus vector containing the KLF4 gene but not the OCT and SOX genes (preferably SeV18+KLF4 / TSΔF). In this case, it is more preferred to further combine this with a Sendai virus vector expressing the MYC gene (preferably SeV(HNL)c-rMYC / TS15ΔF).

[0245] The factors (KLF gene, OCT gene, SOX gene, MYC gene etc.) that are imported can be suitably selected according to the source of the cell to be reprogrammed, and they can be derived from humans or other mammals such as mice, rats, pigs, or primates such as monkeys. In addition, genes and protein sequences do not necessarily have to be wild-type sequences, as long as they can induce reprogramming, they can have mutations. The example of using mutant genes to produce pluripotent stem cells is known (WO 2007 / 69666). For example, encoding has one or a few (such as several, no more than 3, no more than 5, no more than 10, no more than 15, no more than 20 or no more than 25) amino acid additions, deletions, substitutions and / or insertions, and genes that can induce the amino acid sequence of reprogramming can be used in the present invention. In addition, as long as the biological activity (ability to induce reprogramming) is maintained, polypeptides can be used, for example, in which one to several residues (e.g., 2, 3, 4, 5, 6, 10, 15, or 20 residues) of amino acids are deleted or added at the N-terminus and / or C-terminus, or in which one to several residues (e.g., 2, 3, 4, 5, 6, 10, 15, or 20 residues) of amino acids are substituted. Variants that can be used include, for example, fragments, analogs, and derivatives of native proteins, as well as fusion proteins of native proteins with other polypeptides (e.g., those with added heterologous signal peptides or antibody fragments). Specifically, polypeptides are included that contain an amino acid sequence with one or more amino acid substitutions, deletions, and / or additions in the wild-type amino acid sequence and have biological activity equivalent to that of the wild-type protein (e.g., activity to induce reprogramming). When a fragment of a wild-type protein is used, typically, the fragment contains a continuous region that is greater than 70%, preferably greater than 80%, greater than 85%, more preferably greater than 90%, greater than 95%, or greater than 98% of the wild-type polypeptide (mature form in the case of a secreted protein).

[0246] Amino acid sequence variants can be prepared, for example, by introducing mutations into the DNA encoding the native polypeptide (Walker and Gaastra, eds. Techniques in Molecular Biology (MacMillan Publishing Company, New York, 1983); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492, 1985; Kunkel et al., Methods Enzymol. 154:367-382, 1987; Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, NY), 1989; US Pat. No. 4,873,192). Guidance for making amino acid substitutions without affecting biological activity includes the report by Dayhoff et al. (Dayhoff et al., in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), 1978).

[0247] There is no particular limitation on the number of amino acids to be modified, but for example, it should be less than 30%, preferably less than 25%, more preferably less than 20%, more preferably less than 15%, more preferably less than 10%, less than 5%, or less than 3% of the total amino acids in the naturally occurring mature polypeptide, and for example, less than 15 amino acids, preferably less than 10 amino acids, more preferably less than 8 amino acids, more preferably less than 5 amino acids, and more preferably less than 3 amino acids. When amino acid substitutions are made, it is expected that the activity of the protein will be maintained by substituting amino acids with similar side chain properties. Such substitutions are referred to as conservative substitutions in the present invention. Examples of conservative substitutions include substitutions between amino acids in the following groups: basic amino acids (e.g., lysine, arginine, histidine), acidic amino acids (e.g., aspartic acid, glutamic acid), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched-chain amino acids (e.g., threonine, valine, isoleucine), and aromatic amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine). Furthermore, substitutions between amino acids that have a positive relationship in the BLOSUM62 substitution matrix (S. Henikoff and J.G. Henikoff, Proc. Acad. Natl. Sci. USA 89: 10915-10919, 1992) can be cited.

[0248] The modified protein exhibits high homology to the amino acid sequence of the wild-type protein. High homology refers to amino acid sequences having, for example, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 93% or greater, 95% or greater, or 96% or greater identity. Amino acid sequence identity can be confirmed using, for example, the BLASTP program (Altschul, SF et al., J. Mol. Biol. 215:403-410, 1990). For example, a search can be performed on the BLAST website of NCBI (National Center for Biotechnology Information) using default parameters (Altschul SF et al., Nature Genet. 3: 266-272, 1993; Madden, TL et al., Meth. Enzymol. 266: 131-141, 1996; Altschul SF et al., Nucleic Acids Res. 25: 3389-3402, 1997; Zhang J. & Madden TL, Genome Res. 7: 649-656, 1997). For example, the Blast2 sequence program (Tatiana A et al., FEMS Microbiol Lett. 174: 247-250, 1999) for comparing two sequences can be used to create an alignment of the two sequences and determine sequence identity. Gaps and mismatches are treated similarly, and the identity value is calculated, for example, relative to the entire amino acid sequence of the native cytokine (the mature form after secretion). Specifically, the ratio of the number of identical amino acids to the total number of amino acids in the wild-type protein (the mature form in the case of a secreted protein) is calculated.

[0249] Furthermore, silent mutations can be introduced into genes, leaving the encoded amino acid sequence unchanged. In particular, in AT-rich genes, replacing five or more consecutive A or T nucleotides with G or C without altering the encoded amino acid sequence can achieve stable, high gene expression.

[0250] Examples of modified proteins or proteins used for reprogramming include proteins encoded by nucleic acids that hybridize under stringent conditions with a portion or all of the coding region of a gene encoding a wild-type protein and have activity (reprogramming-inducing activity) equivalent to that of the wild-type protein. In hybridization, for example, a probe can be prepared from either a nucleic acid containing the coding region sequence of the wild-type protein gene or its complementary sequence, or from a nucleic acid serving as a target for hybridization, and identification can be performed by detecting whether it hybridizes with the other nucleic acid. Stringent hybridization conditions include, for example, hybridization in a solution containing 5xSSC, 7% (w / v) SDS, 100 μg / ml denatured salmon sperm DNA, and 5xDenhardt's solution (1xDenhardt's solution containing 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% Ficoll) at 60°C, preferably 65°C, and more preferably 68°C, followed by washing with shaking in 2xSSC, preferably 1xSSC, more preferably 0.5xSSC, and even more preferably 0.1xSSC for 2 hours at the same temperature as for hybridization.

[0251] Examples of genes particularly preferred for inducing cell reprogramming generally include: F-box protein 15 (Fbx15, NM_152676, NM_015798), Nanog (NM_024865, AB093574), ERAS (ES cell-expressed Ras; NM_181532, NM_181548), DPPA2 (NM_138815, NM_028615), OCT3 / 4 (also known as POU5F1; NM_00 2701, NM_203289, NM_013633, NM_001009178), SOX2 (NM_003106, NM_011443, XM_574919), TCL1A (T-cell leukemia / lymphoma 1A; NM_021966, NM_009337), KLF4 (Kruppel-like factor 4; NM_004235, NM_010637), calnexin β1 (calnexin-related protein beta 1; NM_001904, NM_007614; including S33Y mutants), c-MYC (NM_002467, NM_010849; including T58A mutants), and Glis1 genes (Maekawa et al., Nature, 474:225-229, 2011; NM_147193, NM_147221), as well as genes of other members of the gene family to which these genes belong. It has been reported that when these genes are introduced, the proportion of colonies exhibiting induced pluripotent stem cell morphology is higher than when four genes (OCT3 / 4, SOX2, KLF4, and c-MYC) are introduced (WO2007 / 69666). Therefore, in addition to the Sendai viral vectors of the present invention that carry the KLF gene, the OCT gene, and the SOX gene, and vectors that contain the KLF gene but do not contain the OCT gene and the SOX gene, Sendai viral vectors that further carry any one or a combination of the aforementioned genes in addition to the KLF gene, the OCT gene, and the SOX gene, Sendai viral vectors that carry genes other than the OCT gene and the SOX gene in addition to the KFL gene, or Sendai viral vectors that carry any one or a combination of the aforementioned genes can be used to induce cell reprogramming in the present invention, and are particularly advantageous for inducing pluripotent stem cells. The various viral vectors can be combined at the time of use. Furthermore, they can be pre-formed into a kit or mixed together to form a composition. Furthermore, the present invention also includes methods for introducing one or more Sendai viral vectors containing any (or all) of the aforementioned genes in any combination, in addition to vectors that sequentially contain the KFL gene, the OCT gene, and the SOX gene, and vectors that contain the KLF gene but do not contain the OCT gene and the SOX gene, as well as kits and compositions containing these vectors.

[0252] It is preferred to combine the vector of the present invention for expressing the KLF gene, the OCT gene, and the SOX gene with a vector containing the KLF gene but not the OCT gene and the SOX gene, and particularly with a vector expressing the MYC and / or Glis1 gene (Takahashi, K. and Yamanaka S., Cell 126, 663-676, 2006; Lowry WE et al., Proc Natl Acad Sci USA, 105(8):2883-8, 2008; Masaki, H. et al., Stem Cell Res. 1:105-115, 2008; Maekawa et al., Nature, 474:225-229, 2011; WO2007 / 69666). Here, the SOX protein, KLF protein, MYC protein, and OCT protein, and their genes refer to proteins and genes belonging to members of the SOX family, the KLF family, the MYC family, and the OCT family, respectively. It has been reported that by adjusting the expression of one or more members of these four families, pluripotent stem cells can be induced from various differentiated cells. For example, it has been reported that for the SOX family, any one of SOX1, SOX2, SOX3, SOX15, and SOX17 can be used to induce pluripotent stem cells (WO2007 / 69666). In addition, for the KLF family, either KLF4 or KLF2 can be used to induce pluripotent stem cells (WO2007 / 69666). For the MYC family, not only wild-type c-MYC but also T58A mutants or N-MYC and L-MYC can be used to induce pluripotent stem cells (WO2007 / 69666; Blelloch R. et al., Cell Stem Cell, 1: 245-247, 2007). In this way, since the genes of the family can be selected and then used in a variety of ways, the above-mentioned four family genes can be appropriately selected to induce reprogramming.

[0253] Specifically, the KLF family includes KLF1 (NM_006563, NM_010635), KLF2 (NM_016270, NM_008452), KLF4 (NM_004235, NM_010637), and KLF5 (NM_001730, NM_009769); the MYC family includes c-MYC (NM_002467, NM_010849, containing T58A mutant), N-MYC (NM_005378, NM_008709), and L-MYC (NM_005376, NM_005806); the OCT family includes OCT1A (NM_002697, NM_198934), OCT3 / 4 (NM_002701 , NM_203289, NM_013633, NM_001009178), and OCT6 (NM_002699, NM_011141); the SOX family includes SOX1 (NM_005986, NM_009233), SOX2 (NM_003106, NM_011443, XM_574919), S OX3 (NM_005634, NM_009237), SOX7 (NM_031439, NM_011446), SOX15 (NM_006942, NM_009235), SOX17 (NM_022454, NM_011441), and SOX18 (NM_018419, NM_009236). A Sendai virus vector carrying any of these genes in the sequence of the present invention can be used to induce cell dedifferentiation in the present invention, and is particularly preferably used to induce pluripotent stem cells. The most preferred KLF genes, OCT genes, and SOX genes are OCT3 / 4 genes, SOX2 genes, and KLF4 genes, respectively.

[0254] MYC family genes are not essential for the induction of pluripotent stem cells, and pluripotent stem cells can be induced using only genes from three families other than MYC (Nakagawa M. et al., Nat Biotechnol. 26(1):101-6, 2008; Wering M. et al., Cell Stem Cell 2(1):10-2, 2008). Without expressing the MYC gene, for example, the use of p53 siRNA and UTF1 can significantly improve the induction efficiency of pluripotent stem cells (Y. Zhao et al., Cell Stem Cell, 3(5):475-479, 2008; N. Maherali, and K. Hochedlinger, Cell Stem Cell, 3(6):595-605, 2008). In addition, there are reports that pluripotent stem cells can be induced using only genes from three families other than the KLF family (Park IH et al., Nature, 451(7175):141-6, 2008). In addition, there are reports that by combining the use of a G9a histone methyltransferase inhibitor (BIX-01294; Kubicek, S. et al., Mol. Cell 25, 473-481, 2007), pluripotent stem cells can be induced from embryonic NPCs using only three genes, namely the KLF gene, the SOX gene, and the MYC gene (Shi Y et al., Cell Stem Cell, 2(6):525-8, 2008). Viral vectors encoding each gene can be prepared separately. They can be used in combination when used. They can be combined arbitrarily or all together as a kit, or mixed to form a composition. In addition to the Sendai virus vectors of the present invention that carry the KLF gene, the OCT gene, and the SOX gene, and the Sendai virus vectors that contain the KLF gene but do not contain the OCT gene and the SOX gene, the present invention also relates to kits or compositions that contain any combination (or all) of genes other than these three genes and compounds. The genes can be suitably loaded onto recombinant vectors. Furthermore, a portion of the recombinant vectors contained in the kit can also be replaced with proteins, synthetic compounds, etc. having comparable functions.

[0255] In the combination of genes described above, other genes can be further combined to improve the induction efficiency of reprogramming. Examples of such genes include TERT (NM_198253, NM_009354) and / or SV40 large T antigen (NC_001669.1, Fiers, W. (05-11-1978) Nature 273: (5658) 113-120) (Park IH. et al., Nature, 451 (7175): 141-6, 2008). One or more genes selected from HPV16 E6, HPV16 E7, and Bmil (NM_005180, NM_007552) can be further combined. In addition, one or any combination of genes selected from Fbx15 (Mol Cell Biol. 23(8):2699-708, 2003), Nanog (Cell 113:631-642, 2003), Eras (Nature 423, 541-545, 2003), DPPA2 (Development 130:1673-1680, 2003), TCL1A (Development 130:1673-1680, 2003), and β-catenin (Nat Med 10(1):55-63, 2004) can be expressed. Furthermore, a combination of the following may be used: ECAT1 (AB211062, AB211060), DPPA5 (NM_001025290, NM_025274, XM_236761), DNMT3L (NM_013369, NM_019448), ECAT8 (AB211063, AB211061), GDF3 (NM_020634, NM_008108), SOX15 (NM_006942, NM_009235), DPPA4 (NM_018189, NM_028610), One or more genes among FTHL17 (NM_031894, NM_031261), SALL4 (NM_020436, NM_175303), Rex-1 (NM_174900, NM_009556), Utf1 (NM_003577, NM_009482), DPPA3 (NM_199286, NM_139218, XM_216263), STAT3 (NM_139276, NM_213659), and GRB2 (NM_002086, NM_008163).A combination of genes including the NANOG gene (NM_024865, AB093574) and the LIN28 gene (NM_024674) in addition to the KLF gene, the OCT gene, and the SOX gene is useful for inducing pluripotent stem cells (Yu J. et al., Science, 318(5858):1917-20, 2007). Furthermore, it is preferably combined with the MYC gene (Liao J et al., Cell Res. 18(5):600-3, 2008). The induction of pluripotent stem cells can be promoted by additionally expressing these genes (WO2007 / 69666). In the case of mature B cells, for example, the myeloid transcription factor C / EBPα (CCAAT / enhancer binding protein α) (NM_004364) can be expressed ectopically, or the expression of the B cell transcription factor Pax5 (paired box 5; NM_016734) can be inhibited to promote reprogramming (Hanna J, Cell. 133(2):250-64, 2008). These factors can also be expressed using the non-chromosomal integrative viral vectors of the present invention. In addition, some of the recombinant vectors contained in the kit can be replaced with proteins, synthetic compounds, etc. having equivalent functions.

[0256] In addition, except expressing above-mentioned factor, for example, by combining with adding compound, the efficiency of reprogramming can also be improved.For example, bFGF (basic fibroblast growth factor) and / or SCF (stem cell factor) can promote the induction of pluripotent stem cells, and can replace the function (WO2007 / 69666) of c-MYC in the induction of pluripotent stem cells. In addition, MAP kinase inhibitor (PD98056) can also be used for setting up the purposes (WO2007 / 69666) such as pluripotent stem cells closer to ES cells. In addition, there is report that DNA methylase (Dnmt) inhibitor and / or histone deacetylase (HDAC) inhibitor can improve the induction efficiency (Huangfu D etc., Nat Biotechnol. (online publication June 22, 2008, doi:10.1038 / nbt1418) of pluripotent stem cells; Nat.Biotechnol.26,795-797 (2008)). For example, by combining HDAC (VPA), only the introduction of two genes, OCT4 and SOX2, can induce pluripotent stem cells (Huangfu, D. et al., Nat Biotechnol. 2008 26 (11): 1269-75). The vector of the present invention can be used in combination with the administration of these compounds. As a Dnmt inhibitor, for example, 5-azacytidine can be used; as an HDAC inhibitor, for example, N-suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA), valproic acid inhibitor (VPA) can be used. In addition, when using 5-azacytidine, the combined use of glucocorticoids (dexamethasone) can improve efficiency.

[0257] For reprogramming cells, for example, (1) the Sendai virus vector of the present invention containing the KLF gene, the OCT gene and the SOX gene in sequence, and the Sendai virus vector containing the KLF gene but not the OCT gene and the SOX gene, (2) a combination of the vector of (1) and a Sendai virus vector for expressing the MYC gene or the Glis1 gene, (3) a combination produced by further adding a Sendai virus vector for expressing the NANOG gene and a Sendai virus vector for expressing LIN28 to (1) and / or (2), (4) a combination of the vector of any one of (1) to (4) and a vector carrying the above-mentioned other desired reprogramming factors and / or desired reprogramming inducing compounds, etc. can be introduced into cells. In the case of combining and introducing multiple vectors and / or compounds, the introduction is preferably carried out simultaneously. Specifically, it is preferred that all vectors and / or compounds encoding reprogramming factors are added within 48 hours, preferably within 36 hours, more preferably within 24 hours, 18 hours, 12 hours, 10 hours, 8 hours, 6 hours, 3 hours, 2 hours, or 1 hour after the initial addition of the vector or compound. The dose of the vector can be appropriately prepared, and the MOI is, for example, 0.1 or more, preferably 0.3 or more, 0.5 or more, 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more, and 100 or less, preferably 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, or 5 or less. Infection is preferably performed at an MOI of 0.3 to 100, more preferably 0.5 to 50, more preferably 1 to 40, more preferably 1 to 30, more preferably 2 to 30, for example, 3 to 10. The induced pluripotent stem cells form flat colonies very similar to ES cells and express alkaline phosphatase. Furthermore, the induced pluripotent stem cells may express undifferentiated cell markers such as Nanog, OCT4, and / or SOX2. Furthermore, the induced pluripotent stem cells preferably express TERT and / or exhibit telomerase activity. The present invention also relates to methods for producing cells that express alkaline phosphatase, and preferably also express the undifferentiated cell markers Nanog and / or TERT, as well as the use of the Sendai virus vectors described above for producing these cells and for producing pharmaceutical agents for inducing these cells.

[0258] According to the present invention, pluripotent stem cells can be generated from desired cells (including adult skin cells and neonatal foreskin cells). According to the present invention, colonies of pluripotent stem cells can be induced from at least certain types of cells (e.g., adult skin cells and / or neonatal foreskin cells), for example, at a concentration of 0.03×10 -5 Above, 0.1×10 -5 Above, 0.3×10 -5 Above, 0.5×10-5 Above, 0.8×10 -5 Above, or 1×10 -5 More than (for example 1×10 -5 ~1×10 -3 ) is preferably 1.5×10 -5 Above, 1.7×10 -5 Above, 2.0×10 -5 Above, 2.5×10 -5 Above, 3×10 -5 Above, 4×10 -5 Above, 5×10 -5 Above, 8×10 -5 Above, 1×10 -4 Above, 2×10 -4 Above, 3×10 -4 Above, 5×10 -4 Above, 8×10 -4 Above, 1×10 -3 Above, 1.5×10 -3 Above, 2×10 -3 Above, 5×10 -3 , 1×10 -2 Above, 1.5×10 -2 Above, 2×10 -2 Above, 2.5×10 -2 Above, 3×10 -2 Above, 4×10 -2 Above, or 5×10 -2 For example, the present invention can be used to induce pluripotent stem cells from human peripheral blood mononuclear cells to produce at least 1×10 -2 (0.01%), preferably at least 3×10 -2 , 5x10 -2 , 8x10 -2 , 1x10 -1 , 2x10 -1 , 3x10 -1 , 4x10 -1 , or 5x10 -1 ; and can induce pluripotent stem cells from human peripheral blood mononuclear cells, with at least 1×10 -3 (0.001%), preferably at least 3×10 -3 , 5x10 -3 , 8x10 -3 , 1x10 -2 , 2x10 -2 , 3x10 -2 , 4x10 -2 , or 5x10-2 The MOI can be appropriately established as described above; for example, it can be performed at 5, 10, or 30. For at least a given cell, the efficiency of emergence of pluripotent stem cell colonies by the vector containing the KLF gene, OCT gene, and SOX gene in sequence and the Sendai virus vector containing the KLF gene but not the OCT gene and SOX gene of the present invention is, for example, 1.2 times or more, preferably 1.3 times or more, 1.5 times or more, 1.7 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, or 5 times or more, compared to a case where the Sendai virus vector expressing the KLF gene, OCT gene, and SOX gene of the present invention is used but a Sendai virus vector containing the KLF gene but not the OCT gene and SOX gene is not used. Compared to conventional methods, the method of the present invention exerts a significant effect because the induction efficiency of pluripotent stem cells is significantly high when cultured at 37°C after vector introduction, and the vector is rapidly removed after induction of the pluripotent stem cells.

[0259] There are no particular limitations on the differentiated cells to be induced for reprogramming, and any desired somatic cells can be used. It has been demonstrated that pluripotent stem cells can be generated not only from cells derived from mouse embryos but also from differentiated cells collected from the tail of adult mice, or from hepatocytes and gastric mucosal cells, suggesting that such generation is independent of cell type or differentiation state (WO2007 / 069666; Aoi T. et al., Science [published online February 14, 2008]; Science. 2008; 321(5889):699-702). In addition, in humans, it has been confirmed that pluripotent stem cells can be induced from a variety of cells, including adult facial skin-derived fibroblasts, adult synovial cells, neonatal foreskin-derived fibroblasts, adult mesenchymal stem cells, adult palm skin cells, and embryonic cells (Takahashi K et al. (2007) Cell 131: 861-872; Park IH et al., Nature, 451(7175): 141-6, 2008). Furthermore, there are reports that pluripotent stem cells can also be induced from terminally differentiated cells such as pancreatic β cells and B lymphocytes (Stadtfeld M et al., Curr Biol. 2008 May 21. [PubMed, PMID: 18501604]; Curr Biol. 2008; 18(12): 890-4; Hanna J et al., Cell. 133(2): 250-64, 2008). These insights suggest that the induction of pluripotent stem cells does not depend on the cells used as the source. The method of the present invention can be applied to the process of inducing pluripotent stem cells from these desired somatic cells. Specifically, the differentiated cells used as the object of reprogramming include fibroblasts, synovial cells, mucosal cells of the oral cavity or stomach, hepatocytes, bone marrow cells, tooth germ cells, blood cells (such as lymphocytes and leukocytes), and other desired cells. The blood cells can be peripheral blood cells. Fibroblasts, monocytes, mononuclear cells, etc. are also preferred. In addition, the cells can be cells derived from, for example, embryos, newborns, children, adults, or the elderly. In addition, there is no particular limitation on the source of the animal, including mammals, such as rodents such as humans and non-human primates (monkeys, etc.), mice, rats, and non-rodents such as cattle, pigs, and goats.

[0260] Cells from which the vector has been removed can be appropriately selected from the colonies of cells after reprogramming is complete. For example, cells from which the vector has been naturally removed can be selected. For this purpose, for example, negative selection can be performed using antibodies specific for viral vectors (e.g., anti-HN antibodies). In addition, when using temperature-sensitive vectors, the vector can be easily removed by culturing at normal temperature (e.g., approximately 37°C, particularly 36.5°C to 37.5°C, preferably 36.6°C to 37.4°C, and more preferably 36.7°C to 37.3°C), or by culturing at slightly higher temperatures (e.g., 37.5-39°C, preferably 38-39°C or 38.5-39°C). When SeV(PM) / TSΔF is used, or when a vector is obtained by introducing more mutations such as TS7, TS12, TS13, TS14, or TS15 into SeV(PM) / TSΔF, passage leads to natural loss of the vector. Examples of preferred vectors include, but are not limited to, combinations of SeV(PM)KOS / TS12ΔF and SeV18+KLF4 / TSΔF. Furthermore, in this case, SeV(HNL)c-rMYC / TS12ΔF, SeV(HNL)c-rMYC / TS13ΔF, SeV(HNL)c-rMYC / TS15ΔF, and the like can be used to introduce the MYC gene. The combination of SeV(PM)KOS / TS12ΔF, SeV18+KLF4 / TSΔF, and SeV(HNL)c-rMYC / TS15ΔF is particularly preferred. Furthermore, not loading the MYC gene onto a Sendai virus vector carrying the KLF gene, OCT gene, and SOX gene has the advantage that reprogramming can be performed without using the MYC gene associated with carcinogenesis. This is also advantageous because a fourth factor other than the KLF gene, OCT gene, and SOX gene can be freely selected from c-MYC, L-MYC, Glis1, and the like.

[0261] The cells produced by the method of the present invention can be used to cause differentiation into various tissues or cells, and can be used for desired inspection, research, diagnosis, testing, treatment, etc. For example, it is expected that the stem cells induced can be used for stem cell therapy. For example, somatic cells collected from the patient can be used to induce reprogramming (reprogramming), and then, the somatic stem cells or other somatic cells obtained by differentiation induction are transplanted to the patient. There are no particular restrictions on the method for cell differentiation induction, for example, differentiation can be induced by retinoic acid treatment, various proliferation factors-cytokine treatment, or the treatment performed by hormones. In addition, the obtained cells can be used to detect the effect of the desired drug or compound, thereby enabling the screening of drugs or compounds. Example

[0262] Hereinafter, the present invention will be specifically described with reference to examples; however, the present invention should not be construed as being limited to the examples. All documents and other references cited herein are incorporated as part of this specification.

[0263] [Example 1]

[0264] Construction of pSeV(PM) / TSΔF

[0265] The method used to generate the Sendai virus vector pSeV(PM) / TSΔF used in this example is shown below. As used herein, "(PM)" refers to the insertion of the reprogramming gene between the P and M genes, and "(HNL)" refers to the insertion of the reprogramming gene between the HN and L genes. Furthermore, as used herein, "TS" refers to the presence of G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511 mutation in the P protein, and N1197S and K1795E mutations in the L protein; "ΔF" indicates a deletion of the F gene. Hereinafter, "SeV(PM) / TSΔF" refers to a Sendai virus vector (strain Z) with an F gene deletion, containing G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein. This vector has an insertion site (NotI site) for gene transfer immediately downstream of the P gene (between the P and M genes). However, these examples are illustrative only and the present invention is not limited to these examples.

[0266] pSeV(PM) / TSΔF was constructed as follows. PCR was performed (94°C for 3 minutes, 25 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 12 minutes, followed by 72°C for 7 minutes) using Litmus SalINheIfrgPmutMtsHNtsΔF-GFPdelGFP (International Publication No. WO2010 / 008054) as a template, along with PMNOTI-F (5'-GAAATTTCACCTAAGCGGCCGCAATGGCAGATATCTATAG-3') (SEQ ID NO: 10) and PMNOTI-R (5'-CTATAGATATCTGCCATTGCGGCCGCTTAGGTGAAATTTC-3') (SEQ ID NO: 11). The resulting approximately 11-kb PCR product was treated with DpnI, and Escherichia coli DH5α (ToYoBo Code No. DNA-903) was transformed with 20 μL of this reaction solution. A plasmid with a NotI sequence was selected by digestion with NotI. A clone with the correct sequence was then selected by sequencing. Thus, LitmusSalINheIfrgPmutMtsHNts(PM)-dF was obtained. LitmusSalINheIfrgPmutMtsHNts(PM)-dF was then digested with SalI and NheI, and the resulting fragment (approximately 8 kbp) was ligated to a fragment (approximately 8 kbp) collected after digestion with SalI / NheI of the pSeV / ΔSalINheIfrgLmut plasmid (International Publication No. WO2003 / 025570), which has two mutations in the L gene. This resulted in pSeV(PM) / TS F. This vector has an insertion site (NotI site) for gene transfer between the P and M genes. The Sendai virus generated from the transcription product of this vector is called SeV(PM) / TSΔF.

[0267] [Example 2]

[0268] Construction of pSeV18+KLF4 / TSΔF

[0269] The KIF4 gene was amplified from the Jurkat cDNA library by PCR using PrimeStar HS DNA polymerase (Takara Bio Inc., Catalogue, catalog No. R010A) and inserted into the NotI site of the BlueScript plasmid vector to obtain the plasmid pBS-KS-KLF4 (WO2010 / 008054). pBS-KS-KLF4 was digested with NotI (37°C, 3 hours) and separated by electrophoresis on a 1% agarose gel. The approximately 1.5 k bp band was excised and purified using the Qiaquick Gel Extraction Kit (Quiagen, Catalogue). The NotI fragment containing the KLF4 gene was cloned into the NotI site of the pSeV18+ / TSΔF vector, and clones with the correct sequence were selected by sequencing to obtain SeV18+KLF4 / TSΔF. The Sendai virus generated from the transcription product of the pSeV18+ / TSΔF vector is called SeV18+ / TSΔF, and the Sendai virus generated from the transcription product of the pSeV18+KLF4 / TSΔF vector is called SeV18+KLF4 / TSΔF.

[0270] [Example 3]

[0271] Construction of pSeV(PM) / TS12ΔF

[0272] PCR was performed using PrimeStar (94°C for 3 minutes, 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 15 minutes, followed by 72°C for 5 minutes and incubation at 4°C) using pSeV(PM) / TSΔF as a template and primers F3208 (5'-AGAGAACAAGACTAAGGCTACC-3' (SEQ ID NO: 12)) and R3787 (5'-ACCTTGACAATCCTGATGTGG-3' (SEQ ID NO: 13)). The amplified band of approximately 600 bp was purified using the QIAquick PCR purification kit. PCR was performed using PrimeStar (94°C for 3 minutes, 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 1.5 minutes, followed by 72°C for 5 minutes and at 4°C for ∞) using pSeV18+Oct3 / 4 / TS12ΔF (WO 2010 / 008054; WO 2012 / 029770) as a template and primers F2001 (5'-CCATCAACACTCCCCAAGGACC-3' (SEQ ID NO: 14)) and R3390 (5'-AGACGTGATGCGTTTGAGGCCC-3' (SEQ ID NO: 15)). The amplified band of approximately 1.4 kbp was purified using the QIAquick PCR purification kit. These PCR products were mixed and PCR was performed using PrimeStar and F2001 and R3787 primers (94°C for 3 minutes, followed by 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 2 minutes, followed by 72°C for 5 minutes and 4°C at ∞). The PCR product was purified using the QIAquick PCR purification kit, digested with SalI and NotI, and separated by agarose gel electrophoresis. A band of approximately 1.6 kbp was then excised and purified using the QIAquick Gel Extraction Kit. pSeV(PM) / TSΔF was digested with SalI and NotI and separated by agarose gel electrophoresis. A band of approximately 14.8 kbp was then excised and purified using the QIAquick Gel Extraction Kit. The two SalI and NotI-digested and purified products were ligated, and a clone with the correct sequence was selected by sequencing to obtain pseV(PM) / TS12ΔF. This vector has an insertion site (NotI site) for gene introduction between the P and M genes. The Sendai virus generated from the transcription product of this vector was named SeV(PM) / TS12ΔF.

[0273] [Example 4]

[0274] Construction of SeV(PM)KOS / TS12ΔF

[0275] The plasmid used to generate the SeV / TS12ΔF vector carrying the KLF4-OCT3 / 4-SOX2 genes was generated as follows. pSeV(PM) / TSΔF was digested with NotI and purified using the QIAquick PCR Purification Kit (QIAGEN, catalog number 28106), followed by BAP treatment. This was then purified using the QIAquick PCR Purification Kit. pSeV(PM)KOS / TSΔF was digested with NotI, separated by 1% agarose gel electrophoresis, and the approximately 3.6 kbp band was excised and purified using the Qiaquick Gel Extraction Kit (QIAGEN, catalog number 28706). Elution was performed using 100 μL of the elution buffer provided with the kit. The NotI fragment containing the KLF4, OCT3 / 4, and SOX2 genes was cloned into the NotI site of the pSeV(PM) / TS12ΔF vector, and a clone with the correct sequence was selected by sequencing. This resulted in pSeV(PM)KOS / TS12ΔF. The Sendai virus generated from the transcription product of this vector was designated SeV(PM)KOS / TS12ΔF.

[0276] [Example 5]

[0277] Generation of pSeV(HNL)c-rMyc / TS15ΔF

[0278] Litmus38TSΔF-P2 was digested with SalI-NheI. ΔGFP and pSeV / TSΔF-linker L1361CL1558I (WO2010 / 008054; WO2012 / 029770) were separated by agarose gel electrophoresis, and the 8.0 kbp and 8.3 kbp bands were excised and purified, respectively. These purified fragments were ligated to obtain pSeV(HNL) / TS15ΔF (a plasmid encoding the antigenome of SeV(HNL) / TS15ΔF). The NotI site of this pSeV(HNL) / TS15ΔF was introduced with a NotI fragment containing the c-rMyc gene, which was excised and purified from pBS-KS-c-rMyc by NotI digestion, to obtain pSeV(HNL)c-rMyc / TS15ΔF. The nucleotide and amino acid sequences of c-rMyc are shown in SEQ ID NOs: 8 and 9. c-rMyc has a378g, t1122c, t1125c, a1191g, and a1194g mutations. The Sendai virus generated from the transcription product of this vector is designated SeV(HNL)c-rMyc / TS15ΔF.

[0279] [Example 6]

[0280] Generation of induced pluripotent stem cells (iPS cells) using Sendai virus vectors that maintain the cell reprogramming-1 gene

[0281] Human neonatal foreskin-derived fibroblasts (BJ) from ATCC (CRL-2522; www.atcc.org) were cultured at 37°C in a 5% CO2 incubator in DMEM (GIBCO-BRL, 11995) containing 10% FBS (Cell Culture Bioscience, Cat. No. 171012), penicillin (100 u / ml) and streptomycin (100 μg / ml; Nacalai Tesque, Code 26253-84) (hereinafter referred to as 10% FBS / PS / DMEM). 5 After overnight culture, the cells were infected with the vector under the following conditions.

[0282] Condition 1:

[0283] SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF+SeV18+KLF4 / TSΔF

[0284] SeV(PM)KOS / TS12ΔF MOI=30

[0285] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0286] SeV18+KLF4 / TSΔF MOI=3

[0287] Condition 2:

[0288] SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF+SeV18+KLF4 / TSΔF

[0289] SeV(PM)KOS / TS12ΔF MOI=30

[0290] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0291] SeV18+KLF4 / TSΔF MOI=10

[0292] Condition 3: SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF+SeV18+ / TSΔF

[0293] SeV(PM)KOS / TS12ΔF MOI=30

[0294] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0295] SeV18+ / TSΔF MOI=3

[0296] Condition 4: SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF+SeV18+ / TSΔF

[0297] SeV(PM)KOS / TS12ΔF MOI=30

[0298] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0299] SeV18+ / TSΔF MOI=10

[0300] Condition 5: SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF

[0301] SeV(PM)KOS / TS12ΔF MOI=30

[0302] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0303] Condition 6: No vehicle administration group (control group)

[0304] After the above vectors were administered to the cells, they were cultured at 37°C in a 5% CO2 incubator. Evaluation was performed with n = 2 cells. The culture medium was replaced almost daily with 10% FBS / PS / DMEM. On the 6th day of infection, 1.0 × 10 cells were detached using 0.25% trypsin-EDTA solution. 4 The cells introduced with the vector were added to 1.25×10 5 100 cells of mitomycin-treated feeder cells (e.g., MEFs) are cultured in a CO2 incubator. The next day, the culture medium is replaced from 10% FBS / PS / DMEM to a primate ES cell culture medium (ReproCell, RCHEMD001) supplemented with 4 ng / ml bFGF, and the cells are cultured at 37°C in a 5% CO2 incubator. The culture medium is replaced every day or every two days. The culture medium can be a conditioned medium for the feeder cells.

[0305] On day 28 of infection, the efficiency of iPS cell induction was observed by staining the activity of alkaline phosphatase, a marker of undifferentiated ES cells, using NBCT / BCIP (PIERCE; NBT / BCIP, 1-Step, #34042). Figure 1 ). Compared with the test group without SeV18+KLF4 / TSΔF vector addition (condition 5 above), the test group with SeV18+KLF4 / TSΔF vector addition (conditions 1 and 2 above) showed improvement in iPS cell induction efficiency in a manner dependent on the amount of SeV18+KLF4 / TSΔF vector added. Figure 2 The graph shows the average induction efficiency for n = 2. When the SeV18+KLF4 / TSΔF vector was added at an MOI of 3, the efficiency of iPS cell induction improved by approximately 6.9-fold, and when it was added at an MOI of 10, it improved by approximately 12-fold. When the SeV18+ / TSΔF vector was added at an MOI of 3, the efficiency of iPS cell induction improved by approximately 3.2-fold, and when it was added at an MOI of 10, it improved by approximately 6-fold.

[0306] [Example 7]

[0307] Generation of induced pluripotent stem cells (iPS cells) using Sendai virus vectors containing the cell reprogramming-2 gene

[0308] Human neonatal foreskin-derived fibroblasts (BJ) from ATCC (CRL-2522; www.atcc.org) were cultured at 37°C in a 5% CO2 incubator in DMEM (GIBCO-BRL, 11995) containing 10% FBS (Cell Culture Bioscience, Cat. No. 171012), penicillin (100 u / ml) and streptomycin (100 μg / ml; Nacalai Tesque, Code 26253-84) (hereinafter referred to as 10% FBS / PS / DMEM) at a concentration of 5 × 10 5 The cells were cultured overnight. Then, the cells were infected with the vector under the following conditions. Evaluation was performed with n=2.

[0309] Condition 1:

[0310] SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF+SeV18+KLF4 / TSΔF

[0311] SeV(PM)KOS / TS12ΔF MOI=5

[0312] SeV(HNL)c-rMYC / TS15ΔF MOI=5

[0313] SeV18+KLF4 / TSΔF MOI=5

[0314] Condition 2:

[0315] SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF+SeV18+KLF4 / TSΔF

[0316] SeV(PM)KOS / TS12ΔF MOI=30

[0317] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0318] SeV18+KLF4 / TSΔF MOI=10

[0319] Condition 3: SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF

[0320] SeV(PM)KOS / TS12ΔF MOI=5

[0321] SeV(HNL)c-rMYC / TS15ΔF MOI=5

[0322] Condition 4: SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF

[0323] SeV(PM)KOS / TS12ΔF MOI=30

[0324] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0325] Condition 5: Group 1 without vehicle administration (control group 1)

[0326] Condition 6: Group 2 without vehicle administration (Control Group 2)

[0327] After the above vectors were administered to the cells, conditions 1, 2, and 5 were cultured at 37°C in a 5% CO2 incubator, while conditions 3, 4, and 6 were cultured at 35°C in a 5% CO2 incubator. The culture medium was replaced almost daily with 10% FBS / PS / DMEM. On the 6th day of infection, 1.0×10 cells were detached using 0.25% trypsin-EDTA solution. 4 The cells introduced with the vector were added to 1.25×10 5100 cells of mitomycin-treated feeder cells (e.g., MEFs) are cultured at 37°C in a 5% CO2 incubator. The next day, the culture medium is replaced from 10% FBS / PS / DMEM to a primate ES cell culture medium (ReproCell, RCHEMD001) supplemented with 4 ng / ml bFGF, and the cells are cultured at 37°C in a 5% CO2 incubator. The culture medium is replaced every day or every two days. This culture medium can be the conditioned medium of the feeder cells.

[0328] On day 28 of infection, the efficiency of iPS cell induction was observed by staining the activity of alkaline phosphatase, a marker of undifferentiated ES cells, using NBCT / BCIP (PIERCE; NBT / BCIP, 1-Step, #34042). Figure 3 ). Compared with the test groups without SeV18+KLF4 / TSΔF vector addition (conditions 3 and 4 above), the test groups with SeV18+KLF4 / TSΔF vector addition (conditions 1 and 2 above) showed high efficiency of iPS cell induction. In the groups without vector administration (conditions 5 and 6 above), no iPS cell induction was observed. Figure 4 The graph shows the average value of the results obtained with n=2.

[0329] [Example 8]

[0330] Generation of induced pluripotent stem cells (iPS cells) by Sendai virus vectors that maintain the cell reprogramming-3 gene

[0331] Human adult skin-derived fibroblasts (HDFs) (Applications, Inc. 106-05A-1388) were cultured at a density of 5 × 10 cells / mL in DMEM (GIBCO-BRL, 11995) containing 10% FBS (Cell Culture Bioscience, Cat. No. 171012), penicillin (100 u / ml) and streptomycin (100 μg / ml; Nacalai Tesque, Code 26253-84) (hereinafter referred to as 10% FBS / PS / DMEM) at 37°C in a 5% CO2 incubator. 5 The cells were cultured overnight. Then, the cells were infected with the vector under the following conditions. Evaluation was performed with n=2.

[0332] Condition 1:

[0333] SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF+SeV18+KLF4 / TSΔF

[0334] SeV(PM)KOS / TS12ΔF MOI=5

[0335] SeV(HNL)c-rMYC / TS15ΔF MOI=5

[0336] SeV18+KLF4 / TSΔF MOI=5

[0337] Condition 2:

[0338] SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF+SeV18+KLF4 / TSΔF

[0339] SeV(PM)KOS / TS12ΔF MOI=30

[0340] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0341] SeV18+KLF4 / TSΔF MOI=10

[0342] Condition 3: SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF

[0343] SeV(PM)KOS / TS12ΔF MOI=30

[0344] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0345] Condition 4: SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF

[0346] SeV(PM)KOS / TS12ΔF MOI=5

[0347] SeV(HNL)c-rMYC / TS15ΔF MOI=5

[0348] Condition 5: Group 1 without vehicle administration (control group 1)

[0349] Condition 6: Group 2 without vehicle administration (Control Group 2)

[0350] After the above vectors were administered to the cells, conditions 1, 2, and 5 were cultured at 37°C in a 5% CO2 incubator, while conditions 3, 4, and 6 were cultured at 35°C in a 5% CO2 incubator. The culture medium was replaced almost daily with 10% FBS / PS / DMEM. On the 6th day of infection, 1.0×10 cells were detached using 0.25% trypsin-EDTA solution. 5The cells introduced with the vector were added to 1.25×10 5 100 cells of mitomycin-treated feeder cells (e.g., MEFs) are cultured at 37°C in a 5% CO2 incubator. The next day, the culture medium is replaced from 10% FBS / PS / DMEM to a primate ES cell culture medium (ReproCell, RCHEMD001) supplemented with 4 ng / ml bFGF, and the cells are cultured at 37°C in a 5% CO2 incubator. The culture medium is replaced every day or every two days. The culture medium can be a conditioned medium for the feeder cells.

[0351] On day 28 of infection, the efficiency of iPS cell induction was observed by staining the activity of alkaline phosphatase, a marker of undifferentiated ES cells, using NBCT / BCIP (PIERCE; NBT / BCIP, 1-Step, #34042). Figure 5 ). Compared with the test groups without SeV18+KLF4 / TSΔF vector addition (conditions 3 and 4 above), the test groups with SeV18+KLF4 / TSΔF vector addition (conditions 1 and 2 above) showed high efficiency of iPS cell induction. In the groups without vector administration (conditions 5 and 6 above), no iPS cell induction was observed. Figure 6 The graph shows the average value of the results obtained with n=2.

[0352] [Example 9]

[0353] Generation of induced pluripotent stem cells (iPS cells) by Sendai virus vectors containing the cell reprogramming-4 gene

[0354] Human fetal lung cell-derived fibroblasts (MRC-5; ATCC, CCL-171) were cultured at 2×10 cells / mL in DMEM (GIBCO-BRL, 11995) containing 10% FBS (Cell Culture Bioscience, Cat. No. 171012), penicillin (100 u / ml) and streptomycin (100 μg / ml; Nacalai Tesque, Code 26253-84) at 37°C in a 5% CO2 incubator. 5 The cells were cultured overnight. Then, the cells were infected with the vector under the following conditions. Evaluation was performed with n=2 cells.

[0355] Condition 1:

[0356] SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF+SeV18+KLF4 / TSΔF

[0357] SeV(PM)KOS / TS12ΔF MOI=5

[0358] SeV(HNL)c-rMYC / TS15ΔF MOI=5

[0359] SeV18+KLF4 / TSΔF MOI=5

[0360] Condition 2:

[0361] SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF+SeV18+KLF4 / TSΔF

[0362] SeV(PM)KOS / TS12ΔF MOI=30

[0363] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0364] SeV18+KLF4 / TSΔF MOI=10

[0365] Condition 3: SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF

[0366] SeV(PM)KOS / TS12ΔF MOI=5

[0367] SeV(HNL)c-rMYC / TS15ΔF MOI=5

[0368] Condition 4: SeV(PM)KOS / TS12ΔF+SeV(HNL)c-rMYC / TS15ΔF

[0369] SeV(PM)KOS / TS12ΔF MOI=30

[0370] SeV(HNL)c-rMYC / TS15ΔF MOI=30

[0371] Condition 5: Group 1 without vehicle administration (control group 1)

[0372] Condition 6: Group 2 without vehicle administration (Control Group 2)

[0373] After the above vectors were administered to the cells, conditions 1, 2, and 5 were cultured at 37°C in a 5% CO2 incubator, while conditions 3, 4, and 6 were cultured at 35°C in a 5% CO2 incubator. The culture medium was replaced almost daily with 10% FBS / PS / DMEM. On the 6th day of infection, 1.0×10 cells were detached using 0.25% trypsin-EDTA solution. 5 The cells introduced with the vector were added to 1.25×10 5 100 cells of mitomycin-treated feeder cells (e.g., MEFs) are cultured at 37°C in a 5% CO2 incubator. The next day, the culture medium is replaced from 10% FBS / PS / DMEM to a primate ES cell culture medium (ReproCell, RCHEMD001) supplemented with 4 ng / ml bFGF, and the cells are cultured at 37°C in a 5% CO2 incubator. The culture medium is replaced every day or every two days. The culture medium can be a conditioned medium for the feeder cells.

[0374] On day 28 of infection, the efficiency of iPS cell induction was observed by staining the activity of alkaline phosphatase, a marker of undifferentiated ES cells, using NBCT / BCIP (PIERCE; NBT / BCIP, 1-Step, #34042). Figure 7 ). Compared with the test groups without SeV18+KLF4 / TSΔF vector addition (conditions 3 and 4 above), the test groups with SeV18+KLF4 / TSΔF vector addition (conditions 1 and 2 above) showed high efficiency of iPS cell induction. In the groups without vector administration (conditions 5 and 6 above), no iPS cell induction was observed. Figure 8 The graph shows the average value of the results obtained with n=2.

[0375] [Example 10]

[0376] In vivo assessment of pluripotency of induced pluripotent stem cells

[0377] In vivo pluripotency was confirmed by teratoma formation in immunocompromised mice. BJ cell-derived iPS cell clone BJ-1215K#1 or HDF cell-derived iPS cell clone HDF-1215K#1 was subcutaneously inoculated into SCID mice, and tumor mass formation was confirmed after about one month. After about two months, samples were collected, fixed in 10% formalin, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin, and three germ layer differentiation ( Figure 9 ).

[0378] [Example 11]

[0379] Karyotype analysis

[0380] Karyotype analysis of the 9th passage BJ cell-derived iPS cell clone BJ-1215K#1, HDF cell-derived iPS cell clone HDF-1215K#1, and MRC-5 cell-derived iPS cell clone MRC5-1215K#1 was performed at the request of Nihon Gene Research Laboratories Inc. The results showed that they had 46 chromosomes and a normal karyotype ( Figure 10 ).

[0381] [Example 12]

[0382] Induction of iPS cells from mouse fibroblasts

[0383] Fibroblasts from 129+TER / SvJcl mice were cultured at 5×10 5Cells / well were seeded into 6-well plates and cultured overnight in a CO2 incubator (37°C, 5% CO2). Trypsin-EDTA solution was used to detach the cells in the wells for cell counting measurement, and the number of cells per well was measured. The amount of vector was calculated based on the cell count. Each of the SeV(PM)KOS / TS12ΔF vector, SeV(HNL)c-rMYC / TS15ΔF vector, and SeV18+KLF4 / TSΔF vector was added to 10% FBS / PS / DMEM culture medium at an MOI of 5, and the vector solution was prepared to a final solution of 1 ml. The culture solution was removed from the 6-well plate, followed by addition of the prepared vector solution and culture in a CO2 incubator for approximately 24 hours (37°C, 5% CO2). The cell culture solution was then removed, 10% FBS / PS / DMEM culture medium (2 mL / well) was added, and culture was carried out in a CO2 incubator for approximately 24 hours (37°C, 5% CO2). The cell culture solution was removed and ES cell culture medium was added (by mixing 250 ml of ES cell culture medium (Neurobasal medium; Life technologies, Cat. No. 21103-049), 250 ml of DMEM / F12, GlutaMAX (Life technologies, Cat. No. 10565-018), 10 ml of B27 (Life technologies, Cat. No. 17504044), 5 ml of N2 (Life technologies. Cat. No. 17502-048), 3.5 μl of 2-mercaptoethanol (Sigma, Cat. No. M3148-100ML), 2.5 ml of L-glutamic acid (Life technologies, Cat. No. 25030-0841), 5 ml of penicillin-streptomycin (Nacalais)). Tesque, Cat.No.26253-84), 100 μl of PD0325901 (prepared at 5 mM; Funakoshi, Cat.No.Axon1408), and 100 μl of CT 99021 (prepared at 15 mM; Funakoshi, Cat.No.Axon1386) were used to prepare 2i medium. LIF (Millipore, Cat.No.ESG1106) was added to the 2i medium at a final concentration of 1000 U / ml, and this was used as a mouse ES cell culture medium (hereinafter referred to as 2i / LIF medium) and cultured in a CO2 incubator (37°C, 5% CO2). Hereinafter, culture was performed while replacing the culture medium with 2i / LIF medium until the 5th day of vector infection. On the 5th day of infection, the feeder cells were inoculated at 1.4×10 5Cells / well / 2ml 10% FBS / PS / DMEM were seeded into 6-well plates coated with 0.1% gelatin solution and plated at 7x10 5 Cells / dish / 10 ml 10% FBS / PS / DMEM were seeded into 10 cm dishes and cultured in a CO2 incubator (37°C, 5% CO2). On the 6th day of infection, the vector-infected cells were detached using trypsin-EDTA solution, 2i / LIF medium was added to the prepared feeder cells, and the cells were plated at 1x10 4 cells / well and 1x10 5 Cells / well were plated into 6-well plates or plated at a ratio of 1x10 4 cells / dish and 1x10 5 The cells were plated in 10 cm culture dishes at a ratio of 10 cells / dish and cultured in a CO2 incubator (37°C, 5% CO2). Thereafter, iPS cell induction was performed using 2i / LIF medium with daily medium changes. On day 21 of infection, alkaline phosphatase staining was performed ( Figure 11 ). iPS cell-like alkaline phosphatase-positive cells were measured, and the resulting induction efficiency was approximately 0.1%.

[0384] [Example 13]

[0385] Obtaining mouse iPS cell clones

[0386] On the 16th day of infection, 1×10 4 cells / dish and 1x10 5 6 clones and 18 clones were picked out from 10 cm dishes inoculated with cells / dish, and they were passaged and cultured. The removal of Sendai virus was confirmed by staining with anti-SeV antibody ( Figure 12 The results showed that 9 Sendai virus vector-negative clones were obtained (clone numbers: 5-2, 5-6, 5-7, 5-11, 5-13, 5-14, 5-15, 5-16 and 5-17).

[0387] [Example 14]

[0388] Gene expression analysis by immunostaining

[0389] The mouse iPS cells induced in Example 12 and confirmed to be Sendai virus vector-negative in Example 13 were evaluated by immunostaining using anti-NANOG antibody and anti-OCT4 antibody ( Figure 13The results showed that NONOG and OCT4 staining in three iPS cell clones (5-2, 5-7, and 5-17) were similar to those in mouse ES cells used as a positive control. Negative control mouse fibroblasts did not stain for NANOG and OCT4. Thus, the evaluated iPS cells were shown to express NANOG and OCT4 genes at the protein level.

[0390] [Example 15]

[0391] Assessment of pluripotency

[0392] The mouse iPS cells induced in Example 12 and confirmed to be Sendai virus vector-negative in Example 13 were evaluated for their pluripotency.

[0393] Embryoid bodies were formed by culturing in serum-free medium without LIF. Figure 14 ). Same as mouse ES cells.

[0394] After embryoid body formation, cells were passaged on gelatin-coated plates and cultured in serum-free medium without LIF. On day 4, cell nuclei were stained by GATA (a transcription factor that is a marker of endoderm) staining ( Figure 15 ).

[0395] Culture was continued in serum-free medium without LIF and passaged to gelatin-coated culture plates. On day 10, tissue believed to be pulsating cardiac muscles was observed. On day 14, cells were fixed and immunostained with an antibody against α-actinin ( Figure 16 ). The cells were stained throughout. Among them, strongly stained cells were observed in some areas.

[0396] On day 4 of culture in 2i medium without LIF and passaged on gelatin-coated plates, differentiation into βIII tubulin-positive ( Figure 17 ) and tyrosine hydroxylase positive ( Figure 18 These results indicate that iPS cells with three germ layer differentiation ability can be induced from mouse cells by using the Sendai virus vector of the present invention.

[0397] [Example 16]

[0398] Induction of iPS cells from human monocyte-1

[0399] The induction of iPS cells from human CD14+ monocytes was carried out. The monocytes used were purchased from LONZA (Cat.No.2W-400C). Monocytes were cultured in IMDM (hereinafter referred to as 10% FBS / PS / IMDM) containing 10% fetal bovine serum and penicillin-streptomycin. The frozen monocytes were thawed in a 37°C water bath, transferred to a 50ml Falcon tube containing 10ml of 10% FBS / PS / IMDM, and gently mixed. After centrifugation at room temperature and 100g for 5 minutes, the supernatant was removed and 10ml of 10% FBS / PS / IMDM was added and mixed. The number of cells was counted using a hemocytometer. The cells were plated at 5x10 5 Cells / well / 2 ml 10% FBS / PS / IMDM were seeded into 6-well plates and cultured in a CO 2 incubator (37° C., 5% CO 2 ).

[0400] After collecting the cells in a tube and centrifuging, the supernatant was removed, 500 μl of 10% FBS / PS / IMDM was added and mixed, and the number of cells was calculated. The amount of vector required was calculated from the number of cells, and the vector solution was prepared as a vector solution with a final volume of 500 μl by mixing 10% FBS / PS / IMDM with three types of vectors: SeV(PM)KOS / TS12ΔF vector, SeV(HNL)c-rMYC / TS15ΔF vector, and SeV18+KLF4 / TSΔF vector, so that the MOI became 5, 10, 30, and 50. The cell solution and vector solution were mixed and added to a 6-well plate and cultured overnight in a CO2 incubator (37°C, 5% CO2). The next day, 10% FBS / PS / IMDM was added at 1 ml / well, and the cells were cultured in a CO2 incubator for two days (37°C, 5% CO2). The culture medium was replaced with 10% FBS / PS / IMDM until the 3rd and 4th days of infection. On the 6th day of infection, cells were detached using a trypsin-EDTA solution, resuspended in 10% FBS / PS / IMDM, added to feeder cells, and cultured in a CO2 incubator (37°C, 5% CO2). The next day, the culture medium was replaced with a primate ES cell culture medium (ReproCell, RCHEMD001) supplemented with 4 ng / ml bFGF. Culture was then continued with the culture medium replaced until the 28th day of vector infection. The status of the cells during the induction process is shown in FIG. Figure 19 On day 28, alkaline phosphatase staining was performed, and the induction of alkaline phosphatase-positive colonies was observed ( Figure 20 The induction efficiency of iPS cells becomes higher in a manner dependent on the amount of vector and is approximately 0.01% to 0.03% ( Figure 21 ).

[0401] [Example 17]

[0402] Induction of iPS cells from human peripheral blood mononuclear cells

[0403] Peripheral blood mononuclear cells were prepared from blood provided by two volunteers (donors 1 and 2) by the Ficoll method. Culture was performed using a culture medium (PBMC+ culture medium) prepared by adding cytokines (100ng / mL SCF, 100ng / mL FLT-3 ligand, 20ng / mL thrombopoietin, 10ng / mL IL-6) to PBMC culture medium (a culture medium prepared by adding StemPro-34 nutrients, L-glutamic acid and penicillin-streptomycin to StemPro-34SFM). Vector infection was performed on the 4th day of culture. The cell number of peripheral blood mononuclear cells was measured, and the amount of vector was calculated so that the MOI became 5, 10, 30 and 50. A vector solution was prepared by adding the calculated vector to the PBMC+ culture medium. After removal of the PBMC culture solution and addition of the vector solution, the cells were cultured in a CO2 incubator (37°C, 5% CO2). On the 3rd day of vector infection, the cells were separated using trypsin-EDTA, resuspended in PBMC+ culture medium, added to feeder cells, and cultured in a CO2 incubator (37°C, 5% CO2). From the next day until the 6th day of infection, the culture medium was replaced with PBMC culture medium, and the cells were cultured in a CO2 incubator (37°C, 5% CO2). On the 7th day of infection, half of the culture medium was removed, and an equal amount of primate ES cell culture medium (ReproCell, RCHEMD001) with 4ng / ml of bFGF was added, and the cells were cultured in a CO2 incubator (37°C, 5% CO2). Thereafter, the culture was completed using primate ES cell culture medium (ReproCell, RCHEMD001) with 4ng / ml of bFGF, and iPS-like cells were observed on the 21st day of infection ( Figure 22 and Figure 23 Then, alkaline phosphatase staining was performed ( Figure 24 ). The calculation results of the induction efficiency of alkaline phosphatase-positive iPS-like cells showed that the induction efficiency reached 0.1%-0.8%, although the induction efficiency varied with the amount of donor and vector ( Figure 25 ).

[0404] [Example 18]

[0405] Induction of iPS cells from human monocyte-2

[0406] Peripheral blood mononuclear cells were prepared from blood provided by two volunteers (donors 3 and 4) by the Ficoll method. From these peripheral blood mononuclear cells, CD14+ cells were separated as starting material by using the EasySep positive selection human CD14 positive selection kit (Veritas, Cat.No.18058). By FACS analysis using anti-CD14 antibodies, about 15% of the peripheral blood mononuclear cells were CD14+ positive cells, and it was confirmed that after separation, the purity was improved to 85% or more. These cells were infected with three types of vectors: SeV (PM) KOS / TS12ΔF vector, SeV (HNL) c-rMYC / TS15ΔF vector and SeV18 + KLF4 / TSΔF vector, so that the MOI became 5, 10, 30 and 50, and iPS cell induction was performed by the same method shown in Example 16. These results confirmed the induction of iPS-like cells positive for alkaline phosphatase staining ( Figure 26 , Figure 27 , Figure 28 ), and the maximum induction efficiency is 0.12% ( Figure 29 ).

[0407] Industrial Applicability

[0408] The present invention significantly improves the efficiency of pluripotent stem cell induction using a vector containing the KLF gene, the OCT gene, and the SOX gene in sequence. It is useful to be able to induce pluripotent stem cells with high efficiency without low-temperature culture, and in particular, the use of a temperature-sensitive vector enables rapid removal of the vector during pluripotent stem cell induction. Using the method of the present invention, it is expected that more physiologically favorable pluripotent stem cells can be obtained because the cells are not subjected to low-temperature stimulation.

Claims

1. A method for improving the efficiency of inducing pluripotent stem cells in a method for inducing pluripotent stem cells without low-temperature culture, the method comprising introducing a temperature-sensitive Sendai virus vector comprising, in sequence, a KLF gene, an OCT gene, and a SOX gene in a single vector, wherein the method comprises further introducing a Sendai virus vector comprising the KLF gene but not comprising the OCT gene and the SOX gene, wherein The temperature-sensitive Sendai virus vector sequentially comprising the KLF gene, the OCT gene, and the SOX gene in one vector is an F gene-deleted Sendai virus vector, which comprises G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, D433A, R434A, K437A, and L511F mutations in the P protein, and N1197S and K1795E mutations in the L protein, wherein the KLF gene, the OCT gene, and the SOX gene are sequentially inserted between the Sendai virus P and M genes, and the KLF gene, the OCT gene, and the SOX gene are KLF4 gene, OCT3 / 4 gene, and SOX2 gene, respectively, and The Sendai virus vector comprising the KLF gene but not the OCT gene and the SOX gene is an F gene-deficient Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, an L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, wherein the KLF gene is inserted upstream of the Sendai virus N gene, and the KLF gene is the KLF4 gene, and The method further comprises introducing a temperature-sensitive Sendai virus vector into which a MYC gene is inserted, wherein the temperature-sensitive Sendai virus vector into which a MYC gene is inserted is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, D433A, R434A, K437A, and L511F mutations in the P protein, and L1361C, L1558I, N1197S, and K1795E mutations in the L protein, wherein the MYC gene is inserted between the Sendai virus HN and L genes, and wherein the MYC gene is c-MYC, c-rMYC, or L-MYC, and The culture was carried out at 37°C.

2. Use of a Sendai virus vector comprising the KLF gene but not the OCT gene and the SOX gene in the manufacture of a medicament for improving the efficiency of pluripotent stem cell induction in a method for inducing pluripotent stem cells without low-temperature culture, wherein the method for inducing pluripotent stem cells comprises introducing a temperature-sensitive Sendai virus vector comprising, in sequence, the KLF gene, the OCT gene, and the SOX gene in a single vector, wherein The temperature-sensitive Sendai virus vector containing the KLF gene, the OCT gene and the SOX gene in sequence in one vector is an F gene-deleted Sendai virus vector, which contains G69E, T116A and A183S mutations in the M protein, A262T, G264R and K461G mutations in the HN protein, D433A, R434A, K437A and L511F mutations in the P protein, and N1197S and K1795E mutations in the L protein, wherein the KLF gene, the OCT gene and the SOX gene are sequentially inserted between the Sendai virus P and M genes, and the KLF gene, the OCT gene and the SOX gene are KLF4 gene, OCT3 / 4 gene and SOX2 gene, respectively, and The Sendai virus vector comprising the KLF gene but not the OCT gene and the SOX gene is an F gene-deficient Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, an L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, wherein the KLF gene is inserted upstream of the Sendai virus N gene, and the KLF gene is the KLF4 gene, and The method further comprises introducing a temperature-sensitive Sendai virus vector into which a MYC gene is inserted, wherein the temperature-sensitive Sendai virus vector into which a MYC gene is inserted is an F gene-deleted Sendai virus vector comprising G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, D433A, R434A, K437A, and L511F mutations in the P protein, and L1361C, L1558I, N1197S, and K1795E mutations in the L protein, wherein the MYC gene is inserted between the Sendai virus HN and L genes, and wherein the MYC gene is c-MYC, c-rMYC, or L-MYC, and Among them, the method of inducing pluripotent stem cells involves culturing at 37°C.

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