Method for producing naive human iPS cells from somatic cells

Abstract

Provided is a method for producing naive induced pluripotent stem cells from human somatic cells, said method comprising the following steps (1) to (3): (1) a step for introducing at least one vector containing an initialization factor into human somatic cells; (2) a step for culturing the somatic cells in the presence of a naive medium; and (3) a step for, after step (2), culturing the obtained cells in the presence of the naive medium under conditions where the amount of the vector per somatic cell is reduced to 30% or less compared to the amount at the start of step 3.

Description

[Technical Field]

[0001] This application relates to a method for producing naive induced pluripotent stem cells from human somatic cells. [Background technology]

[0002] Mouse induced pluripotent stem (iPS) cells and embryonic stem (ES) cells are known to be naive, a highly undifferentiated state similar to that of a preimplantation blastocyst, whereas human iPS cells and human ES cells produced by conventional methods are known to be primed, a more advanced developmental state similar to that of a postimplantation embryo.

[0003] Compared to naive mouse iPS cells, human iPS cells have been criticized for having limited pluripotency, being difficult to genetically manipulate, and exhibiting greater variability in gene expression and pluripotency among cell lines. One of the main reasons for these issues is that human iPS cells are more advanced in development than naive cells, being in a primed state. Therefore, a method for producing human pluripotent stem cells with naive characteristics is urgently needed for the advancement of regenerative medicine.

[0004] To date, the following methods for producing human naive pluripotent stem cells have been reported: (1) a method of converting primed pluripotent stem cells into naive pluripotent stem cells by expressing a specific gene and culturing them in a culture medium containing a specific component (Patent Document 1, Non-Patent Document 1); (2) a method of converting primed pluripotent stem cells into naive pluripotent stem cells by culturing them in a culture medium containing a specific component (Patent Documents 2-4); and (3) a method of producing naive iPS cells by culturing somatic cells (fibroblasts) in a culture medium containing a specific component from a certain point during the reprogramming process (Non-Patent Documents 2-4). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO2016 / 148253 [Patent Document 2] Japanese Patent Publication No. 2015-136349 [Patent Document 3] WO2016 / 179243 [Patent Document 4] WO2017 / 170849 [Non-patent literature]

[0006] [Non-Patent Document 1] Takashima Y et al., Cell, 158:1254-1269, 2014 [Non-Patent Document 2] Theunissen T et al., Cell Stem Cell, 15:471-487, 2014 [Non-Patent Document 3] Kilens S et al., Nature Communications, 9:360-, 2018 [Non-Patent Document 4] Liu X et al., Nature Methods, 14:1055-, 2017 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a method for producing naive induced pluripotent stem cells from human somatic cells. [Means for solving the problem]

[0008] This application provides the following embodiments. [1] A method for producing naive induced pluripotent stem cells from human somatic cells, comprising the following steps (1) to (3); (1) A step of introducing one or more vectors containing reprogramming factors into human somatic cells. (2) A step of culturing the somatic cells in the presence of a naive medium, and (3) After step (2), culturing the obtained cells in the presence of a naive medium under conditions such that the amount of the vector per somatic cell is reduced to 30% or less compared to the start of step 3. [2] The method according to [1], wherein the one or more vectors are negative-strand RNA virus vectors. [3] The method according to [2], wherein the one or more vectors are paramyxovirus vectors. [4] The method according to [3], wherein the one or more vectors are Sendai virus vectors. [5] The method according to any one of [1] to [4], wherein step (2) is started 1 to 10 days after step (1). [6] The method according to any one of [1] to [5], wherein step (2) is performed for 1 to 20 days. [7] The method according to any one of [1] to [6], wherein the one or more vectors are selected from the group consisting of a vector showing temperature sensitivity, a vector containing a target sequence of a microRNA specific to induced pluripotent stem cells, and a vector showing temperature sensitivity and containing a target sequence of a microRNA specific to induced pluripotent stem cells. [8] The method according to [7], wherein the one or more vectors are vectors showing temperature sensitivity. [9] The method according to [8], wherein the one or more vectors are vectors showing temperature sensitivity in a cell culture environment for establishing naive pluripotent stem cells.

[10] The method according to [9], wherein one or more vectors are Sendai virus vectors containing a TS7 mutation (TS mutation (G69E / T116A / A183S mutation in M ​​protein, A262T / G264R / K461G mutation in HN protein, L511F mutation in P protein, and N1197S / K1795E mutation in L protein) in addition to a Y942H / L1361C / L1558I mutation in L protein), a TS12 mutation (TS mutation in addition to a D433A / R434A / K437A mutation in P protein) or a TS15 mutation (TS mutation in addition to a D433A / R434A / K437A mutation in P protein and L1361C / L1558I mutation in L protein).

[11] The method according to any one of [7] to

[10] , wherein step (3) includes culturing the obtained cells at 38°C or higher.

[12] The method according to any one of [7] to

[11] , wherein the vector containing a target sequence of microRNA specific to induced pluripotent stem cells is a negative-strand RNA virus vector, and the target sequence of the microRNA is located in the coding region, 5'UTR, or 3'UTR of an NP gene or a P gene.

[13] The method according to

[12] , wherein the microRNA is miR-367.

[14] The method according to any one of the above [1] to

[13] , wherein the reprogramming factor includes the OCT gene, the SOX gene, the MYC gene, and / or the KLF gene.

[15] The method according to

[14] , wherein the one or more vectors include a vector containing the OCT gene, the SOX gene and the KLF gene, a vector containing the MYC gene and a vector containing the KLF gene.

[16] The method according to

[15] , wherein the vector containing the MYC gene is a Sendai virus vector containing the TS15 mutation.

[17] The method according to

[15] or

[16] , wherein the vector comprising the OCT gene, the SOX gene, and the KLF gene, and the vector comprising the KLF gene, are Sendai virus vectors comprising the TS12 mutation.

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

[17] , wherein in step (3), the amount of the vector per somatic cell is reduced to 30% or less compared to the amount at the start of step 3 within 12 days from the start of step 3.

[19] The method according to any one of the above [1] to

[18] , wherein the naive culture medium contains one or more compounds selected from LIF (Leukemia inhibitory factor), MEK inhibitors, GSK3 inhibitors, cAMP production promoters, TGF-β inhibitors, and PKC inhibitors.

[20] The method according to

[19] , wherein the naive culture medium is a medium selected from the group consisting of t2iLGo, 5iLAF, and tt2iLGo. [twenty one] The method according to any one of the [1] to

[20] , wherein steps (2) and (3) are performed in the absence of feeder cells. [twenty two] The method according to any one of the above [1] to

[21] , wherein the human somatic cells are mononuclear cells or fibroblasts.

[0009] [twenty three] A kit for producing naive induced pluripotent stem cells from human somatic cells, including the following: One or more vectors containing reprogramming factors, wherein the one or more vectors are selected from the group consisting of temperature-sensitive vectors, vectors containing target sequences of microRNAs specific to induced pluripotent stem cells, and vectors that are temperature-sensitive and contain target sequences of microRNAs specific to induced pluripotent stem cells; and Culture medium for naive cells. [twenty four] The kit according to

[23] , wherein one or more vectors are negative-strand RNA virus vectors. [twenty five] The kit according to

[24] , wherein one or more vectors are paramyxovirus vectors.

[26] The kit according to

[25] , wherein one or more vectors are Sendai virus vectors.

[27] The kit according to any one of

[23] to

[26] , wherein one or more of the vectors are temperature-sensitive vectors.

[28] The kit according to

[27] , wherein one or more of the vectors are vectors that exhibit temperature sensitivity in a cell culture environment for establishing naive pluripotent stem cells.

[29] The kit according to

[28] , wherein one or more vectors are Sendai virus vectors containing a TS7 mutation (TS mutation (G69E / T116A / A183S mutation in M ​​protein, A262T / G264R / K461G mutation in HN protein, L511F mutation in P protein, and N1197S / K1795E mutation in L protein) in addition to a Y942H / L1361C / L1558I mutation in L protein), a TS12 mutation (TS mutation in addition to a D433A / R434A / K437A mutation in P protein) or a TS15 mutation (TS mutation in addition to a D433A / R434A / K437A mutation in P protein and L1361C / L1558I mutation in L protein).

[30] The kit according to any one of

[23] to

[29] , wherein the vector containing a target sequence of microRNA specific to induced pluripotent stem cells is a negative-strand RNA virus vector, and the target sequence of the microRNA is located in the coding region, 5'UTR, or 3'UTR of an NP gene or a P gene.

[31] The kit according to

[30] , wherein the microRNA is miR-367.

[32] A kit according to any one of

[23] to

[31] , comprising the reprogramming factor OCT gene, SOX gene, MYC gene and / or KLF gene.

[33] The kit according to

[32] , comprising one or more vectors, a vector containing the OCT gene, the SOX gene and the KLF gene, a vector containing the MYC gene, and a vector containing the KLF gene.

[34] The kit according to

[33] , wherein the vector containing the MYC gene is a Sendai virus vector containing the TS15 mutation.

[35] The kit according to

[33] or

[34] , wherein the vector containing the OCT gene, the SOX gene, and the KLF gene, and the vector containing the KLF gene, are Sendai virus vectors containing the TS12 mutation.

[36] The kit according to any one of

[23] to

[35] , wherein the naive culture medium contains one or more compounds selected from LIF (Leukemia inhibitory factor), MEK inhibitors, GSK3 inhibitors, cAMP production promoters, TGF-β inhibitors, and PKC inhibitors.

[37] The kit according to

[36] , wherein the naive culture medium is a medium selected from the group consisting of t2iLGo, 5iLAF, and tt2iLGo.

[38] The kit according to any one of the above

[23] to

[37] , wherein the human somatic cells are mononuclear cells or fibroblasts.

[39] Culture supernatant of naive induced pluripotent stem cells produced by the method described in any of the above [1] to

[22] .

[40] Cosmetics containing the culture supernatant of naive induced pluripotent stem cells manufactured by any of the methods described in [1] to

[22] above as a raw material. [Effects of the Invention]

[0010] This application provides a method for producing naive induced pluripotent stem cells from human somatic cells. It also provides a vector set and a kit for producing naive induced pluripotent stem cells from human somatic cells. The naive induced pluripotent stem cells obtained by the method of this application are substantially free of vectors containing reprogramming factors and exhibit high phenotypic stability. [Brief explanation of the drawing]

[0011] [Figure 1] Structure of the Sendai virus vector (CytoTune-iPS2.0L) conventionally used to produce naive iPS cells from somatic cells. [Figure 2] A protocol for producing naive iPS cells from human dermal fibroblasts (HDF). The vertical bars indicate an example of the timing for changing the culture medium. [Figure 3A] Phase-contrast microscopy image of HDF-derived naive iPS cells that have been passaged 45 times. [Figure 3B] Immunostaining images of naive iPS cells derived from HDF after 45 passages, using pluripotency markers. The three images side-by-side represent the same field of view. [Figure 4] The effect of high-temperature culture on the genome quantity of Sendai virus vectors. [Figure 5] A protocol for producing naive iPS cells from human peripheral blood mononuclear cells (PBMCs). The vertical bars indicate an example of the timing of medium changes. [Figure 6] Quantitative evaluation of the genomic amount of Sendai virus vector in PBMC-derived naive iPS cells, per passage from day 14. [Figure 7] Principal component analysis integrating single-cell data from each cell type. [Figure 8] Expression patterns of imprinted genes and X chromosome-related genes in each cell type. [Figure 9A] Density bean plot showing global methylation across different cell types. [Figure 9B]Principal component analysis showing global methylation across different cell types. [Figure 9C] The percentage of 5-methylcytosine in total cytosine in HDF-derived naive iPS cells obtained in this example. [Figure 10] Early differentiation potential of the three germ layer components of HDF or PBMC-derived naive iPS cells after long-term passage (P16-45). Reset Naive ESCs (in-house) were used as a control. [Figure 11A] Phase-contrast images and immunofluorescence staining for SeV in naive human iPSCs initialized with CytoTune-iPS 2.0L (nCT2.0L_1) 94 days after SeV infection. Scale bar represents 100 μm. [Figure 11B] qRT-PCR analysis of SeV genome expression in naive iPSCs initialized using CytoTune 2.0 or a 2.0L SeV initialization kit. Values ​​are normalized by GAPDH expression levels and shown as mean ± sd. n=3 for each point. [Figure 11C] qRT-PCR analysis of GAPDH-normalized SeV vector genome expression in naive human iPSCs initialized with CytoTune 2.0 or 2.0L. Data are shown as mean ± sd. n=3 for each point. In this experiment, primers designed to specifically detect each SeV vector and SYBR Green were used. [Figure 11D] Schematic diagram of the SeV vector and modified initialization cocktail. The SeV18+KLF4 / TSΔF(KLF4 / TS) vector in CytoTune-2.0 or the 2.0L initialization kit was replaced with one of the following three modified vectors: SeV18+KLF4 / TS12ΔF(KLF4 / TS12), SeV18+KLF4 / PmiR367T2 / TSΔF(KLF4 / miR / TS), or SeV18+KLF4 / PmiR367T2 / TS12ΔF(KLF4 / miR / TS12). KLF4 / miR / TS12 was newly developed in this embodiment. [Figure 11E]qRT-PCR analysis of hsa-miRNA367-3p expression in HDF and PSC normalized by hsa-miRNA423-3p expression. Data are shown as mean ± sd. n=3. ND indicates no detection after 40 amplification cycles. [Figure 11F] Experimental design for inducing naive human iPSCs from HDF or PBMC using a modified SeV-OSKL cocktail, and for changes in incubation temperature after the generation of naive human iPSCs. [Figure 11G] Representative phase-contrast images of PBMCs 14 days after infection with CytoTune-iPS 2.0 SeV vector. The scale bar represents 200 μm. [Figure 12A] Number of naive human iPSC colonies prepared from HDF on day 14. SeV-KLF4 / TS and three modified SeV-KLF4 vectors were co-infected with SeV-KOS and SeV-LMYC, respectively. Data are shown as mean ± sd. n=3, and * indicates P<0.05. [Figure 12B] Sensitivity of SeV genome detection in cells 14 days after SeV vector infection, as analyzed by qRT-PCR. The X-axis shows the proportion of SeV-positive cells among SeV-negative cells. [Figure 12C] qRT-PCR analysis of SeV genome expression in naive iPSCs derived from HDFs reprogrammed with SeV-KOS, SeV-LMYC, and each SeV-KLF4 vector. Data are shown as mean ± sd. n=3 for each point. [Figure 12D] qRT-PCR analysis of SeV genome expression in HDF-derived naive iPSCs (nOSKL_1, 2) and PBMC-derived naive iPSCs (nOSKL_3, 4) initialized with SeV-KOS, SeV-LMYC, and SeV-KLF4 / miR / TS12 vectors on day 14 and day 34. Data are shown as mean ± sd. n=3. [Figure 12E]Representative phase-contrast images of naive iPSCs on iMEF feeder cells initialized with SeV-KOS, SeV-LMYC, and SeV-KLF4 / miR / TS12 vectors, and naive iPSCs in feeder-free environments. Scale bar indicates 200 μm. [Figure 12F] Cell proliferation rate of the established nOSKL_1-4. [Figure 13A] Relative expression of naive pluripotency markers, normalized by GAPDH expression and analyzed by qRT-PCR. nH9 values ​​are set to 1.0. Data are shown as mean ± sd. n=3. [Figure 13B] Relative expression of primed pluripotency markers, normalized by GAPDH expression and analyzed by qRT-PCR. Data are shown as mean ± sd. n=3. [Figure 13C] Heatmaps of RNA-seq data showing the expression levels of common pluripotency-related marker genes in naive and primed human PSCs, and in primed and naive PSCs. [Figure 13D]References (Takashima, Y. et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158, 1254-1269, doi:10.1016 / j.cell.2014.08.029 (2014).; Theunissen, TW et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell stem cell 15, 471-487, doi:10.1016 / j.stem.2014.07.002 (2014).; Yan, L. et al. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nature structural & molecular biology 20, 1131-1139, doi:10.1038 / nsmb.2660 PCA of RNA-seq data of primed and naive human PSCs in this example compared with reset naive human iPSCs and preimplantation embryo samples from (2013). [Figure 14A] Bean plots showing global DNA methylation levels in primed and naive human PSCs analyzed using DNA methylation arrays. The horizontal lines in the bean plots represent the average methylation beta values. [Figure 14B] Global DNA methylation PCA in primed and naive human PSCs using DNA methylation arrays. [Figure 14C]Representative RNA-FISH images of naive and primed human iPSCs detecting the X-linked genes UTX (which avoids X chromosome inactivation), HUWE1 (which undergoes X chromosome inactivation), and XIST. UTX was analyzed to ensure that only PSCs with a normal X chromosome were included in the quantification. The scale bar represents 20 μm. [Figure 14D] Quantification of RNA-FISH patterns of XIST and HUWE1 in cells expressing both alleles of UTX. 100 cells were analyzed from each cell line. Most naive human iPSCs showed X reactivation indicated by both allele HUWE1 expression. Some cells from nOSKL_3 and 4 showed both allele HUWE1 expression in addition to both allele XIST expression, which is similar to the expression pattern of preimplantation epiblasts. [Figure 15A] A heatmap of genes encoding electron transport chain proteins located in the inner mitochondrial membrane. These genes reflect oxidative phosphorylation activity, are classified by mitochondrial complexes, and are hierarchically clustered. [Figure 15B] Comparison of metabolic capacity between naive and primed iPSCs using an extracellular flux analyzer. [Figure 15C] Quantification of respiratory reserve capacity in naive and primed iPSCs. [Figure 15D] Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in naive and primed iPSCs. [Figure 16A] A schematic diagram of the differentiation into primitive endoderm. [Figure 16B] Representative phase-contrast images of primitive endoderm derived from primed ESCs (H9; left image) and SeV(-) naive iPSCs (nOSKL_4; right image). The scale bar indicates 100 μm. [Figure 16C]Quantification of PDGFRA and ANPEP expression in primed ESCs (H9), naive ESCs (nH9), SeV(-) naive iPSCs (nOSKL_4), and SeV(+) naive iPSCs (nCT2.0L_4) by flow cytometry. [Figure 16D] Median signal value in PDGFRA / ANPEP co-positive cells, as determined by flow cytometry. [Figure 17A] A schematic diagram of differentiation into trophectoderm. [Figure 17B] Representative phase-contrast images of trophectoderm derived from primed ESCs (H9; left image) and SeV(-) naive iPSCs (nOSKL_4; right image). The scale bar indicates 200 μm. [Figure 17C] Quantification of TACSTD2, ENPEP, HAVCR1, and HLA-ABC expression in primed ESCs (H9), naive ESCs (nH9), SeV(-) naive iPSCs (nOSKL_4), and SeV(+) naive iPSCs (nCT2.0L_4) by flow cytometry. [Figure 17D] Percentage of TACSTD2(+) / ENPEP(+) and HAVCR1(+) cells by flow cytometry. Each plot represents a single experiment. [Modes for carrying out the invention]

[0012] In this specification and in the claims, when a number is accompanied by the term “approximately,” it is intended to include a range of ±10% of that value. For example, “approximately 20” includes “18 to 22.” A range of numbers includes all numbers between the two endpoints and the numbers at both endpoints. The “approximately” in relation to a range applies to both endpoints of that range. Thus, for example, “approximately 20 to 30” includes “18 to 33.”

[0013] [vector] In this application, a vector is any vector capable of introducing a gene into a somatic cell, and can be a viral vector or a non-viral vector, for example, a viral vector. In this disclosure, a viral vector is a vector having a genomic nucleic acid derived from the virus, and capable of expressing a transgene by incorporating the transgene into the nucleic acid. The vector in this application is, for example, a non-chromosomal-integrated viral vector. A non-chromosomal-integrated viral vector is a viral vector derived from a virus that can introduce a gene into a target cell, and is a carrier that does not pose a risk of the introduced gene being integrated into the host's chromosome (nuclear-derived chromosome). In this application, a viral vector includes not only infectious viral particles, but also non-infectious viral particles, a viral core, or a complex consisting of a viral genome and a viral protein, and includes complexes that have the ability to express the gene they carry when introduced into a cell. For example, in RNA viruses, a ribonucleoprotein (the core part of the virus) consisting of a viral genome and a viral protein that binds to it can express a transgene in the cell when introduced into a cell (WO00 / 70055). Introduction into cells can be carried out using a transfection reagent or the like as appropriate. Such ribonucleoproteins (RNPs) are also included in viral vectors in this invention.

[0014] In this specification, "no risk of integration into host chromosomes" means that when a viral vector is introduced, the frequency of integration into host chromosomes is sufficiently low. Preferably, the frequency of integration into host chromosomes is 5 × 10 when, for example, a human fibrosarcoma-derived cell line HT1080 (ATCC CCL121) is infected with 10 PFU / cell. -4 The following, more preferably 10 -4 The following, more preferably 10 -5 The following, more preferably 10 -6 The following, more preferably 10 -7The following applies: Non-chromosomal integration viral vectors are preferably RNA viruses. In this disclosure, RNA viruses mean viruses that have an RNA genome and do not have a DNA phase in their life cycle. In this disclosure, RNA viruses do not have reverse transcriptase (i.e., retroviruses are not included). That is, in viral replication, the viral genome is replicated by RNA-dependent RNA polymerase without the use of DNA. Because RNA viruses do not have a DNA phase, the use of RNA viral vectors minimizes the risk of integration into the host chromosome. RNA viruses include single-stranded RNA viruses (including positive-sense and negative-sense RNA viruses) and double-stranded RNA viruses. They also include enveloped viruses and non-enveloped viruses, but vectors derived from enveloped viruses are preferably used. In this application, RNA viruses specifically include viruses belonging to the following families. Arenaviridae family, including Lassa virus Orthomyxoviridae, the family of viruses including influenza viruses. Coronaviridae (coronavirus family), including SARS virus Togaviridae, the family that includes rubella virus. Paramyxoviridae, including mumps virus, measles virus, Sendai virus, and RSV. Picornaviridae, which includes poliovirus, coxsackievirus, and echovirus. Filoviridae, including Marburg virus and Ebola virus Flaviviridae viruses, including yellow fever virus, dengue fever virus, hepatitis C virus, and hepatitis G virus. Bunyaviridae (family including Bunyavirus, Hantavirus, Nairovirus, and the Phlebovirus genus) Rhabdoviridae, the family that includes the rabies virus. Reoviridae

[0015] Non-chromosomal-integrated viral vectors include, for example, negative-strand RNA viral vectors. A negative-strand RNA viral vector is a vector consisting of a virus whose genome contains a negative-strand RNA (the antisense strand relative to the sense strand that codes for viral proteins). Negative-strand RNA is also called negative-strand RNA. A negative-strand RNA virus is, for example, a single-stranded negative-strand RNA virus (also called a non-segmented negative-strand RNA virus). A "single-stranded negative-strand RNA virus" means a virus that has a single-stranded negative-strand [i.e., negative-strand] RNA in its genome. Such viruses include those belonging to families such as Paramyxovirus (Paramyxoviridae; including Paramyxovirus, Morbillivirus, Rubulavirus, and Pneumovirus genera), Rhabdovirus (Rhabdoviridae; including Vesiculovirus, Lyssavirus, and Ephemerovirus genera), and Filovirus (Filoviridae), and are taxonomically classified as the order Mononegavirales (Virus, Vol. 57, No. 1, pp. 29-36, 2007; Annu. Rev. Genet. 32, 123-162, 1998; Fields virology fourth edition, Philadelphia, Lippincott-Raven, 1305-1340, 2001; Microbiol. Immunol. 43, 613-624, 1999; Field Virology, Third edition pp. 1205-1241). 1996).

[0016] Negative-strand RNA viral vectors include, for example, paramyxovirus vectors. Paramyxovirus vectors are viral vectors derived from viruses of the Paramyxoviridae family. For example, the Sendai virus, a Paramyxoviridae virus, can be cited. Other examples include Newcastle disease virus, mumps virus, measles virus, respiratory syncytial virus (RSV), rinderpest virus, distemper virus, monkey parainfluenza virus (SV5), human parainfluenza virus types 1, 2, and 3, influenza virus of the Orthomyxoviridae family, vesicular stomatitis virus of the Rhabdoviridae family, and rabies virus.

[0017] Further examples of viruses that may be used in this application include, for example, Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), phocine distemper virus (PDV), canine distemper virus (CDV), dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MV), rice pest virus (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, the virus is selected from the group consisting of Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), phocinedistemper virus (PDV), canine distemper virus (CDV), dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MV), rice pest virus (RPV), Hendra virus (Hendra), and Nipah virus (Nipah).

[0018] The vectors used in this application are, for example, viruses or derivatives belonging to the Paramyxovirus subfamily (including the genera Respirovirus, Rubravirus, and Morbillivirus), and for example, viruses or derivatives belonging to the genus Respirovirus (also called Paramyxovirus). Derivatives include viruses in which the viral gene has been modified so as not to impair the ability of the virus to deliver genes, and chemically modified viruses. Examples of Respirovirus viruses to which the present invention can be applied include human parainfluenza virus type 1 (HPIV-1), human parainfluenza virus type 3 (HPIV-3), bovine parainfluenza virus type 3 (BPIV-3), Sendai virus (also called mouse parainfluenza virus type 1), and monkey parainfluenza virus type 10 (SPIV-10). The paramyxovirus most preferably is Sendai virus.

[0019] Negative-strand RNA viruses generally contain a complex of RNA and protein (ribonucleoprotein; RNP) inside their envelope. The RNA contained in the RNP is the single-stranded RNA of the negative (-) strand, which is the genome of the negative-strand RNA virus. This single-stranded RNA binds to NP protein, P protein, and L protein to form the RNP. The RNA contained in this RNP serves as a template for transcription and replication of the viral genome (Lamb, RA, and D. Kolakofsky, 1996, Paramyxoviridae: The viruses and their replication. pp.1177-1204. In Fields Virology, 3rd edn. Fields, BN, DM Knipe, and PM Howley et al. (ed.), Raven Press, New York, NY).

[0020] The "NP, P, M, F, HN, and L genes" of negative-strand RNA viruses refer to the genes encoding the nucleocapsid, phospho, matrix, fusion, hemagglutinin-neuraminidase, and large protein, respectively. The nucleocapsid (NP) protein is a protein essential for binding to genomic RNA and for the genomic RNA to have template activity. Generally, the NP gene is sometimes referred to as the "N gene." The phospho (P) protein is a phosphorylated protein, a small subunit of RNA polymerase. The matrix (M) protein plays a role in maintaining the viral particle structure from the inside. The fusion (F) protein is a membrane fusion protein involved in entry into host cells, and the hemagglutinin-neuraminidase (HN) protein is a protein involved in binding to host cells. The large (L) protein is the large subunit of RNA polymerase. Each of the above genes has its own transcriptional regulatory unit, and a single mRNA is transcribed from each gene, which then transcribes into a protein. In addition to the P protein, the P gene translates a non-structural protein (C) that is translated using a different ORF, and a protein (V) that is produced by RNA editing during the reading of the P protein mRNA. For example, the genes of each virus belonging to the Paramyxovirinae subfamily are generally denoted in order from 3' as follows: Respirovirus genus NP / C / VMF HN - L Rubravirus genus NP / VMF HN (SH) L Morbillivirus genus NP / C / VMFH - L

[0021] For example, the accession numbers in the database of the base sequences of each gene of the Sendai virus are as follows: For the N gene: M29343, M30202, M30203, M30204, M51331, M55565, M69046, X17218; For the P gene: M30202, M30203, M30204, M55565, M69046, X00583, X17007, X17008; For the M gene: D11446, K02742, M30202, M30203, M30204, M69046, U31956,X00584, X53056; For the F gene: D00152, D11446, D17334, D17335, M30202, M30203, For M30204, M69046, X00152, X02131, and for HN genes see D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, X56131, and for L genes see D00053, M30202, M30203, M30204, M69040, X00587, X58886. Other examples of viral genes encoded by viruses include, for the N gene, CDV, AF014953; DMV, X75961; HPIV-1, D01070; HPIV-2, M55320; HPIV-3, D10025; Mapuera, X85128; Mumps, D86172; MeV, K01711; NDV, AF064091; PDPR, X74443; PDV, X75717; RPV, X68311; SeV, X00087; SV5, M81442; and Tupaia, AF079780; and for the P gene, CDV, X51869; DMV, Z47758; HPIV-1, M74081; HPIV-3, X04721; HPIV-4a, M55975; HPIV-4b, M55976; Mumps, D86173; MeV, M89920; NDV, M20302; PDV, X75960; RPV, X68311; SeV, M30202; SV5, AF052755;and Tupaia, AF079780; for the C gene: CDV, AF014953; DMV, Z47758; HPIV-1, M74081; HPIV-3, D00047; MeV, 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; MeV,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; HPIV-2, M60182; HPIV-3, X05303, HPIV-4a, D49821; HPIV-4b, D49822; Mumps, D86169; MeV, AB003178; NDV, AF048763; PDPR, Z37017; PDV, AJ224706; RPV, M21514; SeV, D17334; and SV5, AB021962; for the 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; MeV, K01711; NDV, AF204872; PDPR, X74443; PDV, Z36979; RPV, AF132934; SeV, U06433; and SV-5, S76876, and for the L gene CDV, AF014953; DMV, AJ608288; HPIV-1, AF117818; HPIV-2, X57559; HPIV-3, AB012132; Mumps, AB040874; MeV, K01711;Examples include NDV, AY049766; PDPR, AJ849636; PDV, Y09630; RPV, Z30698; and SV-5, D13868. However, multiple strains of each virus are known, and genes consisting of sequences other than those exemplified above exist depending on the strain. Sendai virus vectors containing viral genes derived from any of these genes are useful as vectors for the present invention. Furthermore, with respect to the P protein, its functional site is the region containing the N-binding site, L-binding site, and oligomer formation site on the C-terminal side (320-568 on the C-terminal side of the P protein in the case of SeV) (Blanchard L. et al., Virology. (2004) 319, 201-211.), and in the present invention, it is preferable that the P protein contains at least this region. For example, the vector of the present application includes a nucleotide sequence that has 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the coding sequence of any of the above-mentioned viral genes (for example, in the case of the P gene of SeV, this may be the C-terminal sequence, for example, the amino acid sequence from position 479 to 568 or the amino acid sequence from position 320 to 568). The vector of the present application also includes a nucleotide sequence that encodes an amino acid sequence that has 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence encoded by the coding sequence of any of the above-mentioned viral genes (for example, in the case of the P protein of SeV, this may be the C-terminal sequence, for example, the amino acid sequence from position 479 to 568 or the amino acid sequence from position 320 to 568). Furthermore, the vector of this application includes a nucleotide sequence encoding a polypeptide containing an amino acid sequence in which up to 10, preferably up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or 1 amino acid is substituted, inserted, deleted, and / or added in the amino acid sequence encoded by the coding sequence of any of the above-mentioned viral genes (for example, in the case of the P gene of SeV, this may be the C-terminal sequence, for example, the amino acid sequence from position 479 to 568 or the amino acid sequence from position 320 to 568).

[0022] The sequences referenced by database accession numbers, such as nucleotide sequences and amino acid sequences, described herein refer to sequences at the filing date and priority date of this application, for example, and can be identified as sequences at either the filing date or priority date of this application, preferably as sequences at the filing date of this application. Sequences at each point in time can be identified by referring to the revision history of the database.

[0023] Furthermore, the negative-strand RNA virus of this invention may be derived from natural strains, wild-type strains, mutant strains, laboratory passaged strains, and artificially constructed strains. For example, Sendai virus strain Z can be cited (Medical Journal of Osaka University Vol.6, No.1, March 1955 p1-15). In other words, the virus may be a viral vector having a structure similar to a virus isolated from nature, or it may be a virus artificially modified by genetic engineering. For example, it may be a virus in which any of the genes of the wild-type virus have a mutation or deletion. For example, a virus having a mutation or deletion in at least one gene encoding the viral envelope protein or outer shell protein can be suitably used. Such a viral vector is, for example, a viral vector that can replicate its genome in infected cells but cannot form infectious viral particles. Such a viral vector with impaired transmissibility is highly safe because there is no concern about spreading infection to the surroundings. For example, a negative-strand RNA virus can be used that does not contain at least one gene encoding an envelope protein or spike protein such as F and / or HN, 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 the proteins necessary for genome replication (e.g., N, P, and L proteins), the genome can be amplified in infected cells. To produce a knockout virus, for example, the knockout gene product or a protein that can complement it is exogenously supplied to a virus-producing cell (WO00 / 70055 and WO00 / 70070; Li, H.-O. et al., J. Virol. 74(14) 6564-6569 (2000)). Furthermore, a method is known for recovering the viral vector as non-infectious viral particles (VLPs) without completely compensating for the missing viral protein (WO00 / 70070).Furthermore, when recovering a viral vector as an RNP (for example, an RNP consisting of N, L, and P proteins and genomic RNA), the vector can be manufactured without complementing the envelope protein.

[0024] Furthermore, the viruses referred to in this application are not limited to naturally occurring viruses, but also include, for example, artificially created viruses. For example, the viruses referred to in this application include those in which mutations have been introduced into the nucleic acid sequence to optimize codons, as well as chimeric viruses (including, for example, chimeras between the same type of virus, and chimeric viruses between different types of viruses (e.g., a chimera of PIV and SeV)) (J. Virol. 1995, 69, 849-855).

[0025] Furthermore, in this application, viral proteins such as N, L, and P proteins do not have to be wild-type, as long as they retain the function of expressing genes in introduced cells. For example, modified proteins with appropriately attached peptides such as tags, proteins with modified codons, or proteins in which a portion of the amino acid sequence of the wild-type protein is deleted without losing function can be used as appropriate. In this application, N, L, and P proteins include such modified proteins and deletion-type proteins. For example, if a portion of the C-terminus of the P protein is present, the rest of the region is not essential for viral vector expression.

[0026] A viral vector is, for example, a complex of RNA derived from the negative (-) single-stranded RNA genome of the virus and a protein derived from a viral protein that binds to the negative (-) single-stranded RNA of the virus, and which binds to the RNA, and which expresses the loaded gene when introduced into a cell. A protein that binds to the negative (-) single-stranded RNA means a protein that directly and / or indirectly binds to the negative (-) single-stranded RNA and forms a complex with the negative (-) single-stranded RNA. The complex includes a complex consisting of negative (-) single-stranded RNA derived from a negative-stranded RNA virus and a protein derived from the negative-stranded RNA virus that binds to it (e.g., NP, P, and L proteins). In this application, "derived from a negative-stranded RNA virus" means that the components of the negative-stranded RNA virus (including proteins and RNA) are in their original state or in a modified state. For example, a protein or RNA prepared by modifying the protein or RNA of a negative-stranded RNA virus is a protein or RNA "derived from a negative-stranded RNA virus". The vector of this application may be a viral vector having, for example, envelope proteins (F, HN, and M proteins) and having the structure of a viral particle. Alternatively, it may be an RNP vector that does not have a viral envelope and is the RNP itself.

[0027] In negative-strand RNA viruses, NP, P, and L proteins bind to (-) single-stranded RNA and play essential roles in genomic RNA replication and protein expression (hereinafter, NP, P, and L proteins may be referred to as "genomic RNA-binding proteins"). The NP protein is a protein that binds very strongly to genomic RNA and confers template activity to it. Genomic RNA has template activity for RNA synthesis only when bound to the NP protein; it has no template activity at all when not bound to the NP protein. The P protein binds to genomic RNA as a small subunit of RNA polymerase, and the L protein binds to genomic RNA as a large subunit of RNA polymerase. Therefore, in negative-strand RNA viruses, if even one of the NP, P, or L proteins is missing, genomic RNA replication will not occur.

[0028] The genes contained in the genomic RNA of the vector of this application may be the original virus-derived gene sequences, or they may have mutations introduced into them. For example, a person skilled in the art can introduce minor mutations into each gene on the genomic RNA using known methods, such as those that do not impair the function of each protein. For example, site-specific mutations can be introduced using methods such as PCR or cassette mutation, or random mutations can be introduced using chemical reagents or random nucleotides.

[0029] For example, numerous mutations are known in envelope proteins and spike proteins, including attenuation mutations and temperature-sensitive mutations. Viruses possessing these mutated protein genes can be suitably used in this application. Preferably, vectors with reduced cytotoxicity can be used. The cytotoxicity of a vector can be measured, for example, by quantifying the release of lactate dehydrogenase (LDH) from vector-infected cells. The lower the LDH release, the lower the cytotoxicity. For example, a vector with significantly reduced cytotoxicity compared to the wild type can be used. The degree of reduced cytotoxicity can be determined, for example, by infecting human-derived HeLa cells (ATCC CCL-2) or monkey-derived CV-1 cells (ATCC CCL-70) with an MOI (infectious titer) of 3 and culturing them at 35-37°C (e.g., 37°C) for 3 days, resulting in a significantly lower LDH release in the culture medium compared to the wild type, such as a reduction of 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 50% or more. Mutations that reduce cytotoxicity also include temperature-sensitive mutations.

[0030] Temperature sensitivity, where activity is significantly reduced at normal temperatures (e.g., 37-38°C) compared to low temperatures (e.g., 30-36.5°C), can be determined by measuring the viral replication rate or the expression level of its associated genes at the normal temperature of the viral host. The lower the viral replication rate and / or the expression level of its associated genes compared to non-mutated viruses, the higher the temperature sensitivity.

[0031] The viral vectors that can be used in this application preferably have deletions or mutations in at least one, more preferably at least two, three, four, five, or more viral genes. The deletions and mutations may be introduced in any combination for each gene. Here, the mutations may be functional mutations or temperature-sensitive mutations. Using such modified viral vectors may be useful in reducing cytotoxicity in host cells or facilitating vector removal. For example, the viral vectors preferably used in this application have deletions or mutations in at least two viral genes. Such viruses include those with deletions in 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 deletions in at least one viral gene. The at least two mutated or deleted viral genes are preferably genes that encode envelope constituent proteins. For example, the negative-strand RNA viral vectors of this application preferably lack at least the F gene or the M gene. For example, vectors that delete the F gene and further delete the M and / or HN genes, or that further have mutations (e.g., temperature-sensitive mutations) in the M and / or HN genes, are preferably used in this application. More preferably, the vectors used in this application have deletions or mutations in at least three viral genes (preferably at least three genes encoding envelope constituent proteins; F, HN, and M). Such viral vectors include those with deletions of at least three genes, those with mutations in at least three genes, those with mutations in at least one gene and deletions in at least two genes, and those with mutations in at least two genes and deletions in at least one gene. More preferably, for example, vectors that delete the F gene and further delete the M and HN genes, or that further have mutations (e.g., temperature-sensitive mutations) in the M and HN genes, are preferably used in this application.Furthermore, a vector having, for example, a deletion of the F gene, a further deletion of the M or HN gene, and a mutation (e.g., a temperature-sensitive mutation) in the remaining M or HN gene is preferably used in this application. Such mutant viruses can be produced according to known methods.

[0032] For example, temperature-sensitive mutations in the M gene include amino acid substitutions at any site arbitrarily selected from the group consisting of positions 69 (G69), 116 (T116), and 183 (A183) in the Sendai virus M protein, or at the corresponding site in the negative-strand RNA virus M protein (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246). Viruses having a genome encoding a mutant M protein in which any of the above three sites, preferably any combination of two sites, or more preferably all three sites, are substituted with other amino acids are suitably used in this application.

[0033] Amino acid mutations preferably involve substitution with other amino acids having different chemical properties in the side chain. For example, substitution with amino acids whose BLOSUM62 matrix value (Henikoff, S. and Henikoff, JG (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) is 3 or less, preferably 2 or less, more preferably 1 or less, and more preferably 0. Specifically, for Sendai virus M protein, G69, T116, and A183 can be substituted with Glu(E), Ala(A), and Ser(S), respectively. For other negative-strand RNA virus M proteins, the amino acids at the corresponding sites can also be substituted with Glu(E), Ala(A), and Ser(S), respectively. Furthermore, it is possible to utilize mutations homologous to those in the M protein of the measles virus temperature-sensitive strain P253-505 (Morikawa, Y. et al., Kitasato Arch. Exp. Med. 1991: 64; 15-30). Mutations can be introduced using, for example, oligonucleotides, according to known mutagenesis methods.

[0034] In this specification, the amino acid sequence of Sendai virus is based on the amino acid sequence of the F gene-deficient Sendai virus derived from strain Z, as described in GenBank ACCESSION number: AB855655. Therefore, the Xth amino acid residue of Sendai virus refers to the amino acid residue corresponding to the Xth amino acid residue in the amino acid sequence described in GenBank ACCESSION number: AB855655. In this specification, "corresponding position" to a given position in an amino acid sequence refers to the position in another amino acid sequence that corresponds to a given position in an alignment between one amino acid sequence and another amino acid sequence. Alignment can be performed, for example, using known gene analysis software. Specific examples of gene analysis software include DNASIS from Hitachi Solutions, GENETYX from Genetics, and FASTA, BLAST, and ClustalW, which are publicly available from DDBJ.

[0035] Furthermore, examples of temperature-sensitive mutations in the HN gene include amino acid substitutions at sites arbitrarily selected from the group consisting of positions 262 (A262), 264 (G264), and 461 (K461) of the Sendai virus HN protein, or at corresponding sites in the negative-strand RNA virus HN protein (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246). Viruses having a genome encoding a mutant HN protein in which one of the three sites, preferably any combination of two sites, or more preferably all three sites, are substituted with other amino acids are suitably used in this application. Similarly, amino acid substitutions are preferably made with other amino acids having different chemical properties in the side chain. A preferred example is the substitution of A262, G264, and K461 of the Sendai virus HN protein with Thr (T), Arg (R), and Gly (G), respectively. For other negative-strand RNA virus HN proteins, the corresponding amino acids can be substituted with Thr (T), Arg (R), and Gly (G), respectively. Furthermore, mutations can be introduced into the 464th and 468th amino acids of the HN protein, for example, using the temperature-sensitive vaccine strain Urabe AM9 of the mumps virus as a reference (Wright, KE et al., Virus Res. 2000: 67; 49-57).

[0036] Furthermore, the vector of this application may have mutations in the P gene and / or L gene. Specifically, such mutations include a mutation in the 86th Glu (E86) of the SeV P protein and a substitution of the 511th Leu (L511) of the SeV P protein with another amino acid. Substitutions of corresponding sites are also possible for other negative-strand RNA virus P proteins. As with the above, amino acid substitutions are preferably made to other amino acids with different chemical properties in the side chain. Specifically, examples include substitution of the 86th amino acid with Lys and substitution of the 511th amino acid with Phe. In the L protein, examples include substitution of the 1197th Asn (N1197) and / or the 1795th Lys (K1795) of the SeV L protein with other amino acids, and substitutions of corresponding sites in other negative-strand RNA virus L proteins. As with the above, amino acid substitutions are preferably made to other amino acids with different chemical properties in the side chain. Specifically, examples include substitution of the 1197th amino acid with Ser and substitution of the 1795th amino acid with Glu. Mutations in the P and L genes can significantly enhance the effects of suppressing persistent infectivity, secondary particle release, or cytotoxicity. Furthermore, combining these with mutations and / or deletions of envelope protein genes can dramatically increase these effects. In the L gene, examples include substitutions of the 1214th Tyr (Y1214) and / or 1602nd Met (M1602) amino acids in the SeV L protein, and substitutions of corresponding sites in other negative-strand RNA virus L proteins. As with the above, amino acid substitutions are preferably those with different chemical properties in the side chain. Specifically, examples include substitution of the 1214th amino acid with Phe and substitution of the 1602nd amino acid with Leu. The mutations exemplified above can be combined arbitrarily.

[0037] 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 missing or deleted, as are F gene-deficient or deleted Sendai virus vectors having cytotoxicity similar to or less than these, and / or temperature sensitivity similar to or greater than these. For other negative-strand RNA viruses, vectors in which the corresponding sites are similarly substituted and the F gene is missing or deleted, as well as F gene-deficient or deleted vectors having cytotoxicity similar to or less than these, and / or temperature sensitivity similar to or greater than these, are preferred. To give specific examples of substitutions, for example, for the M protein, substitutions of G69E, T116A, and A183S can be given; for the HN protein, substitutions of A262T, G264R, and K461G can be given; for the P protein, substitution of L511F can be given; and for the L protein, substitutions of N1197S and K1795E can be given.

[0038] Amino acid mutations may involve substitution with other desired amino acids, but preferably, as described above, substitution with amino acids having different chemical properties in their side chains. For example, amino acids can be classified into groups such as basic amino acids (e.g., lysine, arginine, histidine), acidic amino acids (e.g., aspartic acid, glutamic acid), non-charged amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched amino acids (e.g., threonine, valine, isoleucine), and aromatic amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine). An example of an amino acid mutation is substitution with an amino acid from a group other than the group to which that amino acid belongs. Specifically, examples include, but are not limited to, substitution of basic amino acids with acidic or neutral amino acids, substitution of polar amino acids with nonpolar amino acids, substitution of amino acids with a molecular weight greater than the average molecular weight of the 20 natural amino acids with an amino acid with a molecular weight smaller than the average molecular weight, and conversely, substitution of amino acids with a molecular weight smaller than the average molecular weight with an amino acid with a molecular weight larger than the average.

[0039] Furthermore, mutations in the L protein include substitutions of amino acids at any site selected from positions 942 (Y942), 1361 (L1361), and 1558 (L1558) of the SeV L protein, or at the corresponding sites of the negative-strand RNA virus L protein. As with the above, amino acid substitutions are preferably made with amino acids that have different chemical properties in the side chain. Specifically, examples include substitution of the 942nd amino acid with His, substitution of the 1361st amino acid with Cys, and substitution of the 1558th amino acid with Ile. In particular, L proteins in which at least the 942nd or 1558th position is substituted are preferably used. For example, mutant L proteins in which the 1361st position is also substituted with another amino acid in addition to the 1558th position are also preferred. Mutant L proteins in which the 1558th position and / or 1361st position are also substituted with other amino acids in addition to the 942nd position are also preferred. These mutations can increase the temperature sensitivity of the L protein. Furthermore, mutations in the P protein include substitutions of amino acids at any site selected from positions 433 (D433), 434 (R434), and 437 (K437) of the SeV P protein, or at the corresponding sites of the negative-strand RNA virus P protein. As with the above, amino acid substitutions are preferably made with amino acids that have different chemical properties in the side chain. Specifically, examples include substitution of the 433rd amino acid with Ala(A), substitution of the 434th amino acid with Ala(A), and substitution of the 437th amino acid with Ala(A). In particular, P proteins in which all three of these sites are substituted can be suitably used. These mutations can increase the temperature sensitivity of the P protein.

[0040] Temperature-sensitive mutations that may be included in the vectors of this application are described in detail in WO2012 / 029770, WO2010 / 008054, and WO2003 / 025570. Preferably, the P protein is a mutant P protein in which at least three positions, D at position 433, R at position 434, and K at position 437, are substituted with other amino acids. In addition, Sendai virus vectors lacking or deleting the F gene that encode a mutant L protein in which at least L at position 1558 is substituted (preferably a mutant L protein in which at least L at position 1361 is also substituted with other amino acids), as well as Sendai virus vectors lacking or deleting the F gene that have the same or lesser cytotoxicity and / or the same or greater temperature sensitivity, are also suitably used in this application. Each viral protein may have mutations in other amino acids (e.g., 10 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid) in addition to the above mutations. Vectors having the above mutations exhibit high temperature sensitivity.

[0041] The genomic RNA contained in the vector of this invention may contain all envelope protein genes, or it may contain some or all of the envelope protein genes. The envelope protein genes (M gene, F gene, HN gene) contained in the genomic RNA may be wild-type, or they may have temperature-sensitive mutations introduced. Temperature-sensitive mutations of envelope proteins are described in detail in WO2012 / 029770, WO2010 / 008054, and WO2003 / 025570.

[0042] When manufacturing a viral vector, a viral vector containing a desired exogenous envelope protein can be produced by expressing the desired exogenous envelope protein in virus-producing cells. There are no particular restrictions on such proteins; any desired adhesion factors, ligands, receptors, or other proteins that confer infectivity to mammalian cells can be used. Specifically, for example, the G protein (VSV-G) of vesicular stomatitis virus (VSV) can be cited. The VSV-G protein may be derived from any VSV strain; for example, the VSV-G protein derived from the Indiana serotype strain (J. Virology39: 519-528 (1981)) can be used, but is not limited thereto. The viral vector of this application may contain an arbitrary combination of envelope proteins derived from other viruses.

[0043] In this application, temperature sensitivity means that the activity changes depending on the culture temperature. For example, in the case of an expression vector, a temperature-sensitive vector is a vector whose expression level changes depending on the culture temperature, and it is preferable to use one in which the expression level at a certain temperature within the range of 30 to 36.5°C is significantly higher than the expression level at a temperature 0.5 to 5°C higher, preferably 1 to 4°C higher, for example, about 3°C ​​higher. For example, Sendai virus TS7 (G69E / T116A / A183S mutation in the M protein, A262T / G264R / K461G mutation in the HN protein, L511F mutation in the P protein, and N1197S / K1795E mutation in the L protein (collectively referred to as "TS mutations"), in addition to the Y942H / L1361C / L1558I mutation in the L protein), TS12 (TS mutations, in addition to the D433A / R mutation in the P protein) as detailed in WO2012 / 029770 and WO2010 / 008054, Mutations such as the 434A / K437A mutation, TS13 (TS mutation plus D433A / R434A / K437A mutation in the P protein and L1558I mutation in the L protein), TS14 (TS mutation plus D433A / R434A / K437A mutation in the P protein and L1361C mutation in the L protein), and TS15 (TS mutation plus D433A / R434A / K437A mutation in the P protein and L1361C / L1558I mutation in the L protein) are favorable temperature-sensitive mutations.

[0044] Specific examples of vectors include, for instance, F gene deletion type Sendai virus vectors (e.g., Z strain) that have TS7, TS12, TS13, TS14, or TS15 mutations. More specifically, examples include, but are not limited to, vectors such as SeV18+ / TSΔF (WO2010 / 008054, WO2003 / 025570) or SeV(PM) / TSΔF in which mutations of TS7, TS12, TS13, TS14, or TS15 are introduced in addition to the TS mutation. "TSΔF" refers to a condition in which the M protein has mutations G69E, T116A, and A183S; the HN protein has mutations A262T, G264R, and K461G; the P protein has an L511F mutation; and the L protein has N1197S and K1795E mutations, while the F gene is deleted.

[0045] Preferably, the vector of the present invention is a Sendai virus vector derived from the Sendai virus Z strain. The genome sequence of the Z strain is known, and for example, ACCESSION:AB855655 is an example of the genome sequence of an F gene-deficient Sendai virus vector derived from the Z strain. Here, "derived from the Z strain" means that the sequences of the Z strain account for the highest proportion of the Sendai virus genome sequences in the vector's genome. That is, when non-Sendai virus sequences (restriction enzyme recognition sites, mere spacer sequences, sequences derived from other virus species, sequences of the introduced gene, etc.) are excluded from the vector's genome sequence and only Sendai virus sequences are collected, the sequences of the SeV Z strain account for the highest proportion. More preferably, "derived from the Z strain" means that the sequences of the SeV Z strain account for the highest proportion of the negative-strand RNA virus-derived sequences in the vector's genome. In other words, when sequences that are not derived from negative-strand RNA viruses (such as restriction enzyme recognition sites, simple spacer sequences, sequences derived from viruses other than negative-strand RNA viruses, and sequences of the introduced gene) are excluded from the genome sequence of the vector, and only sequences of negative-strand RNA viruses are collected, the sequence of the SeVZ strain accounts for the highest proportion among them. This proportion is preferably 30% or more, more preferably 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Preferably, a Sendai virus vector derived from the Z strain contains only, for example, 1000 bases or less, 500 bases or less, 300 bases or less, 200 bases or less, 100 bases or less, 50 bases or less, 30 bases or less, or 20 bases or less consecutively, Sendai virus genome sequences other than the Z strain (excluding sequences identical to those of the Z strain). More preferably, a Sendai virus vector derived from strain Z does not contain genome sequences of Sendai viruses other than strain Z (excluding sequences identical to those of strain Z) in its genome.

[0046] The vector of this application may be manufactured using known methods. For example, in the case of a negative-strand RNA virus, the specific procedure typically involves (a) transcribing cDNA encoding the negative-strand RNA virus genomic RNA (negative strand) or its complementary strand (positive strand) in cells expressing viral proteins (NP, P, and L) necessary for viral particle formation, and (b) recovering the produced virus. The virus is not limited to infectious particles, but also includes non-infectious particles and RNPs, as long as they retain the ability to express genes when introduced into cells. The viral proteins necessary for virus formation may be expressed from the transcribed viral genomic RNA or supplied from sources other than genomic RNA. For example, they can be supplied by introducing expression plasmids encoding NP, P, and L proteins into cells. If the viral genes necessary for virus formation are missing in the genomic RNA, the viral genes can be expressed separately in virus-producing cells to complement virus formation. In order to express viral proteins or RNA genomes in cells, a vector in which the DNA encoding the proteins or genomic RNA is ligated downstream of a suitable promoter that functions in the host cell is introduced into the host cell. The transcribed genomic RNA replicates in the presence of viral proteins to form virions. When producing knockout viruses lacking genes such as envelope proteins, the missing protein or other viral proteins that can complement its function can also be expressed in virus-producing cells. In this application, vectors lacking at least the F gene or vectors having mutations in the F gene can be suitably used.

[0047] RNPs of this invention can be produced by transcribing the genomic RNA (positive or negative strand) contained in the vector of this invention in the presence of NP, P, and L proteins. RNP formation can be carried out, for example, in BHK-21 or LLC-MK2 cells. The NP, P, and L proteins may be supplied by a viral vector, or by various other methods. For example, as described above, this can be done by introducing an expression vector containing each gene into cells. Alternatively, each gene may be integrated into the chromosome of the host cell. The NP, P, and L genes expressed to form RNPs do not need to be exactly the same as the NP, P, and L genes encoded in the vector's genome. That is, the amino acid sequences of the proteins encoded by these genes do not have to be exactly the same as the amino acid sequences of the proteins encoded by the RNP genome. Mutations may be introduced, or homologous genes from other viruses may be used, as long as they bind to genomic RNA and have transcriptional replication activity to the genome in the cell. Alternatively, wild-type proteins (wild-type NP, P, and / or L proteins) may be expressed.

[0048] For example, the production of the negative-strand RNA virus of this application can be carried out using the following conventional 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. USA 92: 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 information on methods of virus replication and recombinant virus production, please also refer to "Virology Experiments: Specific Topics, Revised Second Edition" (edited by the National Institute of Preventive Health Alumni Association, Maruzen, 1982).

[0049] [Initialization factor] In this application, a reprogramming factor refers to a gene or its product used to induce a more undifferentiated state in a cell, either alone or in conjunction with other factors. This includes, for example, genes or their products used to induce dedifferentiation in differentiated cells. In this application, reprogramming factors include factors essential for nuclear reprogramming and auxiliary factors (cofactors) that increase the efficiency of nuclear reprogramming. In this application, a desired gene for use in nuclear reprogramming may be loaded into the vector. For example, a gene for use in the production of pluripotent stem cells can be loaded. Specifically, reprogramming factors for inducing pluripotent stem cells can be genes that are expressed in ES cells or early embryos but are not expressed or have reduced expression in many differentiated somatic cells (such as ES cell-specific genes). Such genes are preferably genes that encode transcription factors or nuclear proteins. Methods for identifying nuclear reprogramming genes are already known (WO2005 / 80598), and in fact, genes identified using this method have been shown to be useful for reprogramming into pluripotent stem cells (WO2007 / 69666).

[0050] These 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 (EScell). expressed Ras; NM_181532, NM_181548); DNMT3L(DNA (cytosine-5-)-methyltransferase 3-like; NM_013369, NM_019448); ECAT8(AB211063, AB211061); NM_020634, NM_008108)、SOX15(SRY (sex determining region Y)-box 15; NM_006942, NM_009235); NM_028610)、DPPA2(NM_138815, NM_028615)、FTHL17(ferritin, heavy polypeptide-like 17; NM_031894, NM_031261)、SALL4(sal-like 4; NM_020436,17). NM_175303) Oct3 / 4(POU5F1 and ; 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 called Stella, NM_199286, NM_139218, XM_216263), KLF4 (Kruppel-like factor 4; NM_004235, NM_010637), cateninβ1 (cadherin-associated protein beta 1; NM_001904, NM_007614; including S33Y variant), c-Myc (NM_002467, NM_010849; including T58A variant), STAT3 (signal transducer and activator of transcription 3; NM_139276, Examples include NM_213659), GRB2 (growth factor receptor-bound protein 2; NM_002086, NM_008163), and genes of other members of the family to which these genes belong. These genes have been shown to induce pluripotent stem cells when introduced into cells (WO2007 / 69666). These genes may be incorporated individually into separate vectors, or multiple genes may be incorporated together into a single vector. Furthermore, each gene may be incorporated into a single type of vector, or different types of vectors (including chromosome-integrated viral vectors and / or non-viral vectors) may be used in combination with chromosome-non-integrated viral vectors. Moreover, the method for producing initialized cells in this application is not limited to a method in which all gene introduction is performed using viral vectors. That is, the method in this application may use at least one chromosome-non-integrated viral vector, and may include the use of other vectors expressing reprogramming factors (chromosome-integrated viral vectors and / or non-viral vectors) and / or compounds that induce reprogramming.

[0051] Here, reprogramming may be, for example, the induction of pluripotent stem cells from differentiated cells. The vector is used by incorporating a gene that codes for the reprogramming factor. Examples of reprogramming factors include any of the genes listed above or below.

[0052] Examples of genes particularly favored for inducing cell reprogramming 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_002701, NM_203289, NM_013633, NM_001009178), Sox2 (NM_003106, NM_011443, XM_574919), and TCL1A (T-cell leukemia / lymphoma 1A; Examples include NM_021966, NM_009337), KLF4 (Kruppel-like factor 4; NM_004235, NM_010637), cateninβ1 (cadherin-associated protein beta 1; NM_001904, NM_007614; including the S33Y variant), and c-Myc (NM_002467, NM_010849; including the T58A variant), as well as genes of other members of the family to which these genes belong. Individual viral vectors can be used in combination at the time of use.

[0053] One particularly preferred gene combination is one that includes at least four genes: the Sox gene, the KLF gene, the Myc gene, and the Oct 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; WO2007 / 69666). Here, the Sox protein, KLF protein, Myc protein, and Oct protein, as well as their genes, refer to the proteins and genes of members belonging to the Sox family, KLF family, Myc family, and Oct family, respectively. It is sufficient to adjust the expression to include at least one member from each of these four families. For example, any of the Sox family genes—Sox1, Sox2, Sox3, Sox15, or Sox17—can be used. Similarly, for the KLF family, either KLF4 or KLF2 can be used. For the Myc family, not only the wild-type c-Myc, but also the T58A mutant, N-Myc, and L-Myc can be used. In this way, it is possible to select and use a variety of family genes, so reprogramming can be induced by appropriately selecting the four types of family genes mentioned above.

[0054] For example, wild-type c-Myc has been found to have low expression levels from RNA viral vectors such as Sendai virus vectors. However, by introducing one or more, preferably two or more, three or more, four or more, or all five, mutations selected from a378g, t1122c, t1125c, a1191g, and a1194g into wild-type c-Myc, it becomes possible to stably achieve high gene expression from the vector. The insertion site of the gene in the vector can be selected as a desired site.

[0055] For example, the Myc gene may be located at the posterior (5') end of a negative-strand RNA genome, that is, in a position where counting from the 5' end is earlier than counting from the 3' end among multiple protein-coding sequences located on the genome. The Myc gene can be located, for example, at the very 5' end (i.e., the first from the 5' end), or the second or third from the 5' end. The Myc gene may be located, for example, second from the 5' end of the genome, specifically, between the L gene at the very 5' end of the genome and the HN gene. The Myc gene can be subjected to silent mutations as appropriate, without changing the encoded amino acid sequence, by substituting consecutive A or T base sequences.

[0056] A negative-strand RNA viral vector in which the Myc gene is positioned at the posterior (5' end) of the negative-strand RNA genome can be used in combination with other negative-strand RNA viral vectors containing other reprogramming factors. In this case, in the negative-strand RNA viral vector containing other reprogramming factors, those reprogramming factors can be positioned at the anterior (3' end) of the negative-strand RNA genome of each vector, i.e., at a position earlier when counting from the 3' end than when counting from the 5' end among the multiple protein-coding sequences located on the genome. For example, they may be positioned at the very 3' end (i.e., the first from the 3' end), or the second or third from the 3' end. For example, other reprogramming factors (e.g., the Oct gene, Klf gene, and Sox gene) are positioned at the first or second, more preferably the first, position from the 3' end of the genome in each negative-strand RNA viral vector. Specifically, the reprogramming factors can be positioned at the very 3' end of the NP gene on the genome.

[0057] Specifically, the KLF family includes Klf1 (NM_006563, NM_010635), Klf2 (NM_016270, NM_008452), Klf4 (NM_004235, NM_010637), Klf5 (NM_001730, NM_009769), the Myc family includes c-Myc (NM_002467, NM_010849, including the T58A variant), NMyc (NM_005378, NM_008709), L-Myc (NM_005376, NM_005806), and the Oct family includes Oct1A (NM_002697, NM_198934), Oct3 / 4 (NM_002701, This includes NM_203289, NM_013633, NM_001009178), Oct6(NM_002699, NM_011141), and the Sox family includes Sox1(NM_005986, NM_009233), Sox2(NM_003106, NM_011443, XM_574919), Sox3(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). Non-chromosomal-integrated viral vectors carrying any of these genes are useful in the present invention for inducing cell dedifferentiation.

[0058] One or more viral vectors containing any of the Sox, KLF, and Oct genes, or any of the Sox, Myc, and Oct genes, or a combination of the Sox, Myc, and Klf genes, are useful in this invention for inducing cell reprogramming. If the Myc gene is not expressed, p53 siRNA and UTF1 may be used instead, for example. Viral vectors containing each gene may be prepared separately as standalone devices. They can be used in combination at the time of use.

[0059] Furthermore, if one or more of the above genes are already expressed endogenously in the original differentiated cells, for example, the introduction of those genes can be omitted. Viral vectors containing reprogramming factors may be used only as needed. Also, if the endogenous expression of endogenous reprogramming factors is induced by the expression of another gene or by compound treatment, then the introduction of a vector expressing that other gene or compound treatment may be combined to introduce only viral vectors containing reprogramming factors that cannot be induced by that alone. In this application, combining vectors so that at least three types of genes (Oct, Klf, and Sox), at least four types of genes (Oct, Klf, Sox, and Myc), or at least four types of genes (Oct, Sox, Nanog, and Lin28) are expressed endogenously or exogenously includes, for example, not only the state in which a certain reprogramming factor is expressed endogenously in its natural state, but also cases where the expression of an endogenous reprogramming factor can be induced by the introduction of a vector expressing another gene or by compound or protein treatment, and viral vectors are combined to exogenously express only the deficient factors.

[0060] In addition to the four or three combinations described above, combinations containing each of the four genes—Oct, Sox, NANOG (NM_024865, AB093574), and LIN28 (NM_024674)—are also useful. Combinations of these with the Myc and KLF genes are also preferable. Viral vectors carrying any of these genes are useful in this invention for inducing cell dedifferentiation. One or more viral vectors containing any combination (or all) of these genes can also be suitably used in cell reprogramming. As with the above, if the target cells already express some of these genes, a vector expressing those genes may or may not be introduced.

[0061] The gene combinations described above can be further enhanced by adding other genes to increase the efficiency of reprogramming induction. 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). Alternatively, one or more genes selected from the group consisting of HPV16 E6, HPV16 E7, and Bmil (NM_005180, NM_007552) may be added. Additionally, one or any combination of the following may be expressed: 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). In addition, 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), FTHL17(NM_031894, NM_031261), SALL4(NM_020436, NM_175303), Rex-1(NM_174900, One or more genes selected from the group consisting of 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) may be combined.These factors can also be expressed using the chromosomally non-integrated viral vectors described in this invention.

[0062] The factors to be introduced can be appropriately selected according to the origin of the cells to be reprogrammed. They may be of human origin, or of other mammalian origin, such as primates from mice, rats, rabbits, pigs, or monkeys, but are preferably of human origin. Furthermore, the gene and protein sequences do not necessarily have to be wild-type sequences and may have any mutations as long as they can induce reprogramming. For example, genes encoding amino acid sequences in which one or a few (e.g., a few, up to three, up to five, up to ten, up to fifteen, up to twenty, or up to twenty-five) amino acids are added, deleted, substituted, and / or inserted, and which can induce reprogramming, can be used in this application. Also, as long as they maintain biological activity (ability to induce reprogramming), polypeptides 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, and polypeptides in which one to several residues (e.g., 2, 3, 4, 5, 6, 10, 15, or 20 residues) of amino acids are substituted, can also be used. Potential variants include, for example, fragments, analogs, derivatives of native proteins, and fusion proteins with other polypeptides (e.g., those with heterologous signal peptides or antibody fragments attached). Specifically, these include polypeptides that have sequences in which one or more amino acids are substituted, deleted, and / or added to the wild-type amino acid sequence, and that have equivalent biological activity (e.g., activity to induce reprogramming) to the wild-type protein. When using fragments of the wild-type protein, they typically contain 70% or more, preferably 80% or more, 85% or more, more preferably 90% or more, 95% or more, or 98% or more of a continuous region of the wild-type polypeptide (or the mature form in the case of secreted proteins).

[0063] Amino acid sequence variants can be prepared, for example, by introducing mutations into the DNA encoding a natural 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 substituting amino acids without affecting biological activity can be found, for example, by Dayhoff et al. (Dayhoff et al., in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), 1978).

[0064] There are no particular restrictions on the number of amino acids to be modified, but for example, it is within 30% of the total amino acids of the natural mature polypeptide, preferably within 25%, more preferably within 20%, more preferably within 15%, more preferably within 10%, within 5%, or within 3%, and for example, within 15 amino acids, preferably within 10 amino acids, more preferably within 8 amino acids, more preferably within 5 amino acids, and more preferably within 3 amino acids. When substituting amino acids, it is expected that the protein activity will be maintained by substituting them with amino acids that have similar side chain properties. Such substitutions are referred to as conservative substitutions in this application. Conservative substitutions include substitutions between amino acids within each group, such as basic amino acids (e.g., lysine, arginine, histidine), acidic amino acids (e.g., aspartic acid, glutamic acid), non-charged amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched amino acids (e.g., threonine, valine, isoleucine), and aromatic amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine). Furthermore, substitutions between amino acids with positive relationships in the BLOSUM62 substitution matrix (S. Henikoff and JG Henikoff, Proc. Acad. Natl. Sci. USA 89: 10915-10919, 1992) are also examples.

[0065] Modified proteins exhibit high homology to the amino acid sequence of the wild-type protein. High homology refers to amino acid sequences with, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, or 96% or more identity. Amino acid sequence identity can be determined using, for example, the BLASTP program (Altschul, SF et al., J. Mol. Biol. 215: 403-410, 1990). For example, searches can be performed using default parameters on the NCBI (National Center for Biotechnology Information) BLAST webpage (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 blast2sequences program (Tatiana A et al., FEMS Microbiol Lett. 174:247-250, 1999) can be used to create alignments of two sequences and determine their identity. Gaps are treated similarly to mismatches, and for example, the identity value for the entire amino acid sequence of a native cytokine (the mature form after secretion) is calculated. Specifically, the proportion of matching amino acids in the total number of amino acids of the wild-type protein (or the mature form in the case of secreted proteins) is calculated.

[0066] Furthermore, silent mutations can be introduced into genes without altering the encoded amino acid sequence. In particular, in AT-rich genes, stable high gene expression can be achieved by substituting five or more consecutive A or T bases with G or C without changing the encoded amino acid sequence.

[0067] Furthermore, modified proteins or proteins used for reprogramming include proteins encoded by nucleic acids that hybridize under stringent conditions with part or all of the coding region of the gene encoding the wild-type protein, and which have activity equivalent to that of the wild-type protein (activity that induces reprogramming). In hybridization, identification can be achieved, for example, by preparing a probe from either a nucleic acid containing the sequence of the coding region of the wild-type protein gene or its complementary sequence, or the nucleic acid to be hybridized, and detecting whether it hybridizes with the other nucleic acid. Conditions for stringent hybridization include, for example, performing hybridization at 60°C, preferably 65°C, more preferably 68°C, in a solution containing 5x SSC, 7% (W / V) SDS, 100 μg / ml denatured salmon sperm DNA, and 5x Denhardt's solution (1x Denhardt's solution contains 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% Ficol), followed by washing with shaking for 2 hours at the same temperature as the hybridization, in 2x SSC, preferably 1x SSC, more preferably 0.5x SSC, more preferably 0.1x SSC.

[0068] [Method for producing naive induced pluripotent stem cells] This application provides a method for producing naive induced pluripotent stem cells from human somatic cells, comprising the following steps (1) to (3); (1) A step of introducing one or more vectors containing reprogramming factors into human somatic cells. (2) A step of culturing the somatic cells in the presence of a naive medium, and (3) After step (2), the obtained cells are cultured in the presence of a naive medium under conditions in which the amount of the vector per somatic cell is reduced to 30% or less compared to the start of step 3.

[0069] In this disclosure, somatic cells are not particularly limited and any somatic cells can be used. For example, keratinizing epithelial cells (e.g., keratinized epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the tongue surface), exocrine gland epithelial cells (e.g., mammary gland cells), hormone-secreting cells (e.g., adrenal medullary cells), metabolic and storage cells (e.g., hepatocytes), luminal epithelial cells that constitute the interface (e.g., type I alveolar cells), luminal epithelial cells of the inner chain canal (e.g., vascular endothelial cells), ciliated cells with transporting ability (e.g., airway epithelial cells), and extracellular matrix-secreting cells (e.g., fibroblasts). Examples of somatic cells include cells, contractile cells (e.g., smooth muscle cells), blood and immune system cells (e.g., peripheral blood mononuclear cells, umbilical cord blood cells, T lymphocytes), sensory cells (e.g., rod cells), autonomic nervous system neurons (e.g., cholinergic neurons), supporting cells of sensory organs and peripheral neurons (e.g., accompanying cells), central nervous system neurons and glial cells (e.g., astrocytes), pigment cells (e.g., retinal pigment epithelial cells), and their progenitor cells (tissue progenitor cells). There are no particular restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected. Undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells can be used as the origin of somatic cells in this application. Examples of undifferentiated progenitor cells include neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, and other tissue stem cells (somatic stem cells). Human-derived somatic cells are preferred.

[0070] In this disclosure, induced pluripotent stem cells are cells in which pluripotency has been induced by reprogramming somatic cells using known methods. Specifically, this refers to cells in which pluripotency has been induced by reprogramming differentiated somatic cells such as fibroblasts or peripheral blood mononuclear cells by expressing one of several combinations of genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc.

[0071] In this disclosure, naive induced pluripotent stem cells refer to induced pluripotent stem cells that have properties similar to preimplantation embryos, and can be defined as induced pluripotent stem cells that exhibit one or more of the following characteristics: a gene expression pattern equivalent to that of the inner cell mass of a preimplantation embryo (specifically, expression of one or more naive-type specific markers, or naive-type specific intracellular localization of one or more specific gene products), widespread DNA demethylation across the entire genome, X chromosome reactivation, or a multilayered, dome-shaped colony morphology. Examples of naive-type specific markers include TCFP2L1, KLF17, TBX3, PRDM14, SSEA1, CD7, CD75, CD77, CD130, DPPA3, ESRRB, KLF4, and KLF5. Examples of naive-type specific intracellular localization of specific gene products include nuclear localization of the TFE3 protein.

[0072] The naive type of induced pluripotent stem cells can be confirmed by possessing one or more of the above-mentioned characteristics, and preferably by confirming the expression of the naive type-specific marker and / or the naive type-specific intracellular localization of a specific gene product. Furthermore, for speed and simplicity, confirmation may be made by observing a dome-shaped colony morphology.

[0073] Methods for confirming gene expression patterns include well-known methods such as RNA-seq, qPCR, immunostaining, and FCM, with RNA-seq being preferred.

[0074] On the other hand, primed induced pluripotent stem cells (PPTs) are defined as induced pluripotent stem cells that possess properties similar to the epiblast of a post-implantation embryo. They can be defined as PPTs exhibiting one or more of the following characteristics: a gene expression pattern equivalent to that of the epiblast of a post-implantation embryo (specifically, expression of one or more primed-specific markers, or primed-specific intracellular localization of one or more specific gene products), or a monolayered, flattened colony morphology. Examples of primed-specific markers include OTX2 or ZIC2, and an example of primed-specific intracellular localization of a specific gene product is cytoplasmic localization of the TFE3 protein.

[0075] The fact that the manufactured induced pluripotent stem cells are primed can be confirmed by possessing one or more of the above-mentioned characteristics, preferably by confirming the expression of the primed-specific marker and / or the primed-specific intracellular localization of a specific gene product. Furthermore, for speed and simplicity, confirmation may also be made by observing a monolayer, flattened colony morphology.

[0076] In step (1), one or more vectors containing reprogramming factors are introduced into human somatic cells.

[0077] The reprogramming factors listed above can be used as reprogramming factors. For example, the reprogramming factors may include the OCT gene, SOX gene, MYC gene, and KLF gene.

[0078] The above-mentioned vectors can be used as one or more vectors. For example, one or more vectors may be, for example, non-chromosomal-integrated viral vectors, preferably paramyxovirus vectors, and more preferably Sendai virus vectors.

[0079] One or more vectors containing reprogramming factors may include, for example, a vector containing the OCT gene, the SOX gene and the KLF gene, a vector containing the MYC gene, and a vector containing the KLF gene.

[0080] To reprogram cells, the above combination of vectors is introduced into the cells. When introducing multiple vectors in combination, it is preferable to introduce them simultaneously. Specifically, it is preferable to complete the addition of all vectors containing the reprogramming factors 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 adding the first vector or compound. The vector dose can be adjusted as appropriate, but preferably the MOI is 0.3 to 100, more preferably 0.5 to 50, more preferably 1 to 30, more preferably 1 to 10, and more preferably 5 for infection.

[0081] In one embodiment, step (2) is initiated 1 to 15 days, 1 to 10 days, or, for example, 3 to 9 days after step (1). Until step (2) is initiated, human somatic cells can be cultured under appropriate culture conditions.

[0082] In this application, the culture medium can be prepared by appropriately adding necessary factors to the basal medium used for culturing animal cells. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, MEM Zinc Option medium, IMEM Zinc Option medium, Dulbecco's modified Eagle's Medium (DMEM) medium, DMEM / F12 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media of these. The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as albumin, insulin, transferrin, selenium, KnockOut Serum Replacement (KSR) (Invitrogen) (serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecule compounds, antibiotics (e.g., streptomycin, penicillin, puromycin, mitomycin), antioxidants, pyruvate, buffers, inorganic salts, cytokines, and their equivalents. In one embodiment, the basal medium is DMEM medium or DMEM / F12 medium. A commercially available medium may be used as the basal medium; for example, when culturing human hematopoietic cells, a medium such as StemSpan ACF (STEMCELL Technologies) can be used. When culturing human hematopoietic cells, additional factors such as stem cell factor (SCF), thrombopoietin (TPO), Flt3 / Flk2, IL-6, and IL-3 may be added to the basal culture medium.

[0083] In certain embodiments, somatic cells can be cultured together with feeder cells. As used herein, feeder cells refer to cells other than the somatic cells that coexist during the culture of somatic cells. The feeder cells are not particularly limited, and examples thereof include fibroblasts (such as NIH-3T3 cells, MEF cells, STO cells, SNL cells, etc.), fetal kidney cells (such as HEK293 cells, etc.), stromal cells (such as ST2 cells, OP9 cells, PA6 cells, MS-5 cells, etc.), epithelial cells (such as HeLa cells, etc.), placental cells, bone marrow cells, endometrial cells, and the like. The feeder cells are, for example, MEF cells. The feeder cells may be cells derived from the same species as the somatic cells or cells derived from different species. Also, the feeder cells may be treated to inhibit cell proliferation, such as by treatment with a growth inhibitor (such as mitomycin C) or irradiation with radiation (such as X-rays or γ-rays). The concentration of the feeder cells used in the present application is not particularly limited as long as the proliferation of the somatic cells is possible, and is, for example, about 5×10 3 cells / cm 2 ~ about 1×10 5 cells / cm 2 . The somatic cells may be cultured together with the feeder cells from the beginning of the culture or may be cultured together with the feeder cells during the course of the culture. For example, the somatic cells can be cultured together with the feeder cells from the 2nd to the 10th day, or from the 2nd to the 5th day of the culture. In another embodiment, the somatic cells can be cultured in the absence of feeder cells.

[0084] In the culture of somatic cells, the culture temperature is not limited to the following, but may be determined according to the characteristics of the vector within the range of about 30 to 40 °C. The culture is carried out in an atmosphere of air containing CO2, and the CO2 concentration is about 2 to 8%, preferably about 3 to 7%, more preferably about 4 to 6%, and most preferably about 5%. Also, after the step of (1), the culture may be carried out under hypoxic conditions, and the oxygen concentration is about 3 to 10%, preferably about 4 to 8%, more preferably about 4 to 6%, and most preferably about 5%.

[0085] In one embodiment, somatic cells are cultured by two-dimensional culture. When culturing in the presence of feeder cells, for example, feeder cells may be pre-seeded and cultured on the bottom of the culture vessel or on the culture substrate, and after they have grown to cover the bottom of the culture vessel or on the surface of the culture substrate, somatic cells may then be seeded on these feeder cells and cultured.

[0086] Culture vessels for culturing somatic cells are, for example, those treated with a coating agent. Examples of coating agents include gelatin, Matrigel (BD), Synthemax (Corning), collagen, laminin, heparan sulfate proteoglycans, entactin, fragments thereof, and combinations thereof.

[0087] In step (2), the somatic cells are cultured in the presence of a naive culture medium.

[0088] In step (2), the culture temperature is not limited to the following, but should be determined within the range of approximately 30 to 40°C according to the characteristics of the vector. The culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration of approximately 2 to 8%, preferably approximately 3 to 7%, more preferably approximately 4 to 6%, and most preferably approximately 5%. Alternatively, the culture may be carried out under low-oxygen conditions, with an oxygen concentration of approximately 3 to 10%, preferably approximately 4 to 8%, more preferably approximately 4 to 6%, and most preferably approximately 5%.

[0089] In this application, "naive culture medium" means a culture medium used when inducing naive induced pluripotent stem cells from somatic cells. As the basal culture medium for naive cells, the basal culture media described above can be used. For example, an N2B27 medium (a medium prepared by mixing N2 medium, which is DMEM / F12 medium with an N2 supplement, and B27 medium, which is Neurobasal medium with a B27 supplement, in a 1:1 ratio) may be prepared by adding any combination of additives such as non-essential amino acids (NEAAs), L-glutamine, 2-mercaptoethanol, antibiotics (e.g., streptomycin, penicillin, puromycin, mitomycin), and bovine serum albumin (BSA).

[0090] In one embodiment, the naive culture medium contains one or more compounds selected from LIF (Leukemia inhibitory factor), MEK inhibitors, GSK3 inhibitors, cAMP production promoters, TGF-β inhibitors, and PKC inhibitors. The naive culture medium is particularly preferably LIF, and more preferably LIF and a GSK3 inhibitor. The naive culture medium is also preferably substantially free of bFGF.

[0091] The concentration of LIF in the culture medium can range from 1 ng / ml to 100 ng / ml, or from 5 ng / ml to 50 ng / ml, for example, about 10 ng / ml.

[0092] In this application, the MEK inhibitor is not particularly limited, but examples include PD0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide; CAS registry number: 391210-10-9), U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene; CAS registry number: 109511-58-2), PD98059 (2-(2-amino-3-methoxyf Examples include PD184352 (2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; CAS registry number: 212631-79-3). Among these, PD0325901 is preferred. When PD0325901 is used as a MEK inhibitor, its concentration in the culture medium may be 50 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0093] In this application, the GSK3 inhibitor is not particularly limited, but examples include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazole-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitride; CAS Registry No.: 252917-06-9) and BIO (6-bromoindilbine-3'-oxime; CAS Registry No.: 66746) Examples include 3-62-9), Kenpaullone (9-bromo-7,12-dihydroindoro[3,2-d][1]benzazepine-6(5H)-one; CAS registry number: 142273-20-9), and IM-16 (3-(4-fluorophenylethylamino)-1-methyl-4-(2-methyl-1H-indole-3-yl)-1H-pyrrole-2,5-dione; CAS registry number: 1129669-05-1). Among these, CHIR99021 is preferred. When CHIR99021 is used as a GSK3 inhibitor, its concentration in the culture medium may be 50 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0094] In this application, the cAMP production promoter is not particularly limited, but for example, forskolin can be used. When forskolin is used as the cAMP production promoter, its concentration in the culture medium may be 50 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0095] In this application, the TGF-β inhibitor is not particularly limited, but examples include A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide) and SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]-benzamide). Among these, A83-01 is preferred. When A83-01 is used as the TGF-β inhibitor, its concentration in the culture medium may be 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0096] In this application, the PKC inhibitor is not particularly limited, but examples include Go6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indole-3-yl]-4-(1H-indole-3-yl)-1H-pyrrole-2,5-dione; CAS registry number: 133053-19-7) and GF109203X (3-(1-(3-dimethylamino)propyl)-1H-indole-3-yl)-4-(1H-indole-3-yl)-1H-pyrrole-2,5-dione; CAS registry number: 133052-90-1). Among these, Go6983 is preferred. When Go6983 is used as the PKC inhibitor, its concentration in the culture medium may be 50 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM to 3 μM.

[0097] For naive cultures, commercially available media may be used, such as t2iLGo(N2B27+PD0325901(1μM)+CHIR99021(1μM)+LIF+Go6983(2-3μM)), 5iLAF(N2B27+PD0325901(1μM)+CHIR99021(1μM)+SB590885 (0.5μM)+WH-4-023 (1μM)+Y-27632(10μM)+LIF+Activin A), or tt2iLGo(N2B27+PD0325901(1μM)+LIF+Go6983(2μM)+XAV939(2μM)).

[0098] When cells are cultured in the absence of feeder cells, the naive medium can be pre-conditioned with specific cells. These specific cells may be those usable as feeder cells, such as post-irradiated mouse embryonic fibroblasts (iMEFs). The conditioned culture period may be, for example, about 12 hours to about 2 days. The medium can be obtained by removing cellular components by conventional methods after the conditioned culture. In one embodiment, when cells are cultured in the absence of feeder cells, the naive medium may be t2iLGo conditioned with iMEFs.

[0099] In step (2), the culture period for somatic cells may be 1 to 20 days, 3 to 10 days, or, for example, 4 to 6 days.

[0100] In step (3), the obtained cells are cultured in the presence of naive culture medium under conditions in which the amount of vector per somatic cell is reduced to 30% or less compared to the start of step 3.

[0101] In one embodiment, the obtained cells can be cultured together with feeder cells. The cells described above can be used as feeder cells. In another embodiment, the obtained cells can be cultured in the absence of feeder cells.

[0102] In step (3), the naive medium used may be the same as the naive medium used in step (2). The naive medium may contain one or more compounds selected from LIF (Leukemia inhibitory factor), MEK inhibitors, GSK3 inhibitors, cAMP production promoters, TGF-β inhibitors, and PKC inhibitors. For example, it may contain t2iLGo, 5iLAF, or tt2iLGo.

[0103] When cells are cultured in the absence of feeder cells, the naive medium can be pre-conditioned with specific cells. These specific cells may be those usable as feeder cells, such as post-irradiated mouse embryonic fibroblasts (iMEFs). The conditioned culture period may be, for example, about 12 hours to about 2 days. The medium can be obtained by removing cellular components by conventional methods after the conditioned culture. In one embodiment, when cells are cultured in the absence of feeder cells, the naive medium may be t2iLGo conditioned with iMEFs.

[0104] In step (3), the culture temperature is not limited to the following, but should be determined within the range of approximately 30 to 40°C according to the characteristics of the vector. The culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration of approximately 2 to 8%, preferably approximately 3 to 7%, more preferably approximately 4 to 6%, and most preferably approximately 5%. Alternatively, the culture may be carried out under low-oxygen conditions, with an oxygen concentration of approximately 3 to 10%, preferably approximately 4 to 8%, more preferably approximately 4 to 6%, and most preferably approximately 5%.

[0105] In one embodiment, the amount of vector per somatic cell may decrease to 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 3% or less compared to the amount at the start of step 3, within 30 days, 20 days, 15 days, 12 days, 10 days, 8 days, 5 days, 3 days, or 1 day from the start of step 3. For example, the amount of vector per somatic cell may decrease to 30% or less compared to the amount at the start of step 3 within 12 days from the start of step 3.

[0106] In one embodiment, by using one or more vectors, such as a temperature-sensitive vector, a vector containing a microRNA target sequence specific to induced pluripotent stem cells, or a vector that is both temperature-sensitive and contains a microRNA target sequence specific to induced pluripotent stem cells, and culturing the resulting cells in the presence of naive medium, the amount of the vector per somatic cell can be reduced to 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 3% or less compared to the amount at the start of step 3. For example, this reduction can occur within 30 days, 20 days, 15 days, 12 days, 10 days, 8 days, 5 days, 3 days, or 1 day from the start of step 3.

[0107] The above-mentioned vectors can be used as temperature-sensitive vectors. For example, a temperature-sensitive vector may be a vector containing the above-mentioned temperature-sensitive mutation. In one embodiment, a temperature-sensitive vector is a vector that exhibits temperature sensitivity in a cell culture environment for establishing naive pluripotent stem cells. Examples of vectors that exhibit temperature sensitivity in the cell culture environment for establishing naive pluripotent stem cells include, but are not limited to, Sendai virus vectors containing TS7 mutations (G69E / T116A / A183S mutations in the M protein, A262T / G264R / K461G mutations in the HN protein, L511F mutations in the P protein, and N1197S / K1795E mutations in the L protein (collectively referred to as "TS mutations"), in addition to the Y942H / L1361C / L1558I mutation in the L protein), TS12 mutations (TS mutations plus the D433A / R434A / K437A mutation in the P protein), or TS15 mutations (TS mutations plus the D433A / R434A / K437A mutation in the P protein and the L1361C / L1558I mutation in the L protein), as well as other negative-strand RNA virus vectors with similar substitutions at the corresponding sites.

[0108] One or more vectors containing reprogramming factors may include, for example, a vector containing the OCT gene, the SOX gene, and the KLF gene, a vector containing the MYC gene, and a vector containing the KLF gene. The vectors containing the OCT gene, the SOX gene, and the KLF gene, the vectors containing the MYC gene, and the vectors containing the KLF gene may be Sendai virus vectors, for example, Sendai virus vectors containing the TS7, TS12, or TS15 mutation. In one embodiment, the vector containing the MYC gene may be a Sendai virus vector containing the TS15 mutation, and the vectors containing the OCT gene, the SOX gene, and the KLF gene, and the vectors containing the KLF gene may be Sendai virus vectors containing the TS12 mutation.

[0109] In one embodiment, if one or more of the vectors are temperature-sensitive, the cells may be cultured in steps (1) and (2) at temperatures where the expression level of the vector is high, and in step (3) at temperatures where the expression level of the vector is low. For example, in the case of the vectors exemplified above, the cells may be cultured in steps (1) and (2) at 30-36.5°C, 34-36°C, for example, about 35°C, and in step (3) at 37°C or higher, 38°C or higher, 37°C-45°C, 37°C-40°C, for example, about 38°C. As an example, the cells may be cultured in steps (1) and (2) at about 35°C and in step (3) at about 38°C.

[0110] The target sequences of microRNAs specific to induced pluripotent stem cells are not particularly limited as long as they are sequences to which microRNAs specific to induced pluripotent stem cells can bind as targets. Examples of microRNAs specific to induced pluripotent stem cells include miR-367, miR-302, miR-371, miR-372, miR-373, miR-512, miR-517, miR-518, miR-519, miR-520, miR-525, miR-187, miR-299, miR-499, miR-628, and miR-888.

[0111] A microRNA target sequence refers to a sequence to which a microRNA binds as a target. In this application, the microRNA target sequence can be a natural microRNA target sequence or a variant thereof, as long as its expression is suppressed by the binding of the microRNA. The microRNA target sequence may or may not be a sequence that is perfectly complementary to the microRNA. For example, it may contain at least 10 complementary bases, such as 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, or 22 or more, in a continuous or discontinuous manner relative to the microRNA. There is no particular upper limit to the length of the microRNA target sequence, but for example, it is about 30 bases. Preferably, the complementary bases are continuous, or there may be several unpaired bases, such as 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 base. The unpaired bases may be included on the microRNA target sequence side and / or on the microRNA side.

[0112] The microRNA target sequence is preferably a sequence that hybridizes with microRNA under physiological conditions. Physiological conditions are, for example, 150 mM NaCl, 15 mM sodium citrate, pH 7.0, and 37°C. More preferably, the microRNA target sequence is a sequence that hybridizes with microRNA under stringent conditions. Stringent conditions are, for example, 1×SSC (where 1×SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0) or 0.5×SSC at 42°C, more preferably 1×SSC or 0.5×SSC at 45°C, and more preferably 1×SSC or 0.5×SSC at 50°C. In hybridization, for example, either the RNA containing the microRNA sequence or the RNA containing the microRNA target sequence is labeled, and if necessary, the other is immobilized on a membrane or the like to hybridize the two. Hybridization can be carried out in a solution containing, for example, 5x SSC, 7% (W / V) SDS, 100 μg / ml denatured salmon sperm DNA, and 5x Denhardt's solution (1x Denhardt's solution contains 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% Ficol) at, for example, 37°C, 45°C, or 50°C. After incubation for a sufficient time (e.g., 3, 4, 5, or 6 hours or more), washing can be performed under the above conditions, and it can be determined whether the labeled nucleic acid hybridizes by detecting whether the nucleic acid has hybridized.

[0113] Alternatively, the microRNA target sequence preferably exhibits high homology with the complementary sequence of the microRNA sequence. High homology refers to a sequence having, for example, 70% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. The identity of the nucleotide sequence can be determined, for example, using the BLAST program (Altschul, SF et al., J. Mol. Biol. 215: 403-410, 1990). For example, searches can be performed using default parameters on the NCBI (National Center for Biotechnology Information) BLAST webpage (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 blast2sequences program (Tatiana A et al., FEMS Microbiol Lett. 174:247-250, 1999) can be used to create alignments of two sequences and determine their identity. The outer gaps in the complementary sequence of the microRNA sequence are ignored, and the inner gaps are treated similarly to mismatches, for example. The identity value for the entire complementary sequence of the microRNA sequence in alignment (total length of bases including the gaps placed inside the sequence) is then calculated.

[0114] Alternatively, the microRNA target sequence preferably consists of a sequence in which one or more bases are inserted, substituted, and / or deleted from the complementary sequence of the microRNA sequence. Preferably, the microRNA target sequence includes a sequence having insertions, substitutions, and / or deletions of up to 8 bases, up to 7 bases, up to 6 bases, up to 5 bases, up to 4 bases, up to 3 bases, up to 2 bases, or 1 base relative to the complementary sequence of the microRNA sequence.

[0115] Generally, the more mutations introduced into the microRNA target sequence, the more the binding to the microRNA is suppressed, and the lower the expression repression effect becomes. The repression effect can be adjusted by appropriately introducing mutations.

[0116] In one embodiment, when the vector of the present invention is a negative-strand RNA viral vector, the microRNA target sequence is attached to the NP or P gene. When attaching the microRNA target sequence to the NP or P gene, there are no particular restrictions on the attachment site; it can be attached to the coding region or the uncoding region of each gene. When attaching to the coding region, it may be attached to either the N-terminal or C-terminal site, but it is preferable to attach it immediately following the C-terminus. The uncoding region may be either the 5' uncoding region or the 3' uncoding region of the genome, but the 5' uncoding region is preferred. When attaching to the uncoding region, the site can be arbitrarily selected, but for example, when attaching the microRNA target sequence to the 5' uncoding region of the NP gene and / or P gene in the genome, it may be attached to any site between the stop codon and the E (End) sequence of the coding region of the gene to be attached. When attaching to the 3' uncoding region of the genome, it can be attached to any desired site between the S (Start) sequence and the translation start codon of the gene to be attached. The microRNA target sequence can be attached in one copy or multiple copies. Furthermore, not only one type of microRNA target sequence, but multiple types of microRNA target sequences may be added. For example, a sequence can be added in which two or more, for example, three or more, four or more, or five or more, microRNA target sequences of one or more types are arranged in tandem. There is no particular upper limit to the number of microRNA target sequences that can be added in tandem, but for example, it is up to about 10.

[0117] If modifying the P gene inhibits the expression of the C protein encoded by the nucleic acid in the coding region of the P protein, the C protein can be expressed separately from the vector.

[0118] Furthermore, the P protein does not necessarily have to be the full-length form; fragments can be used as appropriate. Only a portion of the C-terminus is essential for the P protein; the rest of the region is not essential for viral vector expression. Specifically, the P protein may be a fragment that holds both the L protein binding site and the N protein: RNA binding site. Examples of L protein binding sites include the amino acid sequence from position 411 to 445 of the SeV P protein, and examples of N protein:RNA binding sites include the SeV P protein (e.g., accession numbers AAB06197.1, P04859.1, P14252.1, AAB06291.1, AAX07439.1, BAM62828.1, BAM62834.1, P04860.1, BAM62840.1, BAD74220.1, P14251.1, BAM62844.1, BAM62842.1, BAM62842.1, BAF73480.1, BAD74226.1, BAF73486.1, Q9DUE2.1, BAC79134.1, NP_056873.1, Examples include the amino acid sequences from position 479 to 568 of ABB00297.1, etc. More specifically, for example, a fragment containing the amino acid sequence from position 320 to 568 of the SeV P protein can be suitably used as a functional P protein in this application. By using a deletion-type P protein, it is expected that the size of the vector can be reduced and that it will be less susceptible to the host immune response.

[0119] When using a P protein that lacks the coding region of the C protein, the C protein may be expressed separately as appropriate, as described above. Here, the C protein includes C', C, Y1, and Y2 proteins (Irie T. et al., PLoS One. (2010) 5:e10719). To express the C protein, the coding sequence of the C protein can be inserted into the vector as appropriate. There are no particular restrictions on the insertion position, but it can be inserted immediately before the P protein (3' side of the coding sequence of the P protein in the genome) or immediately after the P protein (5' side of the coding sequence of the P protein in the genome). When inserting, an EIS sequence may be added as appropriate.

[0120] When a microRNA target sequence is added to an NP gene or P gene, if this microRNA is expressed in cells into which the vector has been introduced, the expression of the vector will decrease accordingly, and the removal of the vector will be promoted. For example, by adding a target sequence of a microRNA that is not expressed at the time of vector introduction but is specifically expressed when a certain differentiated state (or undifferentiated state) is reached, the expression of the vector will be automatically suppressed and the vector will be removed once the objective has been achieved.

[0121] In another embodiment, one or more vectors may be vectors to which degron has been added to the P gene, and the resulting cells may be cultured in the presence of naive medium. This can reduce the amount of vector per somatic cell to 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 3% or less compared to the start of step 3, for example, within 30 days, 20 days, 15 days, 12 days, 10 days, 8 days, 5 days, 3 days, or 1 day from the start of step 3, compared to the start of step 3.

[0122] In this application, "degron" refers to a polypeptide that destabilizes a protein by being attached to it. Degrons include sequences that are stabilized by fusion with small molecules, sequences that are destabilized by binding with small molecules, and sequences that are destabilized regardless of the presence or absence of small molecules. Specifically, examples include the DD-tag derived from FKBP12, known as the mTOR protein (US2009 / 0215169), the DDG-tag derived from dihydrofolate reductase (DHFR) (US2012 / 0178168), the TetR mutant (WO2007 / 032555), the plant-derived auxin-inducible degron (AID) system (WO2010 / 125620), the PEST sequence known as a degradation-promoting sequence (WO99 / 54348), CL1 (WO2004 / 025264), the calpain-derived sequence (JP 2009-136154), and NDS (JP 2011-101639). FKBP12 is known as the mammalian target of rapamycin (mTOR). It is stabilized by binding to small molecules such as rapamycin and shield1, destabilized by their removal, and degraded by the proteasome. DHFR is stabilized by trimethoprim, and TetR mutants are stabilized by doxycycline. PEST sequences are rich in Pro, Glu, Ser, and Thr, and can be destabilized, for example, by adding the C-terminal 422-461 of mouse ornithine decarboxylase (mODC). PEST sequences regulate the half-life of proteins, but desired half-life shortening sequences can be used (Rechsteiner M, et al., Trends Biochem. Sci. 21, 267-271, 1996). PEST sequences are, for example, sequences surrounded at both ends by basic amino acids (H, K, or R) and containing (i) P, (ii) D and E, or (iii) S and E, which bind to ubiquitinase E3. They can be identified, for example, by GENETYX. Sequences that produce a similar effect to PEST include CL1, calpain subsequences, and NDS. AID sequences are destabilized when the plant ubiquitin ligase TIR1 binds to auxin (IAA).

[0123] In this application, the addition of a degron to the P gene means that the P gene codes for a P protein to which the degron has been added. In this application, the above-mentioned degron can be added to the P protein, and preferred degrons include, specifically, mTOR degron, DHFR degron, TetR degron, PEST, and AID. These degrons include natural sequences and those derived therefrom. Particularly preferred degrons include mTOR degron, DHFR degron, TetR degron, and PEST, among which degrons other than the AID sequence are preferred, specifically FKBP12 degron (DD), DHFR degron (DDG), TetR degron, and mODC PEST. Among PESTs, d2 derived from a natural sequence and its variants d1 and d4 are known (WO99 / 54348), and all of these are included in PEST and can be used in this application. The nucleic acid encoding the degron can be appropriately prepared by DNA synthesis.

[0124] Degron can be attached to any desired position on the P protein, for example, to the N-terminus or C-terminus of the P protein. When attached to the N-terminus of the P protein, if the expression of the C protein encoded by the nucleic acid in the coding region of the P protein is inhibited, the C protein can be expressed separately from the vector. When a fragment is used as the P protein instead of the full-length P protein, and the fragment does not contain the coding region of the C protein, degron can be attached to any position, either at the N-terminus or the C-terminus. In this invention, degron is preferably attached to the C-terminus of the P protein. Modified P proteins with degron attached can be prepared by well-known methods. Specifically, the sequence encoding degron can be inserted into the sequence of the viral genome encoding the P protein so that the reading frame matches.

[0125] After introducing a vector in which a degron is attached to the P gene of this invention into cells and expressing the target gene, the vector can be removed as appropriate according to the properties of the degron. For example, when using ligand-controllable degrons such as DD, DDG, and TetR mutants, removal is promoted if no ligand is added, but vector expression can be extended by adding a ligand such as Shield-1. Furthermore, if a degron that functions even without a ligand, such as a PEST sequence, is used, vector removal can be promoted by continuing to culture the cells into which the vector has been introduced.

[0126] The vector to which degron is attached to the P gene of the present invention may be a vector exhibiting the temperature sensitivity described above, a vector containing a microRNA target sequence specific to induced pluripotent stem cells, or a vector exhibiting temperature sensitivity and containing a microRNA target sequence specific to induced pluripotent stem cells.

[0127] In yet another embodiment, one or more vectors may be vectors carrying suicide genes, and the resulting cells may be cultured in the presence of naive culture medium. This can reduce the amount of vector per somatic cell to 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 3% or less compared to the amount at the start of step 3, for example, within 30 days, 20 days, 15 days, 12 days, 10 days, 8 days, 5 days, 3 days, or 1 day from the start of step 3.

[0128] For example, suicide genes incorporated into the vector of the present invention include enzyme genes for converting prodrugs of desired drugs that induce cell death or exhibit cytotoxicity. Specific examples of prodrug and suicide gene combinations include: 5-fluorocytosine and cytosine deaminase • Cyclophosphamide and cytochrome P450 • Fludarabine and E. coli PNP (purine nucleoside phosphorylase) • CB1954 and nitroreductase • Capecitabine and carboxyesterase Examples include the following. Furthermore, suicide genes include genes encoding desired proteins that induce cell death or exhibit cytotoxicity when converted from an inactive to an active form. For example, genes encoding caspases that induce cell death can be suitably utilized. Specifically, a combination of a desired dimerizing agent and a caspase to which its binding domain has been added can be used. An example of a combination of a dimerizing agent and caspase is: AP20187 and iCaspase-9 One could list these:

[0129] For example, a vector containing the suicide gene of this invention is introduced into cells and cultured, after which a prodrug is added. This kills cells in which the vector remains, allowing only cells from which the vector has been eliminated to be obtained.

[0130] The vector carrying the suicide gene of the present invention may be a temperature-sensitive vector as described above, a vector containing a microRNA target sequence specific to induced pluripotent stem cells, or a vector that is both temperature-sensitive and contains a microRNA target sequence specific to induced pluripotent stem cells. The vector carrying the suicide gene of the present invention may also be a vector to which degron has been added to the P gene as described above.

[0131] In step (3), the culture period is not particularly limited, but could be, for example, within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week, for example, within 20 days, within 15 days, within 10 days, within 5 days, or within 3 days. There is no particular lower limit to the culture period, but for example, it is about 1 day. The decrease in the amount of vector can be confirmed by detecting the reporter gene or detecting the virus using antibodies or PCR, and comparing the level with that at the start of step (3).

[0132] Naive induced pluripotent stem cells produced by the method described in this application have a very low residual amount of reprogramming factor-containing vectors, and therefore, their naive properties can be stably maintained even after long-term culture. In the method described in this application, the reprogramming factor-containing vector can be substantially eliminated from the cells approximately 30 days after introduction of the vector. Therefore, naive induced pluripotent stem cells obtained by the method described in this application are substantially free of reprogramming factor-containing vectors and have high phenotypic stability.

[0133] [Vector Set] This application also relates to a vector set for producing naive induced pluripotent stem cells from human somatic cells, Includes one or more vectors containing initialization factors, The present invention provides a vector set in which one or more vectors are selected from the group consisting of temperature-sensitive vectors, vectors containing a microRNA target sequence specific to induced pluripotent stem cells, and vectors that are both temperature-sensitive and contain a microRNA target sequence specific to induced pluripotent stem cells. Examples of reprogramming factors and vectors used in this embodiment are as described above.

[0134] [Composition] The present invention also relates to a composition for producing naive induced pluripotent stem cells from human somatic cells, Includes one or more vectors containing initialization factors, The present invention provides a composition in which one or more vectors are selected from the group consisting of a temperature-sensitive vector, a vector containing a microRNA target sequence specific to induced pluripotent stem cells, and a vector that is both temperature-sensitive and contains a microRNA target sequence specific to induced pluripotent stem cells. Examples of the reprogramming factors and vectors used in this embodiment are as described above.

[0135] The composition of this application can be prepared as a liquid formulation by adding distilled water, pH adjusters, suspending agents, solubilizers, stabilizers, isotonic agents, antioxidants, preservatives, etc., as needed. Examples of pH adjusters include hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, monohydrogen phosphate, and sodium dihydrogen phosphate. Examples of suspending agents include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, and polyoxyethylene sorbitan monolaurate. Examples of solubilizers include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, and polyoxyethylene sorbitan monolaurate. Examples of stabilizers include sodium sulfite, sodium metasulfite, and ether. Examples of isotonic agents include sodium chloride and glucose. Examples of preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

[0136] [kit] The present invention further relates to a kit for producing naive induced pluripotent stem cells from human somatic cells, One or more vectors containing initialization factors, and Includes naive culture medium, The kit is provided in which one or more vectors are selected from the group consisting of a temperature-sensitive vector, a vector containing a microRNA target sequence specific to induced pluripotent stem cells, and a vector that is both temperature-sensitive and contains a microRNA target sequence specific to induced pluripotent stem cells. Examples of reprogramming factors and vectors used in this embodiment are as described above.

[0137] In the kit of the present invention, the one or more vectors may be packaged separately or together. The kit of the present invention may also include a buffer solution, instructions for use, etc.

[0138] [Culture supernatant] This application also provides the culture supernatant of naive induced pluripotent stem cells produced by the method of this application. The culture medium described above for naive stem cells may be used. The culture medium for naive stem cells may contain one or more compounds selected from LIF (Leukemia inhibitory factor), MEK inhibitors, GSK3 inhibitors, cAMP production promoters, TGF-β inhibitors, and PKC inhibitors. For example, it may contain t2iLGo, 5iLAF, or tt2iLGo.

[0139] The culture period to obtain the culture supernatant can be 3 to 72 days, for example, 12 to 48 days. The concentration of naive induced pluripotent stem cells at the start of culture is, for example, 3 × 10⁻⁶. 3 ~1 × 10 5 cells / cm 2 This is possible, for example, 1 × 10 4 ~3×10 4 cells / cm 2 That is the case.

[0140] The culture temperature for obtaining the culture supernatant is not limited to the following, but is approximately 30-40°C, preferably approximately 37°C. The culture is carried out in an atmosphere of CO2-containing air, with a CO2 concentration of approximately 2-8%, preferably approximately 3-7%, more preferably approximately 4-6%, and most preferably approximately 5%. Alternatively, the culture may be carried out under low-oxygen conditions, with an oxygen concentration of approximately 3-10%, preferably approximately 4-8%, more preferably approximately 4-6%, and most preferably approximately 5%.

[0141] The culture supernatant of naive induced pluripotent stem cells can be obtained by separating and removing cellular components after culturing. In this disclosure, the culture supernatant includes not only the culture supernatant obtained by separating and removing cellular components from the culture medium, but also the culture supernatant that has been subjected to various treatments as appropriate (e.g., centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.).

[0142] [cosmetics] The present invention also provides cosmetics containing the culture supernatant of naive induced pluripotent stem cells produced by the present invention as a raw material. The culture supernatant of naive induced pluripotent stem cells produced by the present invention is as described above. The cosmetics of the present invention may be in any dosage form or shape as long as they are used in contact with the skin at the time of use. Specifically, in a broad sense, this includes products such as skin milk, skin cream, foundation cream, massage cream, cleansing cream, shaving cream, cleansing foam, lotion, toner, face mask, lipstick, blush, eyeshadow, nail polish, soap, body wash, hand soap, shampoo, conditioner, hair tonic, treatment, hair cream, hair spray, hair growth products, hair tonics, hair dyes, hair styling products, hair removal products, anti-dandruff products, toothpaste, denture adhesive, mouthwash, permanent wave products, curling agents, styling products, ointments, poultices, tapes, bath additives, antiperspirants, sunscreens, etc. Any type of product that comes into contact with the skin during use is acceptable, but it is especially preferable that it be in a form that can be used as a cosmetic. Furthermore, it also includes products that come into contact with the skin of animals in addition to humans. Furthermore, the cosmetic product of this application may take any form, including solids, liquids, semi-solids, gases, as well as powders, granules, tablets, gels, foams, and many other forms, but is not limited thereto.

[0143] The cosmetic product of this application may contain formulation-permissible carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, preservatives, antiseptics, and physiological saline, etc. Examples of excipients include lactose, starch, sorbitol, D-mannitol, and sucrose. Examples of disintegrants include carboxymethylcellulose and calcium carbonate. Examples of buffers include phosphates, citrates, and acetates. Examples of emulsifiers include gum arabic, sodium alginate, and tragacanth. Examples of suspending agents include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of analgesics include benzyl alcohol, chlorobutanol, and sorbitol. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. [Examples]

[0144] The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way by these examples.

[0145] [material and method] cell culture Human dermal fibroblasts (HDFs) were obtained from the Tokyo Metropolitan Institute of Gerontology based on informed consent. HDFs and post-irradiated mouse embryonic fibroblasts (iMEFs) were maintained in Dulbecco's modified Eagle medium (DMEM, Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS, Japan Bio Serum). PBMCs purchased from Cellular Technology Limited (CTL) were maintained under the conditions recommended by the manufacturer. Primed and naive ES cell (ESC) clones were obtained from WiCELL and Kyoto University. Primed pluripotent stem cells (PSCs) were maintained in StemFit AK02N medium (Ajinomoto) on plates coated with laminin 511-E8 fragment (iMatrix-511, Nippi). Naive PSCs are cultured on iMEF, and t2iLGo culture is performed using N2B27 medium (NDiff227, Takara Bio) containing 1 μM CHIR99021 (Merck), 1 μM PD0325901 (Merck), 10 μg / mL human LIF (Peprotech), and 2.5 μM Go6983 (Merck). 2 The cells were maintained in culture medium. Feeder-free naive induced pluripotent stem cells (iPSCs) were maintained on Matrigel (hESC-qualified, Corning). The culture medium was changed every other day, and 10 μM Y27632 (Wako) was added immediately before each medium change. The naive iPSCs were subculturised every 3-4 days using Accutase (Innovative Cell Technologies) and cultured under constant 5% O2 conditions. Recombinant DNA experiments in this example were performed with the approval of Kyoto University.

[0146] Construction of a modified SeV-KLF4 vector Insertion sequences containing the open reading frame of the human KLF4 gene were constructed from cDNA by PCR using gene-specific forward and reverse primers tagged with NotI, which contained SeV-specific transcriptional regulatory signal sequences. The amplified fragments were inserted into the 18+ region of plasmids containing the genomic sequences of SeV / TS12ΔF, SeV / PmiR367T2 / TSΔF, and SeV / PmiR367T2 / TS12ΔF vectors to construct plasmids pSeV18+KLF4 / TS12ΔF, pSeV18+KLF4 / PmiR367T2 / TSΔF, and pSeV18+KLF4 / PmiR367T2 / TS12ΔF (collectively referred to as "SeV-KLF4 vector plasmids"). The sequence of miR367T2 was TCACCATTGCTAAAGTGCAATTcgatTCACCATTGCTAAAGTGCAATT (SEQ ID NO: 1). The SeV-KLF4 vector was recovered and augmented as follows: First, the SeV-KLF4 vector plasmid and plasmids containing T7 RNA polymerase, NP, P, F5R, and L genes were transduced into 293T cells. The cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and cultured for 1-3 days to generate a seed for the SeV-KLF4 vector. The seed was cloned and augmented using SeV F-expressing LLC-MK2 / F7 / A cells in MEM containing trypsin (2.5 μg / ml).The titer (cell infection units / mL) of the recovered SeV-KLF4 vector was determined by immunohistochemistry using anti-SeV rabbit polyclonal serum, as previously described (Fusaki, N., Ban, H., Nishiyama, A., Saeki, K. & Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings of the Japan Academy, Series B 85, ​​348-362, doi:10.2183 / pjab.85.348 (2009).).

[0147] Reprogramming of human somatic cells into naive iPSCs In the conventional method, HDF or PBMCs were initialized using CytoTune-iPS 2.0 or a 2.0L initialization kit. In the novel method, the KLF4 / TS vector included in the kit was replaced with a new SeV-KLF4 vector (such as KLF4 / TS12, KLF4 / PmiR367T2 / TS, or KLF4 / PmiR367T2 / TS12). The incubation temperature was maintained at 35°C until naive iPSC colonies were generated. After the first passage of the generated iPSCs (usually day 14), the incubation temperature was changed to 38°C and the SeV vector was removed. After the removal of the SeV vector (day 34 if SeV-KLF4 / PmiR367T2 / TS12 was used), the temperature was changed to 37°C. After infection with the SeV vector, the incubator was set to 5% O2.

[0148] First, HDF cells were maintained in DMEM (Nacalai Tesque) supplemented with 10% FBS. On day 0, cells were counted and infected with three SeV vectors: 1) polycistronic KLF4-OCT4-SOX2, 2) CMYC or LMYC, and 3) KLF4 (each with a multiple infection degree of 5). From day 1, the culture medium was changed every other day. On day 5, the cells were re-seed onto iMEF feeder cells. The culture medium was changed to naive medium the following day.

[0149] For PBMC reprogramming, PBMC cultures were started 5 days before SeV vector infection in StemSpan ACF (STEMCELL) containing 100 ng / mL human SCF (R&D), 100 ng / mL human TPO (R&D), 100 ng / mL human Flt3 / Flk2 (R&D), 50 ng / mL human IL-6 (R&D), and 20 ng / mL human IL-3 (R&D). Cells were counted on day 0 and infected with the three SeV vectors using the same reprogramming procedure as HDF. Cells were counted on day 1 and reseeded under the same conditions as HDF reprogramming. From day 2, the same amount of PSC medium as PBMC medium was added every other day. On day 8, the medium was completely replaced with naive medium.

[0150] RNA, DNA, and miRNA extraction, and quantitative real-time PCR (qRT-PCR). Prior to sample collection for nucleic acid extraction, iMEF was removed from naive PSCs by incubation at 37°C for 2 hours on a gelatin-coated dish. Total RNA and DNA were extracted from cell lysates using the AllPrep DNA / RNA Mini Kit (QIAGEN), and genomic DNA was removed by incubation of RNA with the RNase-Free DNase Set (QIAGEN). MicroRNA (miRNA) was extracted using NucleoSpin miRNA (MACHEREY-NAGEL). For qRT-PCR, reverse transcription of 1 μg of DNase-treated RNA was performed using PrimeScript RT Master Mix (Takara) containing oligo dT primers and a random 6-base pair. cDNA was synthesized using TaqMan Advanced miRNA Assays (Applied Biosystems). qRT-PCR was performed on StepOne Plus (Applied Biosystems) or QuantStudio3 (Applied Biosystems) using Fast SYBR Green Master Mix (Applied Biosystems) or TaqMan Fast Advanced Master Mix (Applied Biosystems).

[0151] RNA sequencing and data analysis RNA sequencing libraries were prepared using 100 ng of total RNA as a starting material, following the manufacturer's protocol, with TruSeq Stranded Total RNA Library Prep Gold (Illumina). For the Hiseq2500, clusters were generated using the HiSeq PE Cluster Kit v4-cBot (Illumina) with Illumina cBot. Sequencing was performed using the HiSeq SBS Kit v4 with the HiSeq2500 (2x126 PE mode). Additionally, for sequencing, the NovaSeq 6000 (2x101 PE mode) with the NovaSeq 6000 S1 Reagent Kit v1.5 (Illumina) and the NextSeq 500 (76 SE mode) with the NextSeq 500 / 550 High Output Kit v2.5 were used. FASTQ files were generated from bcl files using bcl2fastq v2.17.1.14 (Illumina) and processed using ENCODE long-rna-seq-pipeline v2.3.4. In short, the sequenced read data was mapped to the human reference genome (GRCh38) using STAR 2.5.1b with gene annotation from GENCODE v24, normalized gene expression data was calculated using RSEM 1.2.23, and gene count data was obtained using featureCounts included in Subread 1.5.1.To characterize our PSC strains, we used the GSE59435 and GSE75868 datasets obtained from GEO, as well as supplementary data from L. Yan et al. (Yan, L. et al. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nature structural & molecular biology 20, 1131-1139, doi:10.1038 / nsmb.2660 (2013)) and Y. Takashima et al. (Takashima, Y. et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158, 1254-1269, doi:10.1016 / j.cell.2014.08.029 (2014)). The expression values ​​of 4,720 genes included in the total data were normalized by the inter-sample quantiles, and the z-score of each gene was used for PCA. For expression heatmaps of PSC markers and oxidative phosphorylation-related genes, quantile-normalized FPKM values ​​were used on a Log2 scale.

[0152] DNA methylation analysis Bisulfite conversion of 500 ng of genomic DNA was performed using the EZ DNA Methylation Kit (Zymo Research), and global DNA methylation status was profiled using either Infinium Human Methylation 450K or EPIC BeadChip Kit (Illumina) according to the manufacturer's protocol. After exporting DNA methylation values ​​using GenomeStudio V2011.1, data processing was performed using the "minfi" package in R 3.6.3. A total of 424,444 probes, common to both 450K and EPIC and not located at known SNP sites, were used for PCA of PSC samples.

[0153] RNA-FISH Dissociated naive iPSCs were incubated on gelatin-coated dishes at 37°C for 2 hours to remove iMEF feeder cells. The cells were then seeded in PSC medium on slides coated with Matrigel. The following day, the cells were fixed in 4% paraformaldehyde at room temperature for 15 minutes. The slides were treated with 0.2M HCl for 20 minutes, permeabilized with 0.2% Triton X-100 for 10 minutes, and dehydrated by digestion in pepsin solution (0.005% in 0.1M HCl) at 37°C for 2–6 minutes. HUWE1 and UTX RNA FISH probes were generated using bacterial artificial chromosomes (BACs) RP11-155O24 and RP11-256P2, respectively. BAC DNA was labeled by nick translation using Cy5-dUTP (RP11-155O24) and Cy3-dUTP (RP11-256P2). Labeled probes and XIST RNA FISH probes (Chromosome Science Labo) were mixed with sonicated salmon sperm DNA and Cot-1 DNA in hybridization solution. The probes were denatured at 85°C for 10 minutes, applied to pre-treated slides, covered with coverslips, and hybridized overnight at 37°C. The slides were then washed with 50% formamide / 2xSSC at 37°C for 20 minutes and with 1xSSC at room temperature for 15 minutes, counterstained with DAPI, and mounted. FISH images were acquired using the CW4000 FISH application program (Leica Microsystems Imaging Solution) with a cooled CCD camera mounted on a Leica DMRA2 microscope.

[0154] Primordial endoderm differentiation derived from naive PSCs For primitive endoderm differentiation, naive PSCs were dissociated and iMEF feeder cells were removed as described above. 500,000 cells were then fed laminin 511-E8 (0.4 μg / cm³) containing early primitive endoderm differentiation medium (Ndiff227, 25 ng / ml FGF4 (Peprotech), 1 ug / ml Heparin (Wako), 10 ng / ml BMP4 (R&D), 10 ng / ml PDGFAA (Peprotech), 1 uM XAV939 (Sigma), 3 μM A83-01 (Wako), 0.1 μM RA (Sigma)). 2 Seeds were seeded in 6 wells coated with iMatrix 511 (Nippi). The following day, the medium was changed with the same medium as above. The day after that, the medium was changed again with the same medium with 10 ng / ml IL-6 (R&D) added. On day 3, cells were detached and collected using Trypsin / EDTA (Nacalai) for 5 minutes, and the expression of PDGFRA and ANPEP was analyzed by flow cytometry.

[0155] trophectoderm (TE) differentiation from naive PSCs For TE differentiation, naive PSCs were dissociated and iMEF feeder cells were removed as described above. 500,000 cells were fed laminin 511-E8 (0.15 μg / cm³) in initial TE differentiation medium (Ndiff227, 2 μM A83-01 (Wako), 2 μM PD0325921 (Sigma), and 10 ng / mL BMP4 (R&D)). 2 Cells were seeded in 6 wells coated with iMatrix 511 (Nippi). The following day, the medium was replaced with Ndiff227, 2 μM A83-01 (Wako), 2 μM PD0325921 (Sigma), and 1 μg / ml JAK inhibitor I (Merck). The medium was replaced again the following day. On day 3, cells were harvested using Accutase (Innovative Cell Technologies) for 30 minutes, and the expression of TACSTD2, ENPEP, HAVCR1, and HLA-ABC was analyzed by flow cytometry.

[0156] Flow cytometry Dissociated cells were stained on ice for 20 minutes with Alexa Fluor 488-labeled TROP-2 (TACSTD2), PE-labeled CD249 (ENPEP), Tim-1 (HAVCR1) biotinylated antibody, and Pacific Blue-labeled HLA-ABC antibody. After washing, APC streptavidin was applied and incubated on ice for 20 minutes. Analysis was performed using BD LSR Fortessa (BD Biosciences) and FACSAria II (BD Biosciences) flow cytometers equipped with FACS Diva software (BD Biosciences). Data were analyzed using FlowJo software (LLC).

[0157] Statistics and Reproducibility The exact value of n is indicated in the description of the relevant figure. Statistical significance was determined by an unpaired Student's two-tailed t-test using Prism software (GraphPad). P < 0.05 was considered significant and is indicated by an asterisk in the figure. Error bars indicate the mean ± sd.

[0158] Data availability The sequencing and array data are registered with GEO, and the accession number is GSE179476.

[0159] [Example 1] Production of the Sendai virus vector (SeV) used in this example The structure of the Sendai virus vector used in this embodiment is shown below. In this specification, "18+" indicates the insertion of a reprogramming factor before the NP gene, "PM" indicates the insertion of a reprogramming factor between the P gene and the M gene, and "HNL" indicates the insertion of a reprogramming factor between the HN gene and the L gene. In this specification, "TS" indicates that the M protein has the mutations G69E, T116A, and A183S, the HN protein has the mutations A262T, G264R, and K461G, the P protein has the mutation L511F, and the L protein has the mutations N1197S and K1795E. In this embodiment, "TS7" indicates that in addition to the TS mutation, the L protein also has the mutations Y942H, L1361C, and L1558I; "TS12" indicates that in addition to the TS mutation, the P protein also has the mutations D433A, R434A, and K437A; and "TS15" indicates that in addition to the TS12 mutation, the L protein also has the mutations L1361C and L1558I. "ΔF" indicates the deletion of the F gene. "KOS" indicates the simultaneous incorporation of KLF4, Oct4, and Sox2. For example, an F gene-deleted Sendai virus vector with a TS12 mutation that incorporates KLF4, Oct4, and Sox2 between the P and M genes is described as PM / KOS / TS12ΔF. Similarly, an F gene-deleted Sendai virus vector with a TS mutation that incorporates KLF4 before the NP gene is described as 18+ / KLF4 / TSΔF.

[0160] Figure 1 shows a Sendai virus vector (CytoTune-iPS2.0L) conventionally used to produce naive pluripotent stem cells from somatic cells. The cDNA it carries is shown in white. The Sendai virus vector used in this example is a system in which, among the Sendai virus vectors shown in Figure 1, "18+ / KLF4 / TS7ΔF", "18+ / KLF4 / TS12ΔF", or "18+ / KLF4 / PmiR367T2 / TSΔF" is used as the KLF4 vector instead of "18+ / KLF4 / TSΔF". Here, "PmiR367T2" indicates that the miR367 recognition sequence is tandem-mounted on the 5' side of the P gene.

[0161] The KOS vector "PM / KOS / TS12ΔF" and the LMYC vector "HNL / LMYC / TS15ΔF" were prepared by conventional methods (WO2012 / 029770 and WO2010 / 008054). In addition, "18+ / KLF4 / TS7ΔF" and "18+ / KLF4 / TS12ΔF" were prepared using the TS7 and TS12 vector skeletons, respectively, in the same manner as "18+ / KLF4 / TSΔF" (WO2010 / 008054). Furthermore, "18+ / KLF4 / PmiR367T2 / TSΔF" was prepared by conventional methods (WO2017 / 082174).

[0162] [Example 2] Production of naive iPS cells from human dermal fibroblasts (HDF) Figure 2 shows the protocol for producing naive iPS cells from human dermal fibroblasts (HDFs).

[0163] Process (1) Human dermal fibroblasts were seeded in the same number of cells for SeV infection and cell counting into gelatin-coated 12-well or 6-well plates one day before infection with the Sendai virus vector. They were cultured at 37°C under normal oxygen conditions (approximately 20%) using Fibroblast medium (DMEM + 10% FBS). On the following day (day 0), the cells seeded for cell counting were detached using a cell detachment solution, and the cell count was measured. A corresponding amount of Sendai virus vector was then used for infection, and the cells were incubated at 35°C under hypoxic conditions (5% O2). As a guideline, the amount of Sendai virus vector used for infection was KOS:MOI5, LMYC:MOI5, KLF4:MOI5, but the conditions were adjusted as needed depending on the cells. The medium was changed to fresh Fibroblast medium after 24 hours (day 1), and again on day 2 and day 4. On day 4, cell culture vessels seeded with iMEF (irradiated mouse embryonic fibroblasts) were prepared. On day 5, cells were detached using a cell detachment solution, and after counting the number of cells, an arbitrary number of cells were seeded onto a cell culture vessel containing iMEF. In this example, culture was carried out under a CO2 concentration of 5%.

[0164] Process (2) From day 6, the medium was changed to a naive culture medium (t2iLGo+Y), and the medium was changed every other day thereafter.

[0165] The composition of t2iLGo+Y medium is shown below. All concentrations listed are final concentrations: NDiff227 (manufactured by Takara Bio Inc.) CHIR99021 (Sigma-Aldrich) 1 μM PD0325901 (manufactured by Sigma-Aldrich) 1μM Go6983 (Sigma-Aldrich) 1.25 μM or 2.5 μM Human recombinant leukemia inhibitory factor (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 10 ng / ml Y27632 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 10uM. Here, Y27632 does not need to be added from the day after subculturing.

[0166] Around day 10, layered, dome-shaped colonies (naive iPS cell colonies) began to appear.

[0167] Process (3) From Day 10 onward, when the dome-shaped colonies in the aforementioned layered culture reached a sufficient size (approximately 50-200 μm in diameter), subculturing was performed, and high-temperature culture (basically 38°C) was started the following day.

[0168] The t2iLGo+Y medium was changed every other day, and the cells were cultured in an incubator with a 5% oxygen concentration. Subculturing was performed every 3-4 days. For cell detachment during subculturing, the medium was removed, the cells were washed once with D-PBS (Nacalai Tesque), and Accutase (Innovative Cell Technologies) was added, followed by incubation at 37°C for 7-10 minutes. If the purpose was solely maintenance, as a guideline, about 1 / 5 of the number of cells obtained after detachment were seeded into a culture vessel of the same area.

[0169] Figure 3A shows a phase-contrast microscope image of cells after 45 passages. Numerous multilayered, dome-shaped colonies were observed, while almost no monolayered, flattened colonies were found. Figure 3B shows images of the dome-shaped colonies from Figure 3A, immunostained with naive-type specific markers (NANOG, KLF17) and a gene product (TFE3) whose intracellular localization differs between naive and primed types. Hoechst shows nuclear staining, and PC shows the phase-contrast microscope image. As shown in Figure 3B, the constituent cells of the dome-shaped colonies were strongly stained for NANOG and KLF17, and the nuclei were intensely stained for TFE3. In other words, induced pluripotent stem cells were obtained that express naive-type specific markers, exhibit naive-type specific intracellular localization of specific gene products (gene expression pattern equivalent to that of the inner cell mass of preimplantation embryos), and further form multilayered, dome-shaped colonies (characteristic colony morphology of the naive type). Therefore, it has been shown that naive iPS cells can be produced from human fibroblasts by the method comprising the steps (1) to (3) described above.

[0170] Next, the intracellular residual amount of the Sendai virus vector used was analyzed. A conventional vector (CytoTune-iPS2.0L) was used as a comparison. The intracellular genome amount of the Sendai virus vector was measured by quantitative PCR, and the results, expressed as relative values ​​with the genome amount at Day 14 set to 1.0, are shown in Figure 4. "Conv. Cocktail" represents CytoTune-iPS2.0L, and "KLF4 / TS7ΔF" represents CytoTune-iPS2.0L with the KLF4 vector replaced by "18+ / KLF4 / TS7ΔF".

[0171] As shown in Figure 4, the vector used in this example had already decreased to below the detection limit by Day 38. This indicates that it rapidly disappeared due to high-temperature culture at 38°C (KLF4 / TS7ΔF_1~3). Furthermore, when "18+ / KLF4 / TS12" or "18+ / KLF4 / TS12 / miR367T2" were used as the KLF vector and cultured at 38°C, it became below the detection limit 8 to 12 days earlier (data not shown).

[0172] In contrast, conventional vectors remained present at a similar level on Day 80 as on Day 14 (Conv. Cocktail_1~3). Since these vectors are known to gradually decrease even at typical culture temperatures (37°C) under normal culture conditions using primed induced pluripotent stem cell culture medium, and almost completely disappear by Day 80, this strongly suggests that the rate of vector disappearance may be affected by different cell culture environments.

[0173] Therefore, to obtain naive pluripotent stem cells that do not contain vector-derived reprogramming factors at an early stage, it is sufficient to use a vector that exhibits temperature sensitivity under culture conditions using naive medium, or a vector containing a microRNA target sequence specific to induced pluripotent stem cells. As an example of such a temperature-sensitive vector, it was shown that Sendai virus vectors containing TS7, TS12, or TS15 mutations can be used.

[0174] [Example 3] Production of naive iPS cells from human peripheral blood mononuclear cells (PBMCs) Figure 5 shows the protocol for producing naive iPS cells from human peripheral blood mononuclear cells (PBMCs).

[0175] Process (1) PBMCs were cultured at 37°C under normal oxygen conditions (approximately 20%) using PBMC medium starting 5 days prior to Sendai virus vector infection. No medium changes were performed. The composition of the PBMC medium is shown below. All concentrations listed are final concentrations: StemSpan ACF (manufactured by STEMCELL Technologies) Recombinant Human SCF (manufactured by R&D Systems) 100 ng / ml Recombinant Human TPO (manufactured by R&D Systems) 100 ng / ml Recombinant Human Flt3 / Flk2 (manufactured by R&D Systems) 100 ng / ml Recombinant Human IL-6 (manufactured by R&D Systems) 50ng / ml Recombinant Human IL-3 (manufactured by R&D Systems) 20ng / ml.

[0176] On the day of infection (day 0), the number of cells was measured, and an amount of Sendai virus corresponding to the number of cells was used for infection. Thereafter, the cells were incubated in a 35°C hypoxic (5% O2) environment. As a guideline, the amount of Sendai virus used for infection was KOS:MOI5, LMYC:MOI5, KLF4:MOI5, but the conditions were adjusted as needed depending on the cells. To increase the virus density during infection, infection was basically carried out in 24 wells or smaller wells. On the same day, cell culture vessels (basically 6 wells) seeded with iMEF were also prepared. 24 hours after infection (day 1), the cells were collected, the virus-containing medium was removed, the number of cells was measured, and an arbitrary number of cells were seeded into the cell culture vessels seeded with iMEF. In this example, culture was carried out under a 5% CO2 concentration condition.

[0177] Process (2) On day 2, half of the PBMC medium was removed and half of the naive medium (t2iLGo+Y) was added. Thereafter, half of the medium was removed and half of the new medium was added in the same manner every other day. On day 8, the medium was completely aspirated and replaced with naive medium. The composition of the naive medium (t2iLGo+Y) is the same as that used when naive iPS cells were produced from human dermal fibroblasts in Example 2.

[0178] Process (3) Around day 10, dome-shaped colonies (naive iPS cell colonies) appeared in a layered structure. Once the colonies reached a sufficient size (approximately 50-200 μm in diameter) after day 10, subculturing was performed, and high-temperature culture (basically 38°C) was started the following day. The maintenance culture and subculturing methods were the same as those used in Example 2 when naive iPS cells were produced from human dermal fibroblasts.

[0179] The amount of Sendai virus vector genome was quantitatively evaluated by qPCR at each passage starting from day 14, when culture was initiated at 38°C (Figure 6). When 18+ / KLF4 / TS7ΔF was used as the KLF4 vector, the vector amount was below the detection limit by day 38, but when 18+ / KLF4 / TS12ΔF or 18+ / KLF4 / PmiR367T2 / TSΔF was used, it was below the detection limit by day 30.

[0180] Therefore, it was shown that naive induced pluripotent stem cells can be produced from human peripheral blood mononuclear cells by the method comprising steps (1) to (3) above, and furthermore, naive iPS cells substantially free of reprogramming factors can be obtained after approximately 30 to 38 days.

[0181] Therefore, it has become clear that naive induced pluripotent stem cells (more specifically, naive iPS cells substantially free of reprogramming factors) can be rapidly produced from various somatic cells of humans by using the method described in this application.

[0182] [Example 4] Trait stability of naive induced pluripotent stem cells The phenotypic stability of naive induced pluripotent stem cells obtained in Examples 2 and 3 after long-term passage was analyzed. Principal component analysis was performed on single-cell data from previously reported naive ES cells (Reset Naive ESC (in-house)), eight clones of HDF-derived prime iPS cells established by the inventors, four clones of HDF-derived naive iPS cells obtained using the present invention after long-term passage (P16-P45), two clones of PBMC-derived naive iPS cells, and human embryos (Figure 7). Naive iPS cells established by the inventors are present in the area enclosed by the red frame, and Reset Naive ESC (in-house) is also present in the same group, indicating that global gene expression is equivalent to that of Reset Naive ESC.

[0183] Based on the same RNA-seq data as in Figure 7, the expression patterns of imprinted genes and X chromosome-related genes in each cell type were compared and verified using SNP analysis (Figure 8). HDF, primed, and naive iPS cells were all derived from HDF cells of the same female. In HDF and primed iPS cells, the expression of imprinted genes, such as MEG3, generally showed an expression pattern from only one allele, but in naive cells, imprinting was lost and an expression pattern from both alleles was observed. Furthermore, in the female-derived HDF and primed iPS cells used in this verification, inactivation of one X chromosome resulted in an expression pattern of X chromosome-related genes from only one allele, but in naive cells, X chromosome reactivation resulted in an expression pattern from both alleles.

[0184] Global methylation of HDF-derived and PBMC-derived naive iPS cells was represented using a methylation array with a density bean plot (Figure 9A) and principal component analysis (Figure 9B) using DNA obtained simultaneously with RNA from the same cells used in RNA-seq in Figure 7. "Primed" refers to primed iPS cells established by the applicant, and "Naive" refers to naive iPS cells. In Figure 9A, the naive cells obtained in this invention all tended to have lower Beta values ​​(=Hypomethylated) compared to primed cells, and showed hypomethylation equivalent to that of Reset Naive ESC (in-house). In Figure 9B, primed and naive cells were clearly separated, indicating that they have different methylation patterns. Furthermore, the proportion of 5-methylcytosine in the total cytosine of HDF-derived naive iPS cells obtained in this example was analyzed using LC-MS / MS (Figure 9C). Naive iPS cells showed a low proportion similar to that of Reset Naive ESCs, suggesting a decrease in the proportion of methylated cytosine in the genome.

[0185] The early differentiation potential of the three germ layer components was compared and analyzed for two clones of HDF-derived naive iPS cells, two clones of PBMC-derived naive iPS cells, and Reset Naive ESCs (in-house) as a control, after long-term passage (P16-45) using RNA-seq and methylation arrays (Figure 10). ES cells / iPS cells were placed in 10 × 10⁶ well plates. 6 Individual cells were seeded and cultured for 24 days in a hypoxic (5% O2) environment using differentiation medium (DMEM + 20% FBS), and the formed embryoid bodies were collected. RNA was extracted from the collected samples, and cDNA was obtained by reverse transcription. The ability to differentiate into the three germ layers was then semi-comprehensively evaluated using the TaqMan hPSC Scorecard Panel (ThermoFisher Scientific). The expression levels of pluripotency markers, ectoderm markers, mesoderm markers, and endoderm markers are shown from left to right in the figure. The gray plots represent Reset Naive ESCs, and the red plots represent naive iPS cells obtained in this invention. All clones showed pluripotency equivalent to or even better than Reset Naive ESCs, particularly in the mesoderm.

[0186] Thus, it was confirmed that naive induced pluripotent stem cells obtained by the method of this invention exhibit a global gene expression pattern, widespread DNA demethylation across the entire genome, X chromosome reactivation, and early differentiation potential of the three germ layer components, similar to that of standard naive induced pluripotent stem cells, even after long-term passage exceeding 180 days. Therefore, it has become clear that true naive induced pluripotent stem cells can be rapidly produced by the method described in this application.

[0187] [Example 5] Development of a modified OSKL-SEV vector initialization system Naive human iPS cells (nCT2.0L_1-4) were established from human dermal fibroblasts (HDF) using a commercially available CytoTune-iPS 2.0L SeV reprogramming kit (containing SeV(PM)KOS / TS12ΔF(SeV-KOS), SeV(HNL)LMYC / TS15ΔF(SeV-LMYC), and SeV18+KLF4 / TSΔF(SeV-KLF4) vectors). At day 94 after SeV introduction, the presence of residual SeV in the established naive human iPSCs was verified by immunohistochemistry (Figure 11A). As shown in the upper right panel of Figure 11A, most cells showed strong positivity, suggesting a high degree of SeV persistence. Next, cells were collected at each passage, and the amount of residual SeV genome was measured by quantitative RT-PCR (qRT-PCR). Whether using commercially available CytoTune-iPS 2.0 (containing SeV-KOS, SeV(HNL)CMYC / TS15ΔF(SeV-CMYC), and SeV-KLF4 vectors; OSKM) or CytoTune-iPS 2.0L (OSKL), the amount of SeV in HDF or human peripheral blood mononuclear cells (PBMCs) initially decreased with each passage, but then tended to increase again, indicating that SeV did not disappear even after repeated passages (Figure 11B). Furthermore, SeV genome expression remained in all clones, and in some cell lines, the expression level was equivalent to or higher than that of the housekeeping gene GAPDH. Quantification of the expression levels of the three remaining SeV vector genomes revealed that SeV-KOS and SeV-CMYC / LMYC vectors remained at particularly high levels among all clones, and the SeV-KLF4 vector remained at particularly high levels in more than half of the clones (Figure 11C).

[0188] We investigated the reasons why these vectors persist. While SeV vectors share viral proteins in mixed infections, the SeV-TSΔF vector, which forms the backbone of the SeV-KLF4 / TS vector, is known to be very temperature-insensitive, unlike the SeV-TS12ΔF or SeV-TS15ΔF vectors, making it difficult to remove from infected cells by increasing temperature. Table 1 shows the presence or absence of point mutations in each gene in the SeV-TSΔF, SeV-TS12ΔF, or SeV-TS15ΔF vectors. [Table 1] Therefore, we hypothesized that the presence of the SeV18+KLF4 / TSΔF(KLF4 / TS) vector in the initialization cocktail is the primary cause of the persistence of all SeV vector genomes.

[0189] To test this hypothesis, the following three modified SeV-KLF4 vectors were applied as substitutes for the SeV-KLF4 / TS vector (Figure 11D): SeV18+KLF4 / TS12ΔF (SeV-KLF4 / TS12), SeV18+KLF4 / PmiR367T2 / TSΔF (SeV-KLF4 / miR / TS), and SeV18+KLF4 / PmiR367T2 / TS12 (SeV-KLF4 / miR / TS12) vectors. The SeV-KLF4 / TS12 vectors were formed based on the SeV-TS12ΔF vector, which has more point mutations in the P gene to enhance temperature sensitivity (Figure 11D). The SeV-KLF4 / miR / TS vector contains an hsa-microRNA-367 (miR-367) target sequence (i.e., not miR-367 itself, but the target sequence of miR-367) inserted in tandem at the 5' end of the P gene in the SeV vector genome (Figure 11D). While miR-367 has been confirmed to be specifically overexpressed in primed human pluripotent stem cells (PSCs), the inventors confirmed that miR-367 is also overexpressed in naive human PSCs compared to HDFs (Figure 11E). Therefore, the receptivity of miR-367 was considered useful for rapid removal of the SeV vector in naive human PSCs. The SeV-KLF4 / miR / TS12 vector incorporates features of both SeV-KLF4 / TS12 and SeV-KLF4 / miR / TS, suggesting that it may further accelerate vector removal (Figure 11D).

[0190] The efficiency of initialization by combinations of these vectors was compared. When using a temperature-sensitive SeV vector having the KLF4 gene in the production of iPSCs from human fibroblasts, it has been reported that iPSCs could not be produced at 37°C except when the multiplicity of infection was high (presumably because most vectors disappeared before the completion of initialization). Therefore, the cells were cultured at 35°C from the time of infection until day 14 when sufficient naive iPSC colonies appeared, and the first passage was performed. After the first passage, the culture temperature was raised to 38°C to remove the SeV vector (Figure 11F). The initialization of HDFs using SeV-KOS, SeV-LMYC, and the modified SeV-KLF4 vector resulted in the appearance of naive iPSC colonies around day 10, which was consistent with the conventional method using the SeV-KLF4 / TS vector.

[0191] There is no report of establishing naive human iPSCs from human peripheral blood mononuclear cells (PBMCs). In fact, the inventors were also unable to establish naive human iPSCs from PBMCs using CytoTune-iPS 2.0 (Figure 11G).

[0192] [Example 6] Verification of SeV disappearance rate, application to feeder-free naive iPSCs The establishment efficiencies when establishing naive human iPS cells from HDFs using each KLF4 vector were compared (Figure 12A). None of the vectors showed a decrease in establishment efficiency compared to the conventional vector (SeV-KLF4 / TS), and the OSKL (OSKM with CMYC replaced by LMYC) cocktail containing SeV-KLF4 / TS12 or SeV-KLF4 / miR / TS12 showed a significantly improved establishment efficiency compared to the conventional vector.

[0193] Next, the SeV vectors remaining in the naive iPSCs produced by these vectors were detected. For the application of iPSC-derived differentiated cells to regenerative medicine, at least 1x10 <​​We confirmed that the method has the sensitivity to detect a single SeV-positive cell from 1,638,400 SeV-negative cells (Figure 12B). Calculations suggest that detection is possible if even one SeV-positive cell is present among 1,638,400 SeV-negative cells, demonstrating significantly higher sensitivity than methods such as immunohistochemistry. This qRT-PCR analysis quantitatively evaluated the remaining amount of SeV genome. After changing the temperature on day 14, the SeV-KLF4 / TS12, SeV-KLF4 / miR / TS, and SeV-KLF4 / miR / TS12 vectors showed significant clearance of the SeV genome, but the decrease in SeV-KLF4 / TS levels was very slow. In particular, SeV-KLF4 / miR / TS12 reached the detection limit of qRT-PCR on day 34, which was the fastest among the SeV-KLF4 vectors (Figure 12C). From these results, we concluded that the SeV-KLF4 / miR / TS12 vector is the best SeV-KLF4 vector in terms of reprogramming efficiency and vector clearance rate.

[0194] Further investigation was conducted on reprogramming using the SeV-KLF4 / miR / TS12 vector. Using SeV-KOS, SeV-LMYC, and the SeV-KLF4 / miR / TS12 vector, naive iPSC clones were successfully generated not only from HDF (nOSKL_1, 2) but also from PBMC (nOSKL_3, 4). On day 34, the disappearance of SeV was confirmed in all nOSKL-iPSC clones (Figure 12D). Interestingly, by using a conditioned medium prepared by contacting irradiated mouse embryonic fibroblasts (iMEF) with t2iLGo medium from the beginning of reprogramming, feeder-free naive iPSCs were successfully established and maintained from HDF (nOSKL_FF_1, 2) (Figure 12E). Next, the cell proliferation rate of nOSKL_1-4 was measured. No significant decrease in cell proliferation rate was observed in any of the clones even after repeated passages (Figure 12F).

[0195] [Example 7] Gene expression of the established naive iPSC Next, we examined the characteristics specific to naive pluripotency of the generated nOSKL-iPSCs. For comparison, we collected primed human iPSCs (OSKL_1-4), primed WA09 H9 embryonic stem cells (ESCs; H9), primed 201B7 iPSCs (201B7), and reset naive H9 ESCs (nH9) that were reset from primed to naive by overexpressing KLF2 and NANOG and maintained under the same conditions as nOSKL-iPSCs. We examined the expression of naive iPSC-specific markers by qRT-PCR (Figure 13A). Here, NANOG is also expressed in primed iPSCs, but it is known to be significantly increased in naive iPSCs. All naive iPSC strains expressed naive iPSC-specific markers at significantly higher levels compared to primed iPSC strains. Similarly, the expression of primed iPSC-specific markers was examined by qRT-PCR (Figure 13B). All naive iPSC strains expressed primed iPSC-specific markers at significantly lower levels compared to primed iPSC strains. RNA-seq was performed on these cell lines to compare the expression of representative pluripotency markers (Figure 13C). nOSKL-iPSC showed pluripotency marker expression almost equivalent to that of nH9.Next, the transcriptome of nOSKL-iPSCs was compared with that of primed PSCs and previously published datasets of reset naive PSCs and human intracellular cell aggregates (Takashima, Y. et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158, 1254-1269, doi:10.1016 / j.cell.2014.08.029 (2014).; Theunissen, TW et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell stem cell 15, 471-487, doi:10.1016 / j.stem.2014.07.002 (2014).; Yan, L. et al. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nature structural & molecular biology 20). 1131-1139, doi:10.1038 / nsmb.2660 (2013). Principal component analysis (PCA) clearly distinguished nOSKL-iPSCs from primed PSCs and revealed that they possessed almost identical characteristics to nH9 (Figure 13D). The naive human iPSC established by the inventors was the closest to the epiblast.

[0196] [Example 8] Verification of methylation and X chromosome reactivation of established naive iPSCs Methylation arrays were performed on the cell lines used in RNA-seq in Example 7 (Figure 14A). nOSKL-iPSCs, including feeder-free naive iPSCs (FF_1-2), showed a global DNA demethylation status compared to primed iPSCs. Furthermore, PCA of the methylation array data clearly separated primed iPSCs from naive iPSCs (Figure 14B).

[0197] X chromosome reactivation was verified by RNA-FISH (Figure 14C). HUWE1 is a gene expressed on the activated X chromosome. XIST is an lncRNA that normally works to inactivate the X chromosome, but in preimplantation epiblasts, which have characteristics similar to naive iPSCs, XIST is positive on both activated X chromosomes, a seemingly contradictory finding (leftmost panel in Figure 14C). As the cells approached primed iPSCs, the gene expression pattern changed to the rightmost panel in Figure 14C, and in primed iPSCs, basically almost all cells showed the gene expression pattern in the rightmost panel in Figure 14C (HUWEI+ / -, XIST- / -). Furthermore, RNA-FISH was performed on the established clones (Figure 14D). Although there was variability among clones, nOSKL-iPSCs clearly showed a pattern similar to preimplantation epiblasts compared to primed iPSCs. These observations confirmed that nOSKL-iPSCs were in a naive state.

[0198] [Example 9] Evaluation of mitochondrial function of established naive iPSCs Based on RNA-seq data from Example 7, the expression of mitochondrial genes related to oxidative phosphorylation was compared (Figure 15A). Naive iPSCs showed higher mitochondrial gene expression levels compared to primed iPSCs, suggesting that mitochondrial functions such as aerobic metabolism were activated. Next, the metabolic capacity of naive iPSCs and primed iPSCs was compared using an extracellular flux analyzer (Figure 15B). Naive iPSCs showed a significantly increased oxygen consumption rate (OCR) after FCCP administration compared to primed iPSCs, suggesting superior respiratory reserve capacity in naive iPSCs. Respiratory reserve capacity, one of the indicators of aerobic metabolic capacity, was quantitatively compared between naive iPSCs and primed iPSCs (Figure 15C). nOSKL-iPSCs showed higher values ​​compared to primed iPSCs, similar to nH9. Furthermore, we examined the extracellular acidification rate (ECAR), an indicator of glycolytic metabolic function, and the optical cell regeneration (OCR), an indicator of aerobic metabolic function (Figure 15D). Naive iPSCs showed higher ECAR and OCR values ​​compared to primed iPSCs, suggesting a high-energy metabolic state.

[0199] [Example 10] Verification of the differentiation ability of established naive iPSCs into primitive endoderm. Naive PSCs resemble preimplantation epiblasts and possess the ability to differentiate into extraembryonic tissues. This characteristic differs from primed PSCs. Primordial endoderm can hardly be differentiated from primed iPSCs, but it can be differentiated from naive iPSCs. Therefore, we investigated the ability of nOSKL-iPSCs to differentiate into primitive endoderm derived from naive PSCs in a specified differentiation medium (Figure 16A). Light micrographs of primed ESCs and nOSKL-iPSCs on day 3 after the start of differentiation induction are shown in Figure 16B. Compared to primed ESCs, nOSKL-iPSCs clearly showed a morphology similar to endodermal cells. Next, we compared and verified the expression levels of PDGFRA and ANPEP, which are primitive endoderm-specific markers, using flow cytometry (Figure 16C). n9 and nOSKL-iPSCs showed high expression of PDGFRA and ANPEP, which are primitive endoderm-specific markers, and demonstrated good differentiation potential into primitive endoderm. Primed PSCs and naive iPSCs with residual SeV established using the conventional SeV-KLF4 / TS vector (CytoTune-iPS 2.0L) showed very low expression levels of PDGFRA and ANPEP, indicating significantly lower differentiation potential into primitive endoderm. The median signal value in PDGFRA / ANPEP co-positive cells was examined (Figure 16D). Primed iPSCs and naive iPSCs with residual SeV were distributed in the lower left of Figure 16D, while nH9 and naive iPSCs established using this method were distributed in the upper right, suggesting that naive iPSCs established using this method have excellent differentiation potential similar to nH9.

[0200] [Example 11] Verification of the differentiation ability of established naive iPSCs into trophectoderm. Trophoectoderm (TE) is also difficult to differentiate from primed iPSCs, but it can be differentiated from naive iPSCs. The inventors investigated the ability of nOSKL-iPSCs to differentiate into trophectoderm (TE) derived from naive PSCs in a specified differentiation medium (Figure 17A). Figure 17B shows light micrographs of primed ESCs and nOSKL-iPSCs on day 3 after the start of differentiation induction. The morphologies of the two were clearly different on day 3 after the start of differentiation induction. Next, the expression levels of trophectoderm-specific markers TACSTD2, ENPEP, HAVCR1, and the negative marker HLA-ABC were compared and verified using flow cytometry (Figure 17C). n9 and nOSKL-iPSCs showed good differentiation ability into TE because they highly expressed HAVCR1, a TE-specific marker, and did not express HLA-ABC. Primed PSCs did not express HAVCR1 at all, and nearly half were HLA-ABC positive, suggesting that they hardly differentiated into TE cells. Naive iPSCs with residual SeV established using the conventional SeV-KLF4 / TS vector (CytoTune-iPS 2.0L) had very low expression levels of TACSTD2, ENPEP, and HAVCR1, indicating significantly low differentiation potential into TE cells, similar to primed PSCs. Flow cytometry was used to examine the proportion of TACSTD2 / ENPEP co-positive and HAVCR1-positive cells (Figure 17D). Primed iPSCs and naive iPSCs with residual SeV were distributed on the left side of Figure 17D, while nH9 and naive iPSCs established using this method were distributed on the right side of Figure 17D, suggesting that naive iPSCs established using this method have excellent differentiation potential similar to nH9. Interestingly, some clones of nOSKL-iPSC expressed TACSTD2 and ENPEP at higher levels than nH9.

[0201] In summary, the inventors developed a remarkably fast SeV genome removal system for generating naive human iPSCs from somatic cells by improving the structure of the SeV-KLF4 vector. As a result, compared to naive human iPSCs produced by conventional methods, they were able to obtain naive human iPSCs that exhibited robust transgene independence in early passage and superior naive-specific differentiation ability. Furthermore, they succeeded in reprogramming PBMCs into naive human iPSCs by changing the combination of reprogramming factors to SeV-OSKL. The inventors also established naive human iPSCs from dermal fibroblasts in a feeder-free environment using iMEF-conditioned medium. These technological innovations will greatly advance research into early development and clinical applications using naive human iPSCs.

Claims

1. A method for producing naive induced pluripotent stem cells from human somatic cells, (1) A step of introducing one or more vectors containing reprogramming factors into human somatic cells. (2) A step of culturing the somatic cells in the presence of a naive medium to obtain cultured cells, and (3) A step in which the cultured cells are cultured under different conditions than those in step (2), wherein the different conditions are such that the amount of vector per cultured cell is reduced to 30% or less compared to the start of step (3). A method comprising, wherein one or more vectors are selected from Sendai virus vectors containing TS12 mutations (TS mutations (G69E / T116A / A183S mutation in M ​​protein, A262T / G264R / K461G mutation in HN protein, L511F mutation in P protein, and N1197S / K1795E mutation in L protein) in addition to the D433A / R434A / K437A mutation in P protein), TS7 mutations (TS mutations in addition to the Y942H / L1361C / L1558I mutation in L protein), or TS15 mutations (TS mutations in addition to the D433A / R434A / K437A mutation in P protein and L1361C / L1558I mutation in L protein).

2. The method according to claim 1, wherein step (2) is started 1 to 10 days after step (1).

3. The method according to claim 1 or 2, wherein step (2) is performed for 1 to 20 days.

4. The method according to any one of claims 1 to 3, wherein step (3) includes culturing the cultured cells at 38°C or higher.

5. The method according to any one of claims 1 to 4, wherein one or more vectors include vectors containing a target sequence of microRNA specific to induced pluripotent stem cells.

6. The method according to claim 5, wherein the target sequence of the microRNA is located in the coding region, 5'UTR, or 3'UTR of an NP gene or a P gene.

7. The method according to claim 6, wherein the microRNA is miR-367.

8. The method according to any one of claims 1 to 7, wherein the reprogramming factor comprises the OCT gene, the SOX gene, the MYC gene, and / or the KLF gene.

9. The method according to claim 8, wherein the one or more vectors include a vector containing the OCT gene, the SOX gene and the KLF gene, a vector containing the MYC gene, and a vector containing the KLF gene.

10. The method according to claim 9, wherein the vector containing the MYC gene is a Sendai virus vector containing the TS15 mutation.

11. The method according to claim 9 or 10, wherein the vector comprising the OCT gene, the SOX gene, and the KLF gene, and the vector comprising the KLF gene, are Sendai virus vectors comprising the TS12 mutation.

12. The method according to any one of claims 1 to 11, wherein in step (3), the amount of the vector per cultured cell decreases to 30% or less compared to the amount at the start of step (3) within 12 days from the start of step (3).

13. The method according to any one of claims 1 to 12, wherein the naive culture medium contains one or more compounds selected from LIF, MEK inhibitors, GSK3 inhibitors, cAMP production promoters, TGF-β inhibitors, and PKC inhibitors.

14. The method according to claim 13, wherein the naive culture medium is a medium selected from the group consisting of t2iLGo, 5iLAF, and tt2iLGo.

15. The method according to any one of claims 1 to 14, wherein steps (2) and (3) are carried out in the absence of feeder cells.

16. The method according to any one of claims 1 to 15, wherein the human somatic cell is a mononuclear cell or a fibroblast.

17. A kit for producing naive induced pluripotent stem cells from human somatic cells, including the following: One or more vectors containing reprogramming factors, wherein the one or more vectors are selected from Sendai virus vectors containing TS12 mutations (TS mutations (G69E / T116A / A183S mutations in M ​​protein, A262T / G264R / K461G mutations in HN protein, L511F mutations in P protein, and N1197S / K1795E mutations in L protein) in addition to the D433A / R434A / K437A mutation in P protein), TS7 mutations (TS mutations in addition to the Y942H / L1361C / L1558I mutation in L protein), or TS15 mutations (TS mutations in addition to the D433A / R434A / K437A mutation in P protein and L1361C / L1558I mutation in L protein); and Culture medium for naive cells.

18. The kit according to claim 17, wherein one or more vectors include vectors containing a target sequence of microRNA specific to induced pluripotent stem cells.

19. The kit according to claim 18, wherein the target sequence of the microRNA is located in the coding region, 5'UTR, or 3'UTR of an NP gene or a P gene.

20. The kit according to claim 19, wherein the microRNA is miR-367.

21. The kit according to any one of claims 17 to 20, wherein the reprogramming factor comprises the OCT gene, the SOX gene, the MYC gene, and / or the KLF gene.

22. The kit according to claim 21, wherein the one or more vectors include a vector containing the OCT gene, the SOX gene and the KLF gene, a vector containing the MYC gene, and a vector containing the KLF gene.

23. The kit according to claim 22, wherein the vector containing the MYC gene is a Sendai virus vector containing the TS15 mutation.

24. The kit according to claim 22 or 23, wherein the vector comprising the OCT gene, the SOX gene, and the KLF gene, and the vector comprising the KLF gene, are Sendai virus vectors comprising the TS12 mutation.

25. The kit according to any one of claims 17 to 24, wherein the naive culture medium comprises one or more compounds selected from LIF, MEK inhibitors, GSK3 inhibitors, cAMP production promoters, TGF-β inhibitors, and PKC inhibitors.

26. The kit according to claim 25, wherein the naive culture medium is a medium selected from the group consisting of t2iLGo, 5iLAF, and tt2iLGo.

27. The kit according to any one of claims 17 to 26, wherein the human somatic cells are mononuclear cells or fibroblasts.

28. Culture supernatant of naive induced pluripotent stem cells produced by the method described in any one of Claims 1 to 16.

29. A cosmetic product comprising the culture supernatant of naive induced pluripotent stem cells produced by the method described in any one of Claims 1 to 16 as a raw material.

30. A method for producing the culture supernatant of naive induced pluripotent stem cells, (1) The process of introducing one or more vectors containing reprogramming factors into human somatic cells; (2) A step of culturing the somatic cells in the presence of a naive medium to obtain cultured cells; (3) A step in which the cultured cells are cultured under different conditions after step (2), wherein the different conditions are such that the amount of vector per cultured cell is reduced to 30% or less compared to the start of step (3); and (4) Step of obtaining the culture supernatant from the cultured cells. A method for producing a culture supernatant, wherein one or more vectors are selected from Sendai virus vectors containing TS12 mutations (TS mutations (G69E / T116A / A183S mutations in M ​​protein, A262T / G264R / K461G mutations in HN protein, L511F mutations in P protein, and N1197S / K1795E mutations in L protein) in addition to the D433A / R434A / K437A mutation in P protein), TS7 mutations (TS mutations in addition to the Y942H / L1361C / L1558I mutation in L protein), or TS15 mutations (TS mutations in addition to the D433A / R434A / K437A mutation in P protein and L1361C / L1558I mutation in L protein).

31. A method for producing cosmetics containing the culture supernatant of naive induced pluripotent stem cells as a raw material, (1) The process of introducing one or more vectors containing reprogramming factors into human somatic cells; (2) A step of culturing the somatic cells in the presence of a naive medium to obtain cultured cells; (3) A step in which the cultured cells are cultured under different conditions than those in step (2), wherein the different conditions are such that the amount of vector per cultured cell is reduced to 30% or less compared to the amount at the start of step (3); (4) A step of obtaining the culture supernatant from the cultured cells; and (5) A step of preparing cosmetics containing the culture supernatant as a raw material. A method for producing cosmetics, comprising, wherein one or more vectors are selected from Sendai virus vectors containing TS12 mutations (TS mutations (G69E / T116A / A183S mutations in M ​​protein, A262T / G264R / K461G mutations in HN protein, L511F mutations in P protein, and N1197S / K1795E mutations in L protein) in addition to the D433A / R434A / K437A mutation in P protein), TS7 mutations (TS mutations in addition to the Y942H / L1361C / L1558I mutation in L protein), or TS15 mutations (TS mutations in addition to the D433A / R434A / K437A mutation in P protein and L1361C / L1558I mutation in L protein).

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