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

Method for producing naive human iPS cells from somatic cells

[0001] The present application relates to a method for producing naive induced pluripotent stem cells from human somatic cells.

[0002] Mouse induced pluripotent stem (iPS) cells and embryonic stem (ES) cells are naive, a highly undifferentiated state similar to that of preimplantation blastocysts, whereas human iPS cells and human ES cells produced by conventional methods are known to have the characteristics of primed, a state closer to that of postimplantation embryos and more advanced in development than naive cells.

[0003] Compared to naive mouse iPS cells, human iPS cells have been shown to have limited pluripotency, are difficult to genetically manipulate, and exhibit significant variability in gene expression and pluripotency among cell lines. One of the main reasons for this is that human iPS cells are primed, which means they are more advanced in development than naive iPS cells. Therefore, for the advancement of regenerative medicine, there is a strong demand for methods to produce human pluripotent stem cells with naive properties.

[0004] To date, the following methods have been reported for producing human naive pluripotent stem cells: (1) a method in which primed pluripotent stem cells are converted into naive pluripotent stem cells by expressing a specific gene and culturing them in a medium containing specific components (Patent Document 1, Non-Patent Document 1); (2) a method in which primed pluripotent stem cells are converted into naive pluripotent stem cells by culturing them in a medium containing specific components (Patent Documents 2 to 4); and (3) a method in which somatic cells (fibroblasts) are reprogrammed and then cultured in a medium containing specific components halfway through the process to produce naive iPS cells (Non-Patent Documents 2 to 4).

[0005] WO2016 / 148253 JP2015-136349 WO2016 / 179243 WO2017 / 170849

[0006] Takashima Y et al., Cell, 158:1254-1269, 2014Theunissen T et al., Cell Stem Cell, 15:471-487, 2014Kilens S et al., Nature Communications, 9:360-, 2018Liu X et al., Nature Methods, 14:1055-, 2017

[0007] The present application aims to provide a method for producing naive induced pluripotent stem cells from human somatic cells.

[0008] The present 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) introducing one or more vectors containing reprogramming factors into human somatic cells, (2) culturing the somatic cells in the presence of a naive medium, and (3) culturing the cells obtained after step (2) in the presence of a naive medium under conditions such that the amount of the vectors per somatic cell is reduced to 30% or less compared to the amount at the start of step (3). [2] The method described in [1] above, wherein the one or more vectors are minus-strand RNA viral vectors. [3] The method described in [2] above, wherein the one or more vectors are paramyxovirus vectors. [4] The method described in [3] above, wherein the one or more vectors are Sendai virus vectors. [5] The method described in any of [1] to [4] above, wherein step (2) is initiated 1 to 10 days after step (1). [6] The method described in any of [1] to [5] above, 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 that exhibits temperature sensitivity, a vector that contains a target sequence of a microRNA specific to induced pluripotent stem cells, and a vector that is both temperature sensitive and contains 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 that exhibit temperature sensitivity. [9] The method according to [8], wherein the one or more vectors are vectors that exhibit temperature sensitivity in a cell culture environment for establishing naive pluripotent stem cells.

[10] The method according to [9] above, wherein the one or more vectors are Sendai virus vectors comprising TS7 mutations (TS 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), as well as Y942H / L1361C / L1558I mutations in the L protein), TS12 mutations (TS mutations, as well as D433A / R434A / K437A mutations in the P protein), or TS15 mutations (TS mutations, as well as D433A / R434A / K437A mutations in the P protein and L1361C / L1558I mutations in the L protein).

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

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

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

[11] above, wherein the vector containing a target sequence of a microRNA specific to induced pluripotent stem cells is a minus-strand RNA viral vector, and the target sequence of the microRNA is present in the translated region, 5'UTR, or 3'UTR of the NP gene or P gene.

[13] The method according to

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

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

[13] above, wherein the reprogramming factors include the OCT gene, the SOX gene, the MYC gene, and / or the KLF gene.

[15] The method according to

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

[17] The method according to

[15] or

[16] , 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 a TS12 mutation.

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

[17] , wherein in step (3), the amount of the vector per somatic cell is reduced to 30% or less of 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 [1] to

[18] above, wherein the naive medium contains one or more compounds selected from the group consisting of LIF (leukemia inhibitory factor), a MEK inhibitor, a GSK3 inhibitor, a cAMP production promoter, a TGF-β inhibitor, and a PKC inhibitor.

[20] The method according to

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

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

[20] above, wherein steps (2) and (3) are performed in the absence of feeder cells.

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

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

[0009]

[23] A kit for producing naive induced pluripotent stem cells from human somatic cells, comprising: one or more vectors containing reprogramming factors, wherein the one or more vectors are selected from the group consisting of a vector exhibiting temperature sensitivity, a vector containing a target sequence of a microRNA specific to induced pluripotent stem cells, and a vector exhibiting temperature sensitivity and containing a target sequence of a microRNA specific to induced pluripotent stem cells; and a naive culture medium.

[24] The kit according to

[23] , wherein the one or more vectors are minus-strand RNA viral vectors.

[25] The kit according to

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

[26] The kit according to

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

[27] The kit according to any one of

[23] to

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

[28] The kit according to

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

[29] The kit according to

[28] above, wherein the one or more vectors are Sendai virus vectors containing TS7 mutations (TS 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) and Y942H / L1361C / L1558I mutations in the L protein), TS12 mutations (TS mutations and D433A / R434A / K437A mutations in the P protein), or TS15 mutations (TS mutations and D433A / R434A / K437A mutations in the P protein and L1361C / L1558I mutations in the L protein).

[30] The kit according to any one of

[23] to

[29] above, wherein the vector containing a target sequence of a microRNA specific to induced pluripotent stem cells is a minus-strand RNA viral vector, and the target sequence of the microRNA is present in the translated region, 5' UTR, or 3' UTR of the NP gene or P gene.

[31] The kit according to

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

[32] The kit according to any one of

[23] to

[31] above, wherein the reprogramming factors include the OCT gene, the SOX gene, the MYC gene, and / or the KLF gene.

[33] The kit according to

[32] above, 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.

[34] The kit according to

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

[35] The kit according to

[33] or

[34] above, 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 a TS12 mutation.

[36] The kit according to any one of

[23] to

[35] above, wherein the naive medium contains one or more compounds selected from LIF (leukemia inhibitory factor), a MEK inhibitor, a GSK3 inhibitor, a cAMP production promoter, a TGF-β inhibitor, and a PKC inhibitor.

[37] The kit according to

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

[38] The kit according to any one of

[23] to

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

[39] A culture supernatant of naive induced pluripotent stem cells produced by the method according to any one of [1] to

[22] above.

[40] A cosmetic product comprising as an ingredient the culture supernatant of naive induced pluripotent stem cells produced by the method according to any one of [1] to

[22] above.

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

[0011] Structure of the Sendai virus vector (CytoTune-iPS2.0L) conventionally used to produce naive iPS cells from somatic cells. Protocol for producing naive iPS cells from human dermal fibroblasts (HDF). The vertical bars indicate an example of the timing of medium changes. Phase-contrast microscopic image of HDF-derived naive iPS cells after 45 passages. Immunostained image for pluripotency markers of HDF-derived naive iPS cells after 45 passages. Three images displayed horizontally represent the same field of view. Effect of high-temperature culture on the genome content of Sendai virus vectors. 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. Quantitative evaluation of the genome content of Sendai virus vectors in PBMC-derived naive iPS cells at each passage from day 14. Principal component analysis integrating single-cell data for each cell type. Expression patterns of imprinted genes and X-chromosome-associated genes in each cell type. Density bean plot showing global methylation in each cell type. Principal component analysis showing global methylation in each cell type. Ratio of 5-methylcytosines to total cytosines in HDF-derived naive iPSCs obtained in this example. Early differentiation potential of three germ layer components in HDF- or PBMC-derived naive iPSCs after long-term passage (P16-45). Reset naive ESCs (in-house) were used as a control. Phase contrast images and immunofluorescent staining with SeV in naive human iPSCs reprogrammed with CytoTune-iPS 2.0L (nCT2.0L_1) 94 days after SeV infection. Scale bar represents 100 μm. qRT-PCR analysis of SeV genome expression in naive iPSCs reprogrammed with CytoTune 2.0 or 2.0L SeV reprogramming kit. Values ​​were normalized to GAPDH expression levels and shown as mean ± SD. n = 3 for each data point. qRT-PCR analysis of SeV vector genome expression normalized to GAPDH expression in naive human iPSCs initialized with CytoTune 2.0 or 2.0L. Data are shown as mean ± SD. n = 3 for each data point.In this experiment, primers and SYBR Green designed to specifically detect each SeV vector were used. Schematic diagram of SeV vectors and modified reprogramming cocktails. The SeV18+KLF4 / TSΔF (KLF4 / TS) vector in the CytoTune-2.0 or 2.0L reprogramming kit was replaced with one of 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 experiment. qRT-PCR analysis of hsa-miRNA367-3p expression in HDFs and PSCs normalized to hsa-miRNA423-3p expression. Data are shown as mean ± SD. n=3. ND indicates no detection even after 40 amplification cycles. Experimental design for induction of naive human iPSCs from HDFs or PBMCs using the modified SeV-OSKL cocktail and change in incubation temperature after naive human iPSC generation. Representative phase-contrast images of PBMCs 14 days after infection with the CytoTune-iPS 2.0 SeV vector. The scale bar indicates 200 μm. The number of naive human iPSC colonies generated from HDFs 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, * indicates P < 0.05. Detection sensitivity of SeV genome in cells 14 days after SeV vector infection, analyzed by qRT-PCR. The x-axis indicates the percentage of SeV-positive cells among SeV-negative cells. 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.qRT-PCR analysis of SeV genome expression in HDF-derived naive iPSCs (nOSKL_1, 2) and PBMC-derived naive iPSCs (nOSKL_3, 4) reprogrammed with SeV-KOS, SeV-LMYC, and SeV-KLF4 / miR / TS12 vectors on days 14 and 34. Data are shown as mean ± sd. n = 3. Representative phase-contrast images of naive iPSCs on iMEF feeder cells and feeder-free naive iPSCs reprogrammed with SeV-KOS, SeV-LMYC, and SeV-KLF4 / miR / TS12 vectors. Scale bar indicates 200 μm. Cell proliferation rate of established nOSKL_1-4. Relative expression of naive pluripotency markers analyzed by qRT-PCR, normalized to GAPDH expression. The value for nH9 is set to 1.0. Data are shown as mean ± sd. n=3. Relative expression of primed pluripotency markers normalized to GAPDH expression and analyzed by qRT-PCR. Data are shown as mean ± sd. n=3. Heatmap of RNA-seq data showing expression levels of common, primed, and naive pluripotency-associated marker genes in naive and primed human PSCs.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 from primed and naive human PSCs compared with reset naive human iPSCs and preimplantation embryo samples from the present study (2013). Bean plot of global DNA methylation levels in primed and naive human PSCs analyzed using a DNA methylation array. The horizontal line in the bean plot represents the mean methylation beta value. PCA of global DNA methylation in primed and naive human PSCs using a DNA methylation array. Representative RNA-FISH images of naive and primed human iPSCs detecting the X-linked genes UTX (which escapes 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. Scale bar indicates 20 μm. Quantification of RNA-FISH patterns of XIST and HUWE1 in cells with biallelic UTX expression.One hundred cells were analyzed for each cell line. Most naive human iPSCs showed X reactivation, indicated by biallelic HUWE1 expression. Some cells from nOSKL_3 and 4 showed biallelic HUWE1 expression as well as biallelic XIST expression, resembling the expression pattern in preimplantation epiblasts. Heatmap of genes encoding electron transport chain proteins present in the inner mitochondrial membrane. These genes reflect oxidative phosphorylation activity and are hierarchically clustered by mitochondrial complex. Comparison of metabolic capacity between naive and primed iPSCs using an extracellular flux analyzer. Quantification of spare respiratory capacity in naive and primed iPSCs. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in naive and primed iPSCs. Schematic diagram of differentiation into primitive endoderm. Representative phase-contrast images of primitive endoderm derived from primed ESCs (H9; left panel) and SeV(-) naive iPSCs (nOSKL_4; right panel). Scale bar represents 100 μm. 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. Median signal values ​​in PDGFRA / ANPEP co-positive cells by flow cytometry. Schematic diagram of trophectoderm differentiation. Representative phase-contrast images of trophectoderm derived from primed ESCs (H9; left panel) and SeV(-) naive iPSCs (nOSKL_4; right panel). Scale bar represents 200 μm. Flow cytometric 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). The percentage of TACSTD2+ / ENPEP+ and HAVCR1+ cells was determined by flow cytometry. Each plot represents a single experiment.

[0012] In this specification and claims, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" is intended to include "18 to 22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" is intended to include "18 to 33."

[0013] [Vector] In the present application, a vector may be any vector capable of introducing a gene into somatic cells, and may be a viral or non-viral vector, such as a viral vector. In the present disclosure, a viral vector is a vector that carries genomic nucleic acid derived from the virus and can express a transgene by incorporating the transgene into the nucleic acid. The vector in the present application may be, for example, a non-chromosomally integrated viral vector. A non-chromosomally integrated viral vector is a viral vector derived from a virus that can introduce a gene into target cells and does not pose a risk of the introduced gene being integrated into the host's chromosome (nuclear-derived chromosome). In the present application, viral vectors include not only infectious viral particles, but also non-infectious viral particles, viral cores, or complexes consisting of a viral genome and viral proteins, which are capable of expressing the gene carried by the vector when introduced into cells. For example, in RNA viruses, ribonucleoproteins (viral core moieties) consisting of the viral genome and the viral proteins that bind to them can express transgenes within cells when introduced into cells (WO 00 / 70055). Introduction into cells can be carried out using an appropriate transfection reagent, etc. Such ribonucleoproteins (RNPs) are also included in the viral vectors of the present application.

[0014] As used herein, "there is no risk of integration into the host chromosome" means that the frequency of integration into the host chromosome when the viral vector is introduced is sufficiently low. Preferably, the frequency of integration into the host chromosome is 5×10 when, for example, the human fibrosarcoma-derived cell line HT1080 (ATCC CCL121) is infected with 10 PFU / cell.－４ or less, more preferably 10 －４ or less, more preferably 10 －５ or less, more preferably 10 －６ or less, more preferably 10 －７The non-chromosomally integrating viral vector is preferably an RNA virus. In this disclosure, RNA virus refers to a virus that has an RNA genome and does not have a DNA phase in its life cycle. In this disclosure, RNA viruses do not have reverse transcriptase (i.e., retroviruses are not included). That is, during viral proliferation, the viral genome is replicated by an RNA-dependent RNA polymerase without the intervention of DNA. Because RNA viruses do not have a DNA phase, the use of RNA viral vectors can minimize the risk of integration into host chromosomes. RNA viruses include single-stranded RNA viruses (including positive-strand and negative-strand 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 of viruses such as Lassa virus) Orthomyxoviridae (family of viruses such as influenza virus) Coronaviridae (family of viruses such as SARS virus) Togaviridae (family of viruses such as rubella virus) Paramyxoviridae (family of viruses such as mumps virus, measles virus, Sendai virus, and respiratory syncytial virus) Picornaviridae (family of viruses such as poliovirus, coxsackievirus, and echovirus) Filoviridae (family of viruses such as Marburg virus and Ebola virus) Flaviviridae (family of viruses such as yellow fever virus, dengue virus, hepatitis C virus, and hepatitis G virus) Bunyaviridae (family of viruses including Bunyavirus, Hantavirus, Nairovirus, and Phlebovirus genera) Rhabdoviridae (family of viruses such as rabies virus) Reoviridae.

[0015] An example of a non-chromosomally integrated viral vector is a minus-strand RNA viral vector. A minus-strand RNA viral vector is a vector consisting of a virus containing minus-strand RNA (antisense strand to the sense strand encoding the viral protein) as its genome. Minus-strand RNA is also called negative-strand RNA. An example of a minus-strand RNA virus is a single-stranded negative-strand RNA virus (also called a non-segmented negative-strand RNA virus). A "single-stranded negative-strand RNA virus" refers to a virus that has single-stranded negative-strand [i.e., minus-strand] RNA in its genome. Such viruses include those belonging to the Paramyxoviridae family (including the genera Paramyxovirus, Morbillivirus, Rubulavirus, and Pneumovirus), the Rhabdoviridae family (including the genera Vesiculovirus, Lyssavirus, and Ephemerovirus), and the Filoviridae family, which are classified into the order Mononegavirales (Viruses, Vol. 57, No. 1, pp. 29-36, 2007; Annu. Rev. Genet. 32, 123-162, 1998; Fields Virology, fourth edition, Philadelphia, Lippincott-Raven, pp. 1305-1340, 2001; Microbiol. Immunol. 43, 613-624, 1999; Field Virology, Third edition, pp. 1205-1241). 1996).

[0016] An example of a negative-strand RNA viral vector is a paramyxoviral vector. Paramyxoviral vectors are viral vectors derived from viruses of the Paramyxoviridae family. For example, Sendai virus, a member of the Paramyxoviridae family, can be mentioned. Other examples include Newcastle disease virus, mumps virus, measles virus, respiratory syncytial virus (RSV), rinderpest virus, distemper virus, simian 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 can be used in the present application include 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), rinderpest 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), rinderpest virus (RPV), Hendra virus (Hendra), and Nipah virus (Nipah).

[0018] The vector used in the present application is, for example, a virus or derivative thereof belonging to the subfamily Paramyxovirinae (including the genera Respirovirus, Rubulavirus, and Morbillivirus), such as a virus or derivative thereof belonging to the genus Respirovirus (also known as Paramyxovirus). Derivatives include viruses whose viral genes have been modified and chemically modified so as not to impair the viral gene transfer ability. Examples of viruses belonging to the genus Respirovirus 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 known as murine parainfluenza virus type 1), and simian parainfluenza virus type 10 (SPIV-10). The most preferred paramyxovirus is Sendai virus.

[0019] Negative-strand RNA viruses generally contain a complex of RNA and protein (ribonucleoprotein; RNP) inside the envelope. The RNA contained in RNP is a negative-strand single-stranded RNA that forms the genome of the negative-strand RNA virus. This single-stranded RNA binds to the NP, P, and L proteins to form RNPs. The RNA contained in these RNPs 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, phosphoprotein, matrix protein, fusion protein, hemagglutinin-neuraminidase, and large proteins, respectively. The nucleocapsid (NP) protein binds to genomic RNA and is essential for the template activity of the genomic RNA. The NP gene is commonly referred to as the "N gene." The phosphoprotein (P) is a phosphorylated protein that is the small subunit of RNA polymerase. The matrix protein (M) functions to maintain the internal structure of the virus particle. The fusion protein (F) is a membrane fusion protein involved in host cell entry, and the hemagglutinin-neuraminidase (HN) protein is a protein involved in binding to host cells. The large protein (L) is the large subunit of RNA polymerase. Each of the above genes has its own transcriptional control unit, and each gene transcribes a separate mRNA and protein. In addition to the P protein, the P gene translates a nonstructural protein (C) that is translated using a different ORF, and a protein (V) that is created by RNA editing during the P protein mRNA reading process. For example, the genes of viruses belonging to the Paramyxovirinae subfamily are generally written as follows, starting from the 3' end: Respirovirus NP / C / VMF HN - L Rubulavirus NP / VMF HN (SH) L Morbillivirus NP / C / VMFH - L

[0021] For example, the database accession numbers for the nucleotide sequences of the genes of 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; and for the F gene, D00152, D11446, D17334, D17335, M30202, M30203, For the HN gene, see D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, X56131; for the L gene, see D00053, M30202, M30203, M30204, M69040, X00587, X58886. Examples of viral genes encoded by other viruses include the N gene of 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 the P gene of CDV, X51869; DMV, Z47758; HPIV-1, M74081; HPIV-3, X04721; HPIV-4a, X04722; 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 for the L gene, and CDV, AF014953; DMV, AJ608288; HPIV-1, AF117818; HPIV-2, X57559; HPIV-3, AB012132; Mumps, X99040. 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 depending on the strain, genes with sequences other than those listed above may exist. Sendai virus vectors carrying viral genes derived from any of these genes are useful as vectors in the present application. Furthermore, the functional site of the P protein is a region containing the N-binding site, L-binding site, and oligomerization site at the C-terminus (in the case of SeV, this region is 320-568 at the C-terminus of the P protein) (Blanchard L. et al., Virology. (2004) 319, 201-211). Therefore, it is preferred that the P protein in the present application contains at least this region. For example, the vector of the present application comprises a nucleotide sequence that is 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the coding sequence of any of the above viral genes (for the SeV P gene, this may be, for example, the C-terminal sequence, e.g., the amino acid sequence of amino acids 479 to 568 or the amino acid sequence of amino acids 320 to 568). Furthermore, the vector of the present application comprises a nucleotide sequence encoding an amino acid sequence that is 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the amino acid sequence encoded by the coding sequence of any of the above viral genes (for the SeV P protein, this may be, for example, the C-terminal sequence, e.g., the amino acid sequence of amino acids 479 to 568 or the amino acid sequence of amino acids 320 to 568). Furthermore, the vector of the present application comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence in which 10 or less, preferably 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid has been substituted, inserted, deleted, and / or added in the amino acid sequence encoded by the coding sequence of any of the above viral genes (for the SeV P gene, this may be, for example, the C-terminal sequence, e.g., the amino acid sequence of amino acids 479 to 568 or the amino acid sequence of amino acids 320 to 568).

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

[0023] The negative-strand RNA virus of the present application may be derived from a natural strain, a wild-type strain, a mutant strain, a laboratory-passaged strain, or an artificially constructed strain. For example, the Sendai virus Z strain can be mentioned (Medical Journal of Osaka University, Vol. 6, No. 1, March 1955, pp. 1-15). That is, the virus may be a viral vector with a structure similar to that of a virus isolated from nature, or a virus artificially modified by genetic recombination. For example, the virus may have a mutation or deletion in any of the genes of a wild-type virus. For example, a virus with a mutation or deletion in at least one gene encoding a viral envelope protein or coat protein can be preferably used. Such a viral vector can replicate its genome in infected cells but cannot form infectious viral particles. Such transmissibility-deficient viral vectors are highly safe because they eliminate the risk of spreading infection to the surrounding area. For example, negative-strand RNA viruses lacking at least one gene encoding an envelope protein such as F and / or HN, or a spike protein, or a combination thereof, can be used (WO00 / 70055 and WO00 / 70070; Li, H.-O. et al., J. Virol. 74(14) 6564-6569 (2000)). If the genomic RNA encodes proteins necessary for genome replication (e.g., N, P, and L proteins), the genome can be amplified in infected cells. Defective viruses can be produced, for example, by exogenously supplying the defective gene product or a protein capable of complementing it to virus-producing cells (WO00 / 70055 and WO00 / 70070; Li, H.-O. et al., J. Virol. 74(14) 6564-6569 (2000)). Additionally, a method for recovering a viral vector as a non-infectious viral particle (VLP) without completely complementing the missing viral protein is also known (WO00 / 70070).Furthermore, when the viral vector is recovered as RNP (for example, RNP consisting of N, L, and P proteins and genomic RNA), the vector can be produced without complementing the envelope protein.

[0024] Furthermore, the viruses of the present application are not limited to naturally occurring viruses, but also include, for example, artificially produced viruses, such as viruses in which mutations have been introduced into nucleic acid sequences for codon optimization, and chimeric viruses (including, for example, chimeras between viruses of the same species and chimeric viruses between viruses of different species (e.g., chimeras between PIV and SeV)) (J. Virol. 1995, 69, 849-855).

[0025] Furthermore, in the present application, viral proteins such as N, L, and P proteins do not have to be wild-type proteins as long as they retain the function of expressing genes in transfected cells. For example, modified proteins with appropriate addition of peptides such as tags, proteins with modified codons, and proteins in which a portion of the amino acid sequence of a wild-type protein has been deleted so as not to lose function can be used as appropriate. In the present application, the N, L, and P proteins also include such modified proteins and deleted proteins. For example, as long as a portion of the C-terminus of the P protein is present, other regions are not essential for the expression of a viral vector.

[0026] A viral vector is, for example, a complex of RNA derived from the negative-strand single-stranded RNA genome of the virus and a protein derived from a viral protein that binds to the negative-strand single-stranded RNA of the virus, and expresses a gene carried by the vector when introduced into a cell. A protein that binds to negative-strand single-stranded RNA refers to a protein that binds directly and / or indirectly to the negative-strand single-stranded RNA and forms a complex with the negative-strand single-stranded RNA. Examples of complexes include those consisting of negative-strand single-stranded RNA derived from a negative-strand RNA virus and proteins derived from a negative-strand RNA virus that bind to the negative-strand single-stranded RNA (e.g., NP, P, and L proteins). In this application, "derived from a negative-strand RNA virus" refers to the components of a negative-strand RNA virus (including proteins and RNA) in their original or partially modified state. For example, a protein or RNA prepared by modifying a protein or RNA of a negative-strand RNA virus is a protein or RNA "derived from a negative-strand RNA virus." The vector of the present application may be, for example, a viral vector that has envelope proteins (F, HN, and M proteins) and takes the form of a viral particle, or an RNP vector that does not have a viral envelope and is an RNP itself.

[0027] In negative-strand RNA viruses, the NP, P, and L proteins bind to negative-strand single-stranded RNA and perform essential functions for genomic RNA replication and protein expression (hereinafter, the NP, P, and L proteins will sometimes be referred to as "genomic RNA-binding proteins"). The NP protein binds very tightly to genomic RNA and confers template activity to the genomic RNA. Genomic RNA only has template activity for RNA synthesis when bound to the NP protein; it has no template activity 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 any 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 the present application may have the same gene sequence as the virus, or may have some 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 that do not impair the function of each protein. For example, site-specific mutations can be introduced using PCR or cassette mutagenesis, or random mutations can be introduced using chemical reagents, random nucleotides, etc.

[0029] For example, numerous mutations, including attenuating mutations and temperature-sensitive mutations, are known in envelope proteins and spike proteins. Viruses carrying these mutant protein genes can be suitably used in the present application. Preferably, vectors with attenuated cytotoxicity can be used in the present application. The cytotoxicity of a vector can be measured, for example, by quantifying lactate dehydrogenase (LDH) release from vector-infected cells. The lower the LDH release, the lower the cytotoxicity. For example, vectors with significantly attenuated cytotoxicity compared to wild-type vectors can be used. The degree of attenuation of cytotoxicity can be measured by infecting human HeLa cells (ATCC CCL-2) or monkey-derived CV-1 cells (ATCC CCL 70) with an MOI (infectivity titer) of 3 and culturing them at 35-37°C (e.g., 37°C) for 3 days, and the amount of LDH release in the culture medium is significantly reduced compared to wild-type vectors, e.g., by 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 growth rate of the virus or the expression level of the gene carried by the virus at the normal temperature of the virus host. The lower the growth rate of the virus and / or the expression level of the gene carried by the virus compared to those without the mutation, the higher the temperature sensitivity is considered to be.

[0031] Viral vectors that can be used in the present application preferably have deletions or mutations in at least one, and more preferably at least two, three, four, five, or more, viral genes. Deletions and mutations may be introduced into each gene in any combination. Here, the mutations may be functionally depleting mutations or temperature-sensitive mutations. The use of such modified viral vectors can be particularly useful in reducing cytotoxicity in host cells and facilitating vector elimination. For example, viral vectors suitable for use in the present application have deletions or mutations in at least two viral genes. Such viruses include those in which at least two viral genes are deleted, those in which at least two viral genes are mutated, and those in which at least one viral gene is mutated and at least one viral gene is deleted. The at least two mutated or deleted viral genes are preferably genes encoding envelope-constituting proteins. For example, the negative-strand RNA viral vectors of the present application preferably lack at least the F gene or the M gene. For example, vectors that lack the F gene and further lack the M and / or HN genes, or that further have mutations (e.g., temperature-sensitive mutations) in the M and / or HN genes, are suitable for use in the present application. Furthermore, more preferably, the vectors used in the present application have deletions or mutations in at least three viral genes (preferably at least three genes encoding envelope-constituting proteins: F, HN, and M). Such viral vectors include those in which at least three genes are deleted, those in which at least three genes are mutated, those in which at least one gene is mutated and at least two genes are deleted, and those in which at least two genes are mutated and at least one gene is deleted. More preferred embodiments include vectors that lack the F gene and further lack the M and HN genes, or those in which the M and HN genes are mutated (e.g., temperature-sensitive mutations) are suitable for use in the present application.Furthermore, vectors that have a deletion of the F gene, a further deletion of the M or HN gene, and a further mutation (e.g., a temperature-sensitive mutation) in the remaining M or HN gene are suitable for use in the present 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 selected from the group consisting of positions 69 (G69), 116 (T116), and 183 (A183) in the Sendai virus M protein, or at corresponding sites in the M protein of minus-strand RNA viruses (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246). Viruses having a genome encoding a mutant M protein in which amino acids at any of the above three sites, preferably any combination of two sites, or more preferably all three sites have been substituted with other amino acids are suitable for use in the present application.

[0033] The amino acid mutation is preferably a substitution with another amino acid having a different side chain chemical property, for example, an amino acid having a value of 3 or less, preferably 2 or less, more preferably 1 or less, and more preferably 0 in the BLOSUM62 matrix (Henikoff, S. and Henikoff, JG (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919). Specifically, in the case of the Sendai virus M protein, G69, T116, and A183 can be substituted with Glu(E), Ala(A), and Ser(S), respectively. In the M proteins of other negative-strand RNA viruses, amino acids at corresponding positions can also be substituted with Glu(E), Ala(A), and Ser(S), respectively. Alternatively, a mutation homologous to the mutation 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) can be used. Mutations can be introduced using, for example, oligonucleotides according to known mutagenesis methods.

[0034] As used herein, the amino acid sequence of the Sendai virus is based on the amino acid sequence of the F gene-deficient Sendai virus derived from the Z strain described in GenBank ACCESSION NO. AB855655. Therefore, the Xth amino acid residue of the Sendai virus refers to the amino acid residue corresponding to the Xth amino acid residue in the amino acid sequence described in GenBank ACCESSION NO. AB855655. As used herein, a "position corresponding to" a given position in an amino acid sequence refers to the position of another amino acid sequence corresponding to a given position in the given amino acid sequence when the given amino acid sequence is aligned with another amino acid sequence. Alignment can be performed using, for example, known genetic analysis software. Specific genetic analysis software includes DNASIS (manufactured by Hitachi Solutions), GENETYX (manufactured by Genetyx), and FASTA, BLAST, and ClustalW (published by DDBJ).

[0035] Temperature-sensitive mutations in the HN gene include, for example, amino acid substitutions at positions 262 (A262), 264 (G264), and 461 (K461) of the Sendai virus HN protein, or at corresponding positions in the HN protein of a negative-strand RNA virus (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246). Viruses having genomes encoding mutant HN proteins in which amino acids at any one of the three positions, preferably any combination of two positions, or more preferably all three positions are substituted with other amino acids are suitable for use in the present application. As above, amino acid substitutions with amino acids having different side chain chemical properties are preferred. In one preferred example, A262, G264, and K461 of the Sendai virus HN protein are substituted with Thr (T), Arg (R), and Gly (G), respectively. In the HN proteins of other negative-strand RNA viruses, the amino acids at the corresponding positions 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 mumps virus vaccine strain Urabe AM9 as a reference (Wright, KE et al., Virus Res. 2000: 67; 49-57).

[0036] The vector of the present application may also have mutations in the P gene and / or L gene. Specific examples of such mutations include a mutation of Glu at position 86 of the SeV P protein (E86) and a substitution of Leu at position 511 of the SeV P protein (L511) with another amino acid. Substitutions at equivalent sites in the P proteins of other negative-strand RNA viruses are also possible. As above, amino acid substitutions with amino acids with different side chain chemical properties are preferred. Specific examples include substitution of Lys at position 86 and Phe at position 511. Regarding the L protein, examples include substitution of Asn at position 1197 of the SeV L protein (N1197) and / or Lys at position 1795 of the SeV L protein (K1795) with another amino acid, and substitutions at equivalent sites in the L proteins of other negative-strand RNA viruses. As above, amino acid substitutions with amino acids with different side chain chemical properties are preferred. Specific examples include substitution of Ser at position 1197 and substitution of Glu at position 1795. Mutations in the P gene and L gene can significantly enhance the effects of persistent infectivity, suppression of secondary particle release, or suppression of cytotoxicity. Furthermore, combining mutations and / or deletions in the envelope protein gene can dramatically increase these effects. Regarding the L gene, examples include substitutions of Tyr at position 1214 (Y1214) and / or Met at position 1602 (M1602) of the SeV L protein with other amino acids, as well as substitutions at corresponding sites in the L proteins of other negative-strand RNA viruses. As mentioned above, amino acid substitutions with amino acids with different side chain chemical properties are preferred. Specific examples include substitution of amino acid 1214 with Phe and substitution of amino acid 1602 with Leu. The above-mentioned mutations can be combined in any combination.

[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 in which the F gene is deleted or lacking, as well as F gene-deleted or F gene-deleted Sendai virus vectors with similar or lesser cytotoxicity and / or similar or greater temperature sensitivity are particularly preferred. For other minus-strand RNA viruses, vectors in which the corresponding sites are similarly substituted and in which the F gene is deleted or lacking, as well as F gene-deleted or ... Specific examples of substitutions include G69E, T116A, and A183S substitutions for the M protein, A262T, G264R, and K461G substitutions for the HN protein, L511F substitutions for the P protein, and N1197S and K1795E substitutions for the L protein.

[0038] The amino acid mutation may be a substitution with another amino acid of interest, but preferably, as described above, a substitution with an amino acid having a different chemical property of the side chain. 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), uncharged polar 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), and the like. However, for a certain amino acid, substitution with an amino acid other than the amino acid of the group to which the amino acid belongs can be exemplified. Specifically, examples include, but are not limited to, substitution of a basic amino acid with an acidic or neutral amino acid, substitution of a polar amino acid with a nonpolar amino acid, substitution of an amino acid with a molecular weight larger than the average molecular weight of the 20 naturally occurring amino acids with an amino acid with a molecular weight smaller than that average, and conversely, substitution of an amino acid with a molecular weight smaller than that average with an amino acid with a molecular weight larger than that average.

[0039] L protein mutations also include substitutions of amino acids at positions arbitrarily selected from positions 942 (Y942), 1361 (L1361), and 1558 (L1558) of the SeV L protein, or at the corresponding positions in minus-strand RNA virus L proteins. As described above, amino acid substitutions with amino acids with different side chain chemical properties are preferred. Specific examples include substitutions of His at position 942, Cys at position 1361, and Ile at position 1558. L proteins with substitutions at at least positions 942 or 1558 are particularly suitable. For example, mutant L proteins with substitutions at positions 1558 and 1361 are also suitable. Furthermore, mutant L proteins with substitutions at positions 1558 and / or 1361 are also suitable. These mutations can increase the temperature sensitivity of the L protein. P protein mutations include substitutions of amino acids at positions arbitrarily selected from positions 433 (D433), 434 (R434), and 437 (K437) of the SeV P protein, or at the corresponding positions in the P protein of negative-strand RNA viruses. As mentioned above, amino acid substitutions with amino acids with different side chain chemical properties are preferred. Specific examples include substitutions of Ala (A) at amino acid position 433, Ala (A) at amino acid position 434, and Ala (A) at amino acid position 437. P proteins with substitutions at all three of these positions are particularly suitable. These mutations can increase the temperature sensitivity of the P protein.

[0040] Temperature-sensitive mutations that can be included in the vectors of the present 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 amino acids (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) are also suitable for use in the present application, as well as Sendai virus vectors lacking or deleting the F gene that exhibit similar or lesser cytotoxicity and / or similar or greater temperature sensitivity. In addition to the above mutations, each viral protein may also contain mutations in other amino acids (e.g., up to 10, 5, 4, 3, 2, or 1 amino acid). Vectors containing the above mutations exhibit high temperature sensitivity.

[0041] The genomic RNA contained in the vector of the present application may contain all of the envelope protein genes, or 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 temperature-sensitive mutations may be introduced. Temperature-sensitive mutations of envelope proteins are described in detail in WO2012 / 029770, WO2010 / 008054, and WO2003 / 025570.

[0042] A viral vector containing a desired exogenous envelope protein can be produced by expressing the protein in virus-producing cells. There are no particular limitations on the type of protein used, and any desired protein, such as an adhesion factor, ligand, or receptor, that confers infectivity to mammalian cells can be used. A specific example is the G protein (VSV-G) of vesicular stomatitis virus (VSV). The VSV-G protein may be derived from any VSV strain, including, but not limited to, the VSV-G protein derived from the Indiana serotype strain (J. Virology 39: 519-528 (1981)). The viral vector of the present application can contain any combination of envelope proteins derived from other viruses.

[0043] In the present 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 is preferably one whose expression level at a 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, preferably 1 to 4°C, e.g., about 3°C ​​higher than the temperature. For example, TS7 of Sendai virus (G69E / T116A / A183S mutations in the M protein, A262T / G264R / K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S / K1795E mutations in the L protein (all of which are referred to as "TS mutations"), in addition to Y942H / L1361C / L1558I mutations in the L protein) and TS12 (TS mutations, in addition to D433A / R mutations in the P protein) Preferred temperature-sensitive mutations include TS13 (TS mutation plus D433A / R434A / K437A mutations in the P protein and L1558I mutation in the L protein), TS14 (TS mutation plus D433A / R434A / K437A mutations in the P protein and L1361C mutation in the L protein), and TS15 (TS mutation plus D433A / R434A / K437A mutations in the P protein and L1361C / L1558I mutations in the L protein).

[0044] Specific examples of vectors include F gene-deleted Sendai virus vectors (e.g., Z strains) carrying TS7, TS12, TS13, TS14, or TS15 mutations. Specific examples include, but are not limited to, SeV18+ / TSΔF (WO2010 / 008054, WO2003 / 025570) and SeV(PM) / TSΔF vectors in which TS7, TS12, TS13, TS14, or TS15 mutations have been introduced in addition to the TS mutation. Note that "TSΔF" refers to a vector that carries G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein, and lacks the F gene.

[0045] Preferably, the vector of the present application is a Sendai virus vector derived from the Sendai virus Z strain. The genomic sequence of the Z strain is known; for example, ACCESSION: AB855655 is an example of the genomic sequence of an F gene-deficient Sendai virus vector derived from the Z strain. Here, "derived from the Z strain" refers to the fact that the Z strain sequence accounts for the highest proportion of the Sendai virus genome sequence in the vector genome. In other words, when non-Sendai virus sequences (restriction enzyme recognition sites, simple spacer sequences, sequences derived from other viruses, sequences of the gene to be introduced, etc.) are excluded from the vector genome sequence and only Sendai virus sequences are collected, SeV Z strain sequences account for the highest proportion of the SeV Z strain sequences. More preferably, "derived from the Z strain" refers to the fact that the SeV Z strain sequences account for the highest proportion of the negative-strand RNA virus-derived sequences in the vector genome. That is, when sequences not derived from minus-strand RNA viruses (e.g., restriction enzyme recognition sites, simple spacer sequences, sequences derived from viruses other than minus-strand RNA viruses, and sequences of genes to be introduced) are excluded from the genomic sequence of a vector, and only minus-strand RNA virus sequences are collected, the SeVZ strain's sequences account for the highest proportion of these sequences. 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. Furthermore, preferably, Sendai virus vectors derived from the Z strain contain only a continuous sequence of a non-Z Sendai virus genomic sequence (excluding sequences identical to those of the Z strain) of, for example, 1,000 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 in the genome. More preferably, a Sendai virus vector derived from the Z strain does not contain any genomic sequence of a non-Z strain Sendai virus (except for the same sequence as the Z strain) in its genome.

[0046] The vectors of the present application can be produced using known methods. For example, in the case of minus-strand RNA viruses, specific procedures typically include (a) transcribing cDNA encoding the minus-strand RNA virus genomic RNA (minus strand) or its complementary strand (plus strand) in cells expressing viral proteins (NP, P, and L) required for virus particle formation, and (b) recovering the resulting viruses. Viruses are not limited to infectious particles and also include non-infectious particles and RNPs, as long as they retain the ability to express genes when introduced into cells. Viral proteins required for virus formation may be expressed from the transcribed viral genomic RNA or may be supplied from sources other than the genomic RNA. For example, expression plasmids encoding the NP, P, and L proteins can be introduced into cells. If the genomic RNA lacks a viral gene required for virus formation, the viral gene can be separately expressed in virus-producing cells to complement virus formation. To express viral proteins or RNA genomes in cells, a vector carrying DNA encoding the protein or genomic RNA ligated downstream of an appropriate promoter functional in the host cell is introduced into the host cell. The transcribed genomic RNA is replicated in the presence of viral proteins to form virions. When producing a defective virus lacking a gene such as an envelope protein, the defective protein or another viral protein capable of complementing its function can be expressed in virus-producing cells. In the present application, a vector lacking at least the F gene or a vector having a mutation in the F gene can be preferably used.

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

[0048] For example, the negative-strand RNA virus of the present application can be produced 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 for propagating viruses and manufacturing recombinant viruses, please also refer to Virology Experiments, Special Topics, Revised Second Edition (edited by the National Institute of Health Alumni Association, Maruzen, 1982).

[0049] [Reprogramming Factors] As used herein, the term "reprogramming factor" refers to a gene or its product used, alone or in combination with multiple factors, to induce the differentiation state of a cell into a more undifferentiated state. This term includes, for example, a gene or its product used to induce dedifferentiation of a differentiated cell. As used herein, "reprogramming factors" include factors essential for nuclear reprogramming and auxiliary factors (cofactors) that increase the efficiency of nuclear reprogramming. In this application, the desired gene to be used for nuclear reprogramming may be carried on a vector. For example, a gene for use in the production of pluripotent stem cells may be carried. Specifically, reprogramming factors for inducing pluripotent stem cells include genes that are expressed in ES cells or early embryos but are not expressed or whose expression is reduced in many differentiated somatic cells (e.g., ES cell-specific genes). Such genes are preferably genes encoding transcription factors, nuclear proteins, etc. Methods for identifying nuclear reprogramming genes are already known (WO 2005 / 80598), and genes identified using this method have been shown to be useful for reprogramming pluripotent stem cells (WO 2007 / 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)、GDF3(growth differentiation factor 3); NM_020634, NM_008108)、SOX15(SRY (sex determining region Y)-box 15; NM_006942, NM_009235)、DPPA4(developmental pluripotency associated 4; NM_018189, NM_028610)、DPPA2(NM_138815, NM_028615)、FTHL17(ferritin, heavy polypeptide-like 17; NM_031894, NM_031261)、ROOM4(room-like 4; NM_020436, NM_175303) Oct3 / 4(POU5F1; NM_002701, NM_203289, NM_013633, NM_001009178)、Sox2(NM_003106, NM_011443, XM_574919);Rex-1(ZFP42 (zinc finger protein 42 homolog); NM_174900, NM_009556)、Utf1(undifferentiated embryonic cell transcription factor 1;NM_003577, NM_009482), TCL1A (T-cell leukemia / lymphoma 1A; NM_021966, NM_009337), DPPA3 (also known as Stella; NM_199286, NM_139218, XM_216263), KLF4 (Kruppel-like factor 4; NM_004235, NM_010637), cateninβ1 (cadherin-associated protein beta 1; NM_001904, NM_007614; including the S33Y mutant), c-Myc (NM_002467, NM_010849; including the T58A mutant), and STAT3 (signal transducer and activator of transcription 3; NM_139276, Examples of such genes 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. It has been shown that introducing these genes into cells can induce pluripotent stem cells (WO2007 / 69666). These genes can be incorporated individually into separate vectors, or multiple genes can be incorporated together into a single vector. Alternatively, each gene can be incorporated into a single vector, or different types of vectors (including chromosomally integrating viral vectors and / or non-viral vectors) can be used in combination with a non-chromosomally integrating viral vector. Furthermore, the method for producing reprogrammed cells of the present application is not limited to methods in which all genes are introduced using viral vectors. That is, the method of the present application requires only the use of at least one non-chromosomally integrating viral vector, and may also include the use of other vectors (chromosomally integrating viral vectors and / or non-viral vectors) that express reprogramming factors and / or compounds that induce reprogramming.

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

[0052] Examples of genes particularly preferred 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; NM_021966, Examples of such vectors include KLF4 (Kruppel-like factor 4; NM_004235, NM_010637), cateninβ1 (cadherin-associated protein beta 1; NM_001904, NM_007614; including the S33Y mutant), and c-Myc (NM_002467, NM_010849; including the T58A mutant), as well as genes of other members of the same family to which these genes belong. Individual viral vectors can be used in combination.

[0053] One particularly preferred gene combination is a combination containing at least four genes: Sox, KLF, Myc, and Oct (Takahashi, K. and Yamanaka, S., Cell 126, 663-676, 2006; Lowry, W. E. 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, Sox, KLF, Myc, and Oct proteins, as well as their genes, refer to proteins and genes belonging to the Sox family, KLF family, Myc family, and Oct family, respectively. Expression of one or more members of each of these four families may be adjusted. For example, any of the Sox family genes, Sox1, Sox2, Sox3, Sox15, and Sox17, may be used. Furthermore, for the KLF family, KLF4 or KLF2 may be used. For the Myc family, not only wild-type c-Myc but also T58A mutants, N-Myc, and L-Myc can be used. Thus, various family genes can be selected and used, and reprogramming can be induced by appropriately selecting the above four family genes.

[0054] For example, wild-type c-Myc has been shown to be expressed at low 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, stable, high-level gene expression from the vector becomes possible. The insertion site of the gene into the vector can be selected as desired.

[0055] For example, the Myc gene may be located at the end (5' end) of the minus-strand RNA genome, i.e., at a position earlier in the protein-coding sequences located on the genome when counting from the 5' end than from the 3' end. The Myc gene may be located, for example, at the 5'-most position (i.e., the first position from the 5' end), or the second or third position from the 5' end. For example, the Myc gene may be located second from the 5' end of the genome, specifically, between the L gene at the 5'-most position on the genome and the HN gene next. Silent mutations can be introduced into the Myc gene as appropriate to replace consecutive A or T base sequences without changing the encoded amino acid sequence.

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

[0057] Specifically, the KLF family includes Klf1 (NM_006563, NM_010635), Klf2 (NM_016270, NM_008452), Klf4 (NM_004235, NM_010637), and Klf5 (NM_001730, NM_009769); the Myc family includes c-Myc (NM_002467, NM_010849, including the T58A mutant), NMyc (NM_005378, NM_008709), and L-Myc (NM_005376, NM_005806); and the Oct family includes Oct1A (NM_002697, NM_198934), Oct3 / 4 (NM_002701, NM_203289, These include Oct6 (NM_002699, NM_011141), and Oct1 (NM_013633, NM_001009178), and Oct6 (NM_002699, NM_011141), while 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). A non-chromosomally integrating viral vector carrying any of these genes is useful in the present application for inducing cell dedifferentiation.

[0058] One or more viral vectors carrying any one of the Sox, KLF, and Oct genes, or any one of the Sox, Myc, and Oct genes, or a combination of the Sox, Myc, and Klf genes, are useful for inducing cell reprogramming in the present application. If the Myc gene is not expressed, p53 siRNA and UTF1, for example, may be used instead. Viral vectors containing each gene may be prepared separately or may be used in combination.

[0059] In addition, if one or more of the above genes are already endogenously expressed in the originally differentiated cells, for example, the introduction of those genes can be omitted. Only the necessary viral vectors containing reprogramming factors may be used as appropriate. Furthermore, if the endogenous expression of an endogenous reprogramming factor is induced by the expression of another gene or compound treatment, the introduction of a vector expressing that other gene or compound treatment may be combined with the introduction of a viral vector containing a reprogramming factor that cannot be induced by that alone. In the present application, combining vectors to endogenously or exogenously express at least three genes (Oct, Klf, and Sox), at least four genes (Oct, Klf, Sox, and Myc), or at least four genes (Oct, Sox, Nanog, and Lin28) refers not only to the state in which a certain reprogramming factor is endogenously expressed in nature, but also to the case in which, when 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, the viral vector is combined with that treatment to exogenously express only the missing factor.

[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 suitable. Viral vectors carrying any of these genes are useful for inducing cell dedifferentiation in the present application. One or more viral vectors containing any combination (or all) of these genes can also be used effectively in cell reprogramming. As mentioned above, if the target cells already express some of these genes, it is not necessary to introduce vectors expressing those genes.

[0061] The above-described gene combinations can be combined with additional 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). Furthermore, one or more genes selected from the group consisting of HPV16 E6, HPV16 E7, and Bmil (NM_005180, NM_007552) may be further combined. Alternatively, one or any combination of proteins selected from the group consisting of 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) may be expressed. 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 non-chromosomally integrating viral vectors of the present application.

[0062] The factor to be introduced may be selected appropriately depending on the origin of the cells to be reprogrammed. It may be derived from humans or other mammals, such as mice, rats, rabbits, pigs, monkeys, and other primates, but is preferably derived from humans. Furthermore, the sequences of genes and proteins do not necessarily have to be wild-type sequences; they may contain any mutations as long as they are capable of inducing reprogramming. For example, genes encoding amino acid sequences in which one or a small number of amino acids (e.g., several, up to 3, up to 5, up to 10, up to 15, up to 20, or up to 25) have been added, deleted, substituted, and / or inserted, and which are capable of inducing reprogramming, can be used in the present application. Furthermore, as long as they maintain their biological activity (ability to induce reprogramming), polypeptides in which one to several amino acid residues (e.g., 2, 3, 4, 5, 6, 10, 15, or 20 residues) have been deleted or added at the N-terminus and / or C-terminus, and polypeptides in which one to several amino acid residues (e.g., 2, 3, 4, 5, 6, 10, 15, or 20 residues) have been substituted, can also be used. Examples of variants that can be used include fragments, analogs, and derivatives of natural proteins, as well as fusion proteins with other polypeptides (e.g., those with the addition of a heterologous signal peptide or antibody fragment). Specifically, these include polypeptides that contain sequences in which one or more amino acids of the wild-type amino acid sequence have been substituted, deleted, and / or added, and that have biological activity equivalent to that of the wild-type protein (e.g., the activity of inducing reprogramming). When a fragment of a wild-type protein is used, it typically contains at least 70%, preferably at least 80%, at least 85%, more preferably at least 90%, at least 95%, or at least 98% of the continuous region of the wild-type polypeptide (the mature form in the case of a secreted protein).

[0063] Amino acid sequence variants can be prepared, for example, by introducing mutations into DNA encoding the native polypeptide (Walker and Gaastra, eds. Techniques in Molecular Biology (MacMillan Publishing Company, New York, 1983); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492, 1985; Kunkel et al., Methods Enzymol. 154:367-382, 1987; Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, NY), 1989; US Pat. No. 4,873,192). Guidance on amino acid substitutions that do not affect biological activity is provided, for example, by Dayhoff et al. (Dayhoff et al., in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), 1978).

[0064] The number of amino acids to be modified is not particularly limited, but is, for example, within 30%, preferably within 25%, more preferably within 20%, more preferably within 15%, more preferably within 10%, 5%, or 3% of the total amino acids of the native mature polypeptide, 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, substitution with amino acids having similar side chain properties is expected to maintain the activity of the protein. Such substitutions are referred to herein as conservative substitutions. Conservative substitutions include, for example, 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), uncharged polar 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). Conservative substitutions also include substitutions between amino acids with a positive correlation in the BLOSUM62 substitution matrix (S. Henikoff and JG Henikoff, Proc. Acad. Natl. Sci. USA 89: 10915-10919, 1992).

[0065] The modified protein exhibits high homology with the amino acid sequence of the wild-type protein. High homology refers to an amino acid sequence that has, 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, for example, using 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 BLAST webpage of the National Center for Biological Technology Information (NCBI) (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), which compares two sequences, can be used to align two sequences and determine sequence identity. Gaps are treated in the same way as mismatches, and the identity value is calculated for the entire amino acid sequence of, for example, a natural cytokine (the mature form after secretion). Specifically, the percentage of matching amino acids in the total number of amino acids in the wild-type protein (or the mature form in the case of a secreted protein) is calculated.

[0066] Silent mutations can also be introduced into genes without changing the encoded amino acid sequence. In particular, in AT-rich genes, substitution of five or more consecutive A or T bases with G or C without changing the encoded amino acid sequence can result in stable, high gene expression.

[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 a gene encoding a wild-type protein and have activity equivalent to that of the wild-type protein (activity to induce reprogramming). In hybridization, for example, a probe can be prepared 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 identification can be performed by detecting whether it hybridizes to the other nucleic acid. Stringent hybridization conditions include, for example, hybridization in a solution containing 5xSSC, 7% (W / V) SDS, 100 μg / ml denatured salmon sperm DNA, and 5x Denhardt's solution (1x Denhardt's solution contains 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% Ficoll) at 60°C, preferably 65°C, and more preferably 68°C, followed by washing with shaking for 2 hours in 2xSSC, preferably 1xSSC, more preferably 0.5xSSC, and more preferably 0.1xSSC at the same temperature as for hybridization.

[0068] [Method for producing naive induced pluripotent stem cells] The present application provides a method for producing naive induced pluripotent stem cells from human somatic cells, comprising the following steps (1) to (3): (1) introducing one or more vectors containing reprogramming factors into human somatic cells; (2) culturing the somatic cells in the presence of a naive medium; and (3) culturing the cells obtained after step (2) 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 amount at the start of step 3.

[0069] In the present disclosure, the somatic cells are not particularly limited, and any somatic cells can be used. Examples include keratinizing epithelial cells (e.g., keratinizing 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 form interface surfaces (e.g., type I alveolar cells), luminal epithelial cells of the inner chain ducts (e.g., vascular endothelial cells), ciliated cells with transport capacity (e.g., airway epithelial cells), cells that secrete extracellular matrix (e.g., fibroblasts), contractile cells (e.g., smooth muscle cells), cells of the blood and immune system (e.g., peripheral blood mononuclear cells, umbilical cord blood, 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., companion cells), neurons and glial cells of the central nervous system (e.g., astrocytes), pigment cells (e.g., retinal pigment epithelial cells), and their precursor cells (tissue precursor cells). There are no particular limitations on the degree of differentiation of the cells or the age of the animal from which the cells are collected, and both undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells can be used as the source of somatic cells in the present application. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells. Somatic cells derived from humans are preferred.

[0070] In the present disclosure, induced pluripotent stem cells refer to cells in which pluripotency has been induced by reprogramming somatic cells using known methods, etc. Specifically, examples of induced pluripotent stem cells include cells in which differentiated somatic cells such as fibroblasts or peripheral blood mononuclear cells have been reprogrammed by expressing any combination of multiple 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., to induce pluripotency.

[0071] In the present disclosure, "naive induced pluripotent stem cells" refers to induced pluripotent stem cells with properties similar to those of 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-specific markers or naive-specific subcellular localization of one or more specific gene products), extensive DNA demethylation across the whole genome, X chromosome reactivation, and stratified, domed colony morphology. Examples of naive-specific markers include TCFP2L1, KLF17, TBX3, PRDM14, SSEA1, CD7, CD75, CD77, CD130, DPPA3, ESRRB, KLF4, and KLF5. Examples of naive-specific subcellular localization of specific gene products include nuclear localization of the TFE3 protein.

[0072] The naive nature of the produced induced pluripotent stem cells can be confirmed by one or more of the above characteristics, preferably by the expression of the naive-type specific markers and / or the naive-type specific intracellular localization of specific gene products.Furthermore, for the sake of speed and convenience, it can also be confirmed by the dome-shaped colony morphology.

[0073] Methods for confirming gene expression patterns include known methods such as RNA-seq, qPCR, immunostaining, and FCM, and preferably RNA-seq can be used.

[0074] On the other hand, primed induced pluripotent stem cells (PPS) refer to induced pluripotent stem cells with properties similar to those of the epiblast of a postimplantation embryo, 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 epiblast of a postimplantation embryo (specifically, the expression of one or more primed-specific markers or the primed-specific intracellular localization of one or more specific gene products) and a monolayered, flat colony morphology. Primed-specific markers include, for example, OTX2 or ZIC2, and primed-specific intracellular localization of specific gene products includes, for example, the cytoplasmic localization of the TFE3 protein.

[0075] The primed nature of the produced induced pluripotent stem cells can be confirmed by one or more of the above characteristics, preferably by confirming the expression of the primed-type specific marker and / or the primed-type specific intracellular localization of a specific gene product.Furthermore, for the sake of speed and convenience, confirmation may also be made by the formation of a monolayer flat colony.

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

[0077] The reprogramming factors may be any of the above-mentioned reprogramming factors, including, for example, the OCT gene, the SOX gene, the MYC gene, and the KLF gene.

[0078] The one or more vectors can be any of the vectors described above. For example, the one or more vectors may be a chromosomally non-integrating virus vector, preferably a paramyxovirus vector, and more preferably a Sendai virus vector.

[0079] The one or more vectors containing the 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-described combination of vectors is introduced into the cells. When multiple vectors are introduced in combination, they are preferably introduced simultaneously. Specifically, it is preferable to complete the addition of all vectors containing 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 the addition of the first vector or compound. The vector dose can be adjusted as appropriate, but infection is preferably carried out at an MOI of 0.3 to 100, more preferably 0.5 to 50, more preferably 1 to 30, more preferably 1 to 10, and more preferably an MOI of about 5.

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

[0082] In the present application, the medium can be prepared by appropriately adding necessary factors to a 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 mixtures thereof. The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If necessary, the basal medium may contain one or more serum substitutes, such as albumin, insulin, transferrin, selenium, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. It may also contain one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics (e.g., streptomycin, penicillin, puromycin, and mitomycin), antioxidants, pyruvate, buffers, inorganic salts, cytokines, and the like. In one embodiment, the basal medium is DMEM medium or DMEM / F12 medium. Commercially available media may also 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, the basal medium may be supplemented with factors such as stem cell factor (SCF), thrombopoietin (TPO), Flt3 / Flk2, IL-6, and IL-3.

[0083] In some embodiments, somatic cells can be cultured with feeder cells. As used herein, "feeder cells" refers to cells other than somatic cells that are allowed to coexist with the somatic cells when they are cultured. Examples of feeder cells include, but are not limited to, fibroblasts (NIH-3T3 cells, MEF cells, STO cells, SNL cells, etc.), embryonic kidney cells (HEK293 cells, etc.), stromal cells (ST2 cells, OP9 cells, PA6 cells, MS-5 cells, etc.), epithelial cells (HeLa cells, etc.), placental cells, bone marrow cells, and endometrial cells. Examples of feeder cells include MEF cells. The feeder cells may be derived from the same species as the somatic cells or from a different species. The feeder cells may be treated to inhibit cell proliferation with a growth inhibitor (e.g., mitomycin C) or irradiation (e.g., X-rays or gamma rays). The concentration of feeder cells used in this application is not particularly limited as long as it allows the proliferation of the somatic cells. For example, the concentration may be about 5 × 10 3 cells / cm 2 ~Approx. 1×10 5 cells / cm 2 Somatic cells may be cultured with feeder cells from the beginning of the culture, or may be cultured with feeder cells from the middle of the culture. For example, somatic cells may be cultured with feeder cells from days 2 to 10 or days 2 to 5 of the culture. In another embodiment, somatic cells may be cultured in the absence of feeder cells.

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

[0085] In some embodiments, somatic cells are cultured in two-dimensional culture. When culturing in the presence of feeder cells, for example, the feeder cells may be seeded and cultured in advance on the bottom of a culture vessel or on a culture substrate, and then grown to fill the bottom of the culture vessel or the surface of the culture substrate. Then, the somatic cells may be seeded and cultured on the feeder cells.

[0086] Culture vessels for culturing somatic cells are typically treated with a coating agent, such as gelatin, Matrigel (BD), Synthemax (Corning), collagen, laminin, heparan sulfate proteoglycan, entactin, fragments thereof, and combinations thereof.

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

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

[0089] In the present application, "naive medium" refers to a medium used when inducing naive induced pluripotent stem cells from somatic cells. The basal medium for the naive medium can be any of the basal media described above, and examples thereof include a medium obtained by adding any combination of additives such as non-essential amino acids (NEAA), L-glutamine, 2-mercaptoethanol, antibiotics (e.g., streptomycin, penicillin, puromycin, mitomycin), and bovine serum albumin (BSA) to N2B27 medium (a 1:1 mixture of N2 medium (DMEM / F12 medium supplemented with N2 supplement) and B27 medium (Neurobasal medium supplemented with B27 supplement).

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

[0091] The concentration of LIF in the medium can be between 1 ng / ml and 100 ng / ml, or between 5 ng / ml and 50 ng / ml, for example, about 10 ng / ml.

[0092] In the present application, examples of MEK inhibitors include, but are not limited to, 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-methoxyphenyl)-2-methyl-2-propanol; CAS Registry Number: 109511-58-2), and PD98059 (2-(2-amino-3-methoxyphenyl)-2-methyl-2-propanol; CAS Registry Number: 109511-58-2). Examples of MEK inhibitors include PD0325901 (2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; CAS Registry Number: 212631-79-3), and PD184352 (2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; CAS Registry Number: 212631-79-3). Of these, PD0325901 is preferred. When PD0325901 is used as the MEK inhibitor, its concentration in the medium may be 50 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

[0093] In the present application, the GSK3 inhibitor is not particularly limited, but examples thereof include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; CAS Registry Number: 252917-06-9), BIO (6-bromoindirubin-3'-oxime; CAS Registry Number: 66746 3-62-9), Kenpaullone (9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one; CAS Registry Number: 142273-20-9), and IM-16 (3-(4-fluorophenylethylamino)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; CAS Registry Number: 1129669-05-1). Among these, CHIR99021 is preferred. When CHIR99021 is used as the GSK3 inhibitor, its concentration in the medium can be 50 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

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

[0095] In the present application, the TGF-β inhibitor is not particularly limited, but examples thereof 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-imidazol-2-yl]-benzamide). Of these, A83-01 is preferred. When A83-01 is used as the TGF-β inhibitor, its concentration in the medium may be 10 nM to 100 μM, or 100 nM to 10 μM, for example, about 1 μM.

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

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

[0098] When cells are cultured in the absence of feeder cells, the naive medium may be preconditioned with specific cells. The specific cells may be cells that can be used as feeder cells, such as irradiated mouse embryonic fibroblasts (iMEFs). The culture period for conditioning may be, for example, about 12 hours to about 2 days. The medium may be obtained by removing cellular components by a conventional method after conditioning. 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 of the somatic cells may be 1 to 20 days, 3 to 10 days, for example, 4 to 6 days.

[0100] In step (3), the obtained cells are cultured 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 amount at the start of step (3).

[0101] In one embodiment, the obtained cells can be cultured 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 may be the same as the naive medium used in step (2). The naive medium may contain one or more compounds selected from the group consisting of leukemia inhibitory factor (LIF), a MEK inhibitor, a GSK3 inhibitor, a cAMP production promoter, a TGF-β inhibitor, and a PKC inhibitor. For example, the naive medium may contain t2iLGo, 5iLAF, or tt2iLGo.

[0103] When cells are cultured in the absence of feeder cells, the naive medium may be preconditioned with specific cells. The specific cells may be cells that can be used as feeder cells, such as irradiated mouse embryonic fibroblasts (iMEFs). The culture period for conditioning may be, for example, about 12 hours to about 2 days. The medium may be obtained by removing cellular components by a conventional method after conditioning. 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 may be determined according to the characteristics of the vector, within the range of about 30 to 40°C, but is not limited thereto. Culture is carried out in a CO2-containing atmosphere, with a CO2 concentration of about 2 to 8%, preferably about 3 to 7%, more preferably about 4 to 6%, and most preferably about 5%. Culture may also be carried out under hypoxic conditions, with an oxygen concentration of about 3 to 10%, preferably about 4 to 8%, more preferably about 4 to 6%, and most preferably about 5%.

[0105] In one embodiment, the amount of vector per somatic cell may 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 relative 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. For example, the amount of vector per somatic cell may be reduced to 30% or less relative to the amount at the start of step 3 within 12 days from the start of step 3.

[0106] In one embodiment, the one or more vectors are temperature-sensitive vectors, vectors containing a target sequence of a microRNA specific to induced pluripotent stem cells, or vectors that are both temperature-sensitive and contain a target sequence of a microRNA specific to induced pluripotent stem cells, and the resulting cells are cultured in the presence of a naive medium, whereby the amount of 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, the amount 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 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 temperature-sensitive vector may be any of the vectors described above. For example, the temperature-sensitive vector may be a vector containing the temperature-sensitive mutation described above. In one embodiment, the 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 a 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"), as well as Y942H / L1361C / L1558I mutations in the L protein), TS12 mutations (TS mutations, as well as D433A / R434A / K437A mutations in the P protein), or TS15 mutations (TS mutations, as well as D433A / R434A / K437A mutations in the P protein and L1361C / L1558I mutations in the L protein), as well as other minus-strand RNA virus vectors with similar substitutions at equivalent sites.

[0108] The one or more vectors comprising reprogramming factors can include, for example, a vector comprising an OCT gene, a SOX gene and a KLF gene, a vector comprising a MYC gene, and a vector comprising a KLF gene.The vector comprising an OCT gene, a SOX gene and a KLF gene, the vector comprising a MYC gene, and the vector comprising a KLF gene can be a Sendai virus vector, for example, a Sendai virus vector comprising a TS7, TS12, or TS15 mutation.In some embodiments, the vector comprising a MYC gene can be a Sendai virus vector comprising a TS15 mutation, and the vector comprising an OCT gene, a SOX gene and a KLF gene, and the vector comprising a KLF gene can be a Sendai virus vector comprising a TS12 mutation.

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

[0110] The target sequence of induced pluripotent stem cell-specific microRNA is not particularly limited, as long as it is a sequence that induced pluripotent stem cell-specific microRNA binds to as a target.Examples of induced pluripotent stem cell-specific microRNA 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 the present application, the microRNA target sequence can be a natural microRNA target sequence or a variant thereof, as long as expression is suppressed by microRNA binding. Furthermore, the microRNA target sequence may or may not be perfectly complementary to the microRNA. For example, the microRNA target sequence may contain at least 10 bases, e.g., 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 bases complementary to the microRNA, either consecutively or non-consecutively. There is no particular upper limit to the length of the microRNA target sequence, but it may be, for example, approximately 30 bases. Preferably, the complementary bases are consecutive, or may contain several unpaired bases, e.g., 5 or less, 4 or less, 3 or less, 2 or less, or 1 unpaired base. The unpaired bases may be contained on the microRNA target sequence side and / or on the microRNA side.

[0112] The microRNA target sequence is preferably a sequence that hybridizes with the microRNA under physiological conditions. Physiological conditions include, 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 the microRNA under stringent conditions. Stringent conditions include, for example, 1xSSC (1xSSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0) or 0.5xSSC at 42°C, more preferably 1xSSC or 0.5xSSC at 45°C, and even more preferably 1xSSC or 0.5xSSC 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, and the two are allowed to hybridize. Hybridization conditions may be, for example, 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% Ficoll), 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 is performed under the above conditions, and the hybridization of the labeled nucleic acid is detected, thereby determining whether or not the nucleic acid hybridizes under these conditions.

[0113] Alternatively, the microRNA target sequence preferably shows high homology with the complementary sequence of the microRNA sequence.High homology refers to, 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 of the nucleotide sequence identity.The identity of the nucleotide sequence can be determined, for example, by 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 BLAST webpage of the National Center for Biological Technology Information (NCBI) (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), which compares two sequences, can be used to align two sequences and determine sequence identity. Gaps outside the base sequence of the complementary sequence of the microRNA sequence are ignored, and internal gaps are treated the same as mismatches, for example, and the identity value is calculated for the entire base sequence of the complementary sequence of the microRNA sequence in the alignment (the total base length including any gaps inside the sequence).

[0114] Alternatively, the microRNA target sequence preferably comprises a sequence in which one or several bases are inserted, substituted, and / or deleted relative to the complementary sequence of the microRNA sequence. Preferably, the microRNA target sequence includes a sequence in which the insertion, substitution, and / or deletion of 8 bases or less, 7 bases or less, 6 bases or less, 5 bases or less, 4 bases or less, 3 bases or less, 2 bases or less, or 1 base is inserted, substituted, and / or deleted relative to the complementary sequence of the microRNA sequence.

[0115] Generally, the more mutations introduced into the microRNA target sequence, the more the binding with the microRNA is suppressed, and the lower the expression suppression effect. It is possible to adjust the suppression effect by introducing appropriate mutations.

[0116] In certain embodiments, when the vector of the present application is a minus-strand RNA viral vector, the microRNA target sequence is added to the NP or P gene. When adding a microRNA target sequence to the NP or P gene, there are no particular limitations on the location of addition; it can be added to the translated or untranslated region of each gene. When adding to the translated region, it may be added to either the N-terminal or C-terminal position, but it is preferable to add it immediately following the C-terminus. The untranslated region may be the 5' untranslated region or the 3' untranslated region of the genome, but the 5' untranslated region is preferred. When adding to the untranslated region, the location may be selected arbitrarily. For example, when adding a microRNA target sequence to the 5' untranslated region of the genome of the NP gene and / or P gene, it may be added to any position between the stop codon and the E (End) sequence in the translated region of the gene to be added. When adding to the 3' untranslated region of the genome, it can be added to any desired position, for example, between the S (Start) sequence and the translation initiation codon of the gene to be added. One or more copies of the microRNA target sequence can be added. In addition, not only one type of microRNA target sequence but also multiple types of microRNA target sequences may be added. As an example, for example, a sequence in which one or multiple types of microRNA target sequences are arranged in tandem, such as two or more, for example, three or more, four or more, or five or more, can be added. There is no particular upper limit to the number of microRNA target sequences added in tandem, but it can be up to about 10, for example.

[0117] When the P gene is modified, 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 may be separately expressed from a vector.

[0118] The P protein does not have to be full-length; an appropriate fragment can be used. Only a portion of the C-terminus is essential for the P protein; other regions are not essential for viral vector expression. Specifically, the P protein may be a fragment that retains the L protein binding site and the N protein:RNA binding site. An example of the L protein binding site is the amino acid sequence from positions 411 to 445 of the SeV P protein. An example of the N protein:RNA binding site is the sequence from positions 411 to 445 of the SeV P protein. An example of the N protein:RNA binding site is the sequence from positions 411 to 445 of 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 of suitable P proteins include the amino acid sequence of positions 479 to 568 of the SeV P protein (e.g., ABB00297.1). More specifically, a fragment comprising the amino acid sequence of positions 320 to 568 of the SeV P protein can be suitably used as a functional P protein in the present application. Use of a deleted P protein is expected to reduce the size of the vector and make it less susceptible to the host's immune response.

[0119] When using a P protein lacking the coding region of the C protein, the C protein may be expressed separately as appropriate, as described above. Here, C proteins include the 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 may be inserted into an appropriate vector. There are no particular restrictions on the insertion position, but it can be inserted just before the P protein (3' side of the coding sequence of the P protein in the genome) or just after the P protein (5' side of the coding sequence of the P protein in the genome). An EIS sequence may be added as appropriate during insertion.

[0120] When a microRNA target sequence is added to the NP or P gene, expression of the microRNA in cells into which the vector has been introduced reduces the expression of the vector, facilitating its elimination. For example, by adding a target sequence for a microRNA that is not expressed at the time of vector introduction but is specifically expressed when the cell reaches a certain differentiated state (or undifferentiated state), the expression of the vector is automatically suppressed and the vector is eliminated once its purpose is achieved.

[0121] In another embodiment, the one or more vectors may be vectors in which a degron has been added to the P gene, and the resulting cells may be cultured in the presence of a naive medium. This may 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, the amount may 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 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.

[0122] As used herein, "degron" refers to a polypeptide that destabilizes a protein when attached to it. Examples of degrons include sequences that stabilize proteins upon fusion with a small molecule, sequences that destabilize proteins upon binding with a small molecule, and sequences that destabilize proteins regardless of the presence or absence of a small molecule. Specific 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), TetR mutants (WO2007 / 032555), the plant-derived auxin-inducible degron (AID) system (WO2010 / 125620), the PEST sequence (WO99 / 54348), known as a degradation-promoting sequence, CL1 (WO2004 / 025264), a calpain-derived sequence (JP2009-136154), and NDS (JP2011-101639). FKBP12 is known as the mammalian target of rapamycin (mTOR) and is stabilized by binding to small molecules such as rapamycin and shield 1, whereas its removal destabilizes it and leads to proteasomal degradation. 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 by adding, for example, the C-terminal residues 422-461 of mouse ornithine decarboxylase (mODC). The PEST sequence regulates the half-life of proteins, and any desired sequence can be used to shorten their half-life (Rechsteiner M, et al., Trends Biochem. Sci. 21, 267-271, 1996). A PEST sequence is, for example, a sequence surrounded by basic amino acids (H, K, or R) at both ends, containing (i) P, (ii) D and E, or (iii) S and E, which binds to the ubiquitination enzyme E3, and can be identified using, for example, GENETYX (Genetyx). Sequences that have the same effect as PEST include CL1, calpain partial sequences, and NDS.The AID sequence is destabilized by the binding of plant ubiquitin ligase TIR1 and auxin (IAA).

[0123] As used herein, a degron added to a P gene means that the P gene encodes a P protein to which the degron has been added. In the present application, the above-described degron can be added to a P protein. Preferred degrons include the mTOR degron, DHFR degron, TetR degron, PEST, and AID. These degrons include naturally occurring sequences and those derived therefrom. Particularly preferred degrons include the mTOR degron, DHFR degron, TetR degron, and PEST. Among these, degrons other than the AID sequence are preferred, specifically the FKBP12 degron (DD), DHFR degron (DDG), TetR degron, and mODC PEST. Known PESTs include d2, derived from the naturally occurring sequence, and its variants d1 and d4 (WO 99 / 54348). All of these are included in the PEST family and can be used in the present application. Nucleic acids encoding degrons can be appropriately prepared by DNA synthesis.

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

[0125] After introducing a vector containing a degron added to the P gene of the present invention into cells and expressing the gene of interest, the vector can be removed appropriately depending on the properties of the degron. For example, when using a ligand-controllable degron such as DD, DDG, or a TetR mutant, removal is promoted without the addition of a ligand, but vector expression can be extended by adding a ligand such as Shield-1. Furthermore, if the degron is functional even without a ligand, such as a PEST sequence, vector removal can be promoted by continuing to culture cells into which the vector has been introduced.

[0126] The vector of the present application in which a degron has been added to the P gene may be a vector exhibiting temperature sensitivity as described above, a vector containing a target sequence of a microRNA specific to induced pluripotent stem cells, or a vector that is both temperature sensitive and contains a target sequence of a microRNA specific to induced pluripotent stem cells.

[0127] In yet another embodiment, the one or more vectors may be vectors carrying a suicide gene, and the resulting cells may be cultured in the presence of a naive medium, thereby reducing the amount of the 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, the amount may 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 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 carried by the vectors of the present invention include prodrug converting enzyme genes for desired drugs that induce cell death or exhibit cytotoxicity. Specific examples of combinations of prodrugs and suicide genes include: 5-fluorocytosine and cytosine deaminase; cyclophosphamide and cytochrome P450; fludarabine and Escherichia coli PNP (purine nucleoside phosphorylase); CB1954 and nitroreductase; and capecitabine and carboxylesterase. Furthermore, suicide genes include genes encoding desired proteins that induce cell death or exhibit cytotoxicity by converting from an inactive to an active form. For example, genes encoding caspases that induce cell death can be used. Specifically, combinations of desired dimerizers and caspases with their binding domains can be used. Examples of combinations of dimerizers and caspases include: AP20187 and iCaspase-9.

[0129] For example, after introducing a vector carrying the suicide gene of the present invention into cells and culturing them, a prodrug can be added, which will kill cells in which the vector remains, allowing only cells in which the vector has disappeared to be isolated.

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

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

[0132] The naive induced pluripotent stem cells produced by the method of the present application have a very low residual amount of vectors containing reprogramming factors, so that the naive properties can be stably maintained even when cultured for a long period of time. In the method of the present application, the vectors containing reprogramming factors can be substantially eliminated from the cells about 30 days after the introduction of the vector. Therefore, the naive induced pluripotent stem cells obtained by the method of the present application are substantially free of vectors containing reprogramming factors and have high phenotypic stability.

[0133] [Vector Set] The present application also provides a vector set for producing naive induced pluripotent stem cells from human somatic cells, comprising 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. Examples of the reprogramming factors and vectors used in this embodiment are as described above.

[0134] [Composition] The present application also provides a composition for producing naive induced pluripotent stem cells from human somatic cells, comprising 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. Examples of the reprogramming factors and vectors used in this embodiment are as described above.

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

[0136] [Kit] The present application further provides a kit for producing naive induced pluripotent stem cells from human somatic cells, comprising one or more vectors containing reprogramming factors and a naive medium, wherein the one or more vectors are selected from the group consisting of temperature-sensitive vectors, vectors containing a target sequence of a microRNA specific to induced pluripotent stem cells, and vectors that are temperature-sensitive and contain a target sequence of a microRNA specific to induced pluripotent stem cells. Examples of the reprogramming factors and vectors used in this embodiment are as described above.

[0137] In the kits of the present application, the one or more vectors may be packaged separately or together. The kits of the present application may also include buffers, instructions, etc.

[0138] [Culture Supernatant] The present application also provides a culture supernatant of naive induced pluripotent stem cells produced by the method of the present application. The culture medium can be the naive medium described above. The naive medium can contain one or more compounds selected from LIF (leukemia inhibitory factor), MEK inhibitor, GSK3 inhibitor, cAMP production promoter, TGF-β inhibitor, and PKC inhibitor. For example, it can contain t2iLGo, 5iLAF, or tt2iLGo.

[0139] The culture period for obtaining the culture supernatant may 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 3 ~1×10 5 cells / cm 2 can be, for example, 1 x 10 4 ~3×10 4 cells / cm 2 is.

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

[0141] After culturing, cellular components can be separated and removed to obtain a culture supernatant of naive induced pluripotent stem cells. In the present disclosure, the culture supernatant includes not only a culture supernatant obtained by separating and removing cellular components from a culture medium, but also a culture supernatant that has been appropriately subjected to various treatments (e.g., centrifugation, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.).

[0142] [Cosmetics] The present application also provides cosmetics containing as an ingredient the culture supernatant of naive induced pluripotent stem cells produced by the method of the present application. The culture supernatant of naive induced pluripotent stem cells produced by the method of the present application is as described above. The cosmetics of the present application may be in any formulation or form as long as they are used by contacting the skin. Specifically, in a broad sense, the term includes, for example, skin milk, skin cream, foundation cream, massage cream, cleansing cream, shaving cream, cleansing foam, skin lotion, lotion, pack, lipstick, blush, eye shadow, nail polish, soap, body shampoo, hand soap, shampoo, rinse, hair tonic, treatment, hair cream, hair spray, hair growth agent, hair care agent, hair dye, hair styling agent, depilatory agent, antidandruff agent, toothpaste, denture adhesive, mouthwash, permanent wave agent, curling agent, styling agent, ointment, cataplasm, bath agent, antiperspirant, sunscreen, etc. Any type of product may be used as long as it comes into contact with the skin during use, but a form that can be used as a cosmetic is particularly preferred. Furthermore, the term also includes products that come into contact with the skin of animals in addition to humans. Furthermore, the cosmetic product of the present application may be in any form, including solid, liquid, semi-solid, gas, as well as many other forms such as powder, granules, tablets, gel, and foam, but is not limited to these.

[0143] The cosmetic product of the present application may contain pharmaceutically acceptable carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, and physiological saline. Examples of excipients include lactose, starch, sorbitol, D-mannitol, and sucrose. Examples of disintegrating agents include carboxymethylcellulose and calcium carbonate. Examples of buffering agents 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 soothing agents 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.

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

[0145] Materials and Methods Cell Culture: Human dermal fibroblasts (HDFs) were obtained with informed consent from the Tokyo Metropolitan Institute of Gerontology. HDFs and post-irradiation mouse embryonic fibroblasts (iMEFs) were maintained in Dulbecco's modified Eagle's 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 manufacturer's recommended conditions. Primed and naive embryonic stem 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 were cultured on iMEFs and treated with t2iLGo medium, which consists of 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 Naive induced pluripotent stem cells (iPSCs) were maintained in medium. Feeder-free naive induced pluripotent stem cells (iPSCs) were maintained on Matrigel (hESC-qualified, Corning). The medium was changed every other day, and 10 μM Y27632 (Wako) was added immediately before each medium change. Naive iPSCs were passaged every 3–4 days using Accutase (Innovative Cell Technologies) and always cultured under 5% O2. Recombinant DNA experiments in this example were performed under the approval of Kyoto University.

[0146] Construction of modified SeV-KLF4 vectors. An insert containing the open reading frame of the human KLF4 gene was generated by PCR from cDNA using NotI-tagged gene-specific forward and reverse primers containing an SeV-specific transcriptional regulatory signal sequence. The amplified fragment was inserted into the 18+ region of plasmids containing the genomic sequences of the SeV / TS12ΔF, SeV / PmiR367T2 / TSΔF, and SeV / PmiR367T2 / TS12ΔF vectors to generate plasmids pSeV18+KLF4 / TS12ΔF, pSeV18+KLF4 / PmiR367T2 / TSΔF, and pSeV18+KLF4 / PmiR367T2 / TS12ΔF (collectively referred to as "SeV-KLF4 vector plasmids"), respectively. The sequence of miR367T2 was TCACCATTGCTAAAGTGCAATTcgatTCACCATTGCTAAAGTGCAATT (SEQ ID NO: 1). The SeV-KLF4 vector was recovered and propagated as follows. First, the SeV-KLF4 vector plasmid and a plasmid carrying the T7 RNA polymerase, NP, P, F5R, and L genes were transfected into 293T cells. The cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and cultured for 1 to 3 days to generate seeds of the SeV-KLF4 vector. The seeds were cloned and propagated in MEM containing trypsin (2.5 μg / ml) using SeV F-expressing LLC-MK2 / F7 / A cells.The titer (cell infectious units / mL) of the recovered SeV-KLF4 vector was determined by immunostaining 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 to naive iPSCs. In the conventional method, HDFs or PBMCs were reprogrammed using the CytoTune-iPS 2.0 or 2.0L reprogramming kit. In the new method, the KLF4 / TS vector included in the kit was replaced with a novel SeV-KLF4 vector (e.g., 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 on day 14), the incubation temperature was changed to 38°C and the SeV vector was removed. After removing the SeV vector (day 34 when using SeV-KLF4 / PmiR367T2 / TS12), the temperature was changed to 37°C. After infection with the SeV vector, the incubator was set to 5% O2.

[0148] Initially, HDFs 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 at a multiplicity of infection of 5). From day 1, the medium was changed every other day. On day 5, cells were replated onto iMEF feeder cells. The following day, the medium was changed to naive medium.

[0149] For PBMC reprogramming, PBMCs were cultured 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) 5 days before SeV vector infection. On day 0, cells were counted and infected with three SeV vectors using the same reprogramming procedure as for HDFs. On day 1, cells were counted and replated under the same conditions as for HDF reprogramming. From day 2, PSC medium (equivalent to the 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, naive PSCs were incubated on gelatin-coated dishes at 37°C for 2 hours to remove iMEFs. Total RNA and DNA were extracted from cell lysates using the AllPrep DNA / RNA Mini Kit (QIAGEN), and RNA was incubated with an RNase-Free DNase Set (QIAGEN) to remove genomic DNA. MicroRNA (miRNA) was extracted using NucleoSpin miRNA (MACHEREY-NAAGEL). For qRT-PCR, reverse transcription of 1 μg of DNase-treated RNA was performed using PrimeScript RT Master Mix (Takara) containing an oligo-dT primer and a random hexanucleotide sequence. cDNA was synthesized using TaqMan Advanced miRNA Assays (Applied Biosystems). qRT-PCR was performed on a 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 generated using TruSeq Stranded Total RNA Library Prep Gold (Illumina) according to the manufacturer's protocol, starting with 100 ng of total RNA. For the Hiseq2500, clusters were generated using the HiSeq PE Cluster Kit v4-cBot (Illumina). Sequencing was performed on the HiSeq2500 (2x126 PE mode) with the HiSeq SBS Kit v4. Sequencing was also performed on 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. FASTQ files were generated from the bcl files using bcl2fastq v2.17.1.14 (Illumina) and processed using the ENCODE long-rna-seq-pipeline v2.3.4. Briefly, sequenced reads were mapped to the human reference genome (GRCh38) using STAR 2.5.1b with gene annotations from GENCODE v24. Normalized gene expression data were calculated using RSEM 1.2.23, and gene count data were obtained using featureCounts in Subread 1.5.1.To characterize our PSC lines, 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 humans. Cell 158, 1254–1269, doi:10.1016 / j.cell.2014.08.029 (2014)). Expression values ​​of 4,720 genes included in all data sets were normalized by quantiles between samples, and z-scores for each gene were 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 the Infinium Human Methylation 450K or EPIC BeadChip Kit (Illumina) according to the manufacturer's protocol. DNA methylation values ​​were exported using GenomeStudio v2011.1, and data processing was performed using the "minfi" package in R 3.6.3. A total of 424,444 probes shared between 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. Cells were then plated in PSC medium on Matrigel-coated slides. The following day, cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature. Slides were treated with 0.2M HCl for 20 minutes, permeabilized with 0.2% Triton X-100 for 10 minutes, digested with pepsin solution (0.005% in 0.1M HCl) for 2–6 minutes at 37°C, and dehydrated. 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 with Cy5-dUTP (RP11-155O24) and Cy3-dUTP (RP11-256P2). The labeled probe and XIST RNA FISH probe (Chromosome Science Lab) were mixed with sonicated salmon sperm DNA and Cot-1 DNA in hybridization solution. The probe was denatured at 85°C for 10 minutes, applied to the pretreated slides, covered with a coverslip, and hybridized overnight at 37°C. Slides were then washed with 50% formamide / 2xSSC for 20 minutes at 37°C and 1xSSC for 15 minutes at room temperature, counterstained with DAPI, and mounted. FISH images were captured using a cooled CCD camera mounted on a Leica DMRA2 microscope with the CW4000 FISH application program (Leica Microsystems Imaging Solution).

[0154] For primitive endoderm differentiation, naive PSCs were dissociated and iMEF feeder cells were removed as described above. 500,000 cells were cultured in early primitive endoderm differentiation medium (Ndiff227, 25 ng / ml FGF4 (Peprotech), 1 μg / ml Heparin (Wako), 10 ng / ml BMP4 (R&D), 10 ng / ml PDGFAA (Peprotech), 1 μM XAV939 (Sigma), 3 μM A83-01 (Wako), 0.1 μM RA (Sigma)) containing laminin 511-E8 (0.4 μg / cm). 2 Cells were seeded onto 6-well plates coated with iMatrix 511 (Nippi). The next day, the medium was replaced with the same medium as above. The day after that, the medium was replaced with the same medium supplemented with 10 ng / ml IL-6 (R&D). 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 cultured in initial TE differentiation medium (Ndiff227, 2 μM A83-01 (Wako), 2 μM PD0325921 (Sigma), and 10 ng / mL BMP4 (R&D)) containing laminin 511-E8 (0.15 μg / cm). 2 The cells were seeded into six-well plates 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 analyzed for the expression of TACSTD2, ENPEP, HAVCR1, and HLA-ABC by flow cytometry.

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

[0157] Statistics and reproducibility. Exact n values ​​are provided in the relevant figure legends. Statistical significance was determined by 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 figures. Error bars indicate the mean ± sd.

[0158] Data availability Sequencing and array data have been deposited in GEO with accession number GSE179476.

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

[0160] Figure 1 shows a Sendai virus vector (CytoTune-iPS2.0L) conventionally used to generate naive pluripotent stem cells from somatic cells. The cDNAs carried are 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, the KLF4 vector "18+ / KLF4 / TS7ΔF," "18+ / KLF4 / TS12ΔF," or "18+ / KLF4 / PmiR367T2 / TSΔF" is used instead of "18+ / KLF4 / TSΔF." Here, "PmiR367T2" indicates that the miR367 recognition sequence is carried in tandem on the 5' side of the P gene.

[0161] The KOS vector "PM / KOS / TS12ΔF" and the LMYC vector "HNL / LMYC / TS15ΔF" were constructed by standard methods (WO2012 / 029770 and WO2010 / 008054). Furthermore, "18+ / KLF4 / TS7ΔF" and "18+ / KLF4 / TS12ΔF" were constructed using the TS7 and TS12 vector backbones, respectively, in a manner similar to that used for "18+ / KLF4 / TSΔF" (WO2010 / 008054). Furthermore, "18+ / KLF4 / PmiR367T2 / TSΔF" was constructed by standard methods (WO2017 / 082174).

[0162] Example 2 Production of naive iPS cells from human dermal fibroblasts (HDF) The protocol for producing naive iPS cells from human dermal fibroblasts (HDF) is shown in FIG.

[0163] Step (1): One day before infection with the Sendai virus vector, human dermal fibroblasts were seeded onto gelatin-coated 12-well or 6-well plates at the same cell numbers for SeV infection and cell counting. They were cultured in Fibroblast medium (DMEM + 10% FBS) at 37°C under normoxia (approximately 20%). The following day (day 0), the cells seeded for cell counting were detached using a cell detachment solution and counted. An appropriate amount of Sendai virus vector was then infected and incubated at 35°C in a hypoxic (5% O2) environment. The dose of Sendai virus vector used for infection was approximately 5 for KOS, 5 for LMYC, and 5 for KLF4, although appropriate conditions were adjusted depending on the cell type. After 24 hours (day 1), the medium was replaced with fresh Fibroblast medium on days 2 and 4. On day 4, cell culture vessels seeded with irradiated mouse embryonic fibroblasts (iMEFs) were prepared. On day 5, cells were detached using a cell detachment solution, and the cell number was counted. After that, an arbitrary number of cells were seeded onto the cell culture vessel seeded with iMEF. In this example, the culture was carried out under a CO2 concentration condition of 5%.

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

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

[0166] From around day 10, stratified, dome-shaped colonies (naive iPS cell colonies) appeared.

[0167] Step (3) After day 10, when the dome-shaped colonies on the layered culture medium reached a sufficient size (diameter of about 50 to 200 μm), they were subcultured, and the next day, high-temperature culture (usually 38° C.) was started.

[0168] The t2iLGo+Y medium was changed every other day and the cells were cultured in an incubator with an oxygen concentration of 5%. They were passaged every 3-4 days. For cell detachment during passage, the medium was removed, the cells were washed once with D-PBS (Nacalai Tesque), Accutase (Innovative Cell Technologies) was added, and the cells were incubated at 37°C for 7-10 minutes. If the only purpose was maintenance, approximately one-fifth of the number of cells obtained after detachment was seeded into a culture vessel of the same surface area.

[0169] Figure 3A shows a phase-contrast microscopy image of cells after 45 passages. Numerous dome-shaped colonies were observed in the stratified form, while few flat colonies were observed in the monolayer. Figure 3B shows the dome-shaped colonies in Figure 3A immunostained for naive-specific markers (NANOG and KLF17) and a gene product (TFE3) whose subcellular localization differs between naive and primed forms. Hoechst staining is used for nuclear staining, and PC indicates a phase-contrast image. As shown in Figure 3B, the constituent cells of the dome-shaped colonies were strongly stained for NANOG and KLF17, while the nuclei were deeply stained for TFE3. Thus, induced pluripotent stem cells were obtained that expressed naive-specific markers, showed naive-specific subcellular localization of specific gene products (a gene expression pattern equivalent to that of the inner cell mass of preimplantation embryos), and formed stratified dome-shaped colonies (a characteristic colony morphology of the naive form). Therefore, it was demonstrated that naive iPS cells can be produced from human fibroblasts by the method comprising the steps (1) to (3).

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

[0171] As shown in Figure 4, the vectors used in this example had already decreased to below the detection limit by Day 38. This indicates that they disappeared rapidly upon incubation at a high temperature of 38°C (KLF4 / TS7ΔF_1-3). Furthermore, when the KLF vectors "18+ / KLF4 / TS12" or "18+ / KLF4 / TS12 / miR367T2" were used and incubation was performed at 38°C, the levels dropped below the detection limit 8 to 12 days earlier (data not shown).

[0172] In contrast, the conventional vector remained at the same level on Day 80 as on Day 14 (Conv. Cocktail_1-3). Under culture conditions using standard primed induced pluripotent stem cell production medium, the vector gradually decreases even at a typical culture temperature (37°C) and is known to have almost completely disappeared by Day 80. This strongly suggests that the rate of vector disappearance may be affected by different cell culture environments.

[0173] Therefore, to quickly obtain naive pluripotent stem cells that do not contain vector-derived reprogramming factors, it is sufficient to use a vector that exhibits temperature sensitivity when cultured in naive medium, or a vector containing a target sequence of a microRNA specific to induced pluripotent stem cells.It has been shown that an example of a vector that exhibits temperature sensitivity is a Sendai virus vector containing a TS7, TS12, or TS15 mutation.

[0174] Example 3 Production of naive iPS cells from human peripheral blood mononuclear cells (PBMCs) A protocol for producing naive iPS cells from human peripheral blood mononuclear cells (PBMCs) is shown in FIG.

[0175] Step (1): PBMCs were cultured in PBMC medium at 37°C under normal oxygen conditions (approximately 20%) starting 5 days before infection with the Sendai virus vector. The medium was not replaced. The composition of the PBMC medium is shown below. All concentrations are final concentrations: StemSpan ACF (STEMCELL Technologies), Recombinant Human SCF (R&D Systems) 100 ng / ml, Recombinant Human TPO (R&D Systems) 100 ng / ml, Recombinant Human Flt3 / Flk2 (R&D Systems) 100 ng / ml, Recombinant Human IL-6 (R&D Systems) 50 ng / ml, and Recombinant Human IL-3 (R&D Systems) 20 ng / ml.

[0176] On the day of infection (day 0), cells were counted and infected with Sendai virus in an amount appropriate for the cell number. Subsequently, the cells were incubated at 35°C in a hypoxic (5% O2) environment. The amount of Sendai virus used for infection was generally MOI 5 for KOS, MOI 5 for LMYC, and MOI 5 for KLF4, but conditions were adjusted accordingly depending on the cell type. To increase virus density, infection was generally performed in 24-well or smaller wells. On the same day, cell culture vessels (basically 6-well) seeded with iMEFs were prepared. 24 hours after infection (day 1), the cells were harvested, the virus-containing medium was removed, and the cell count was performed. An arbitrary number of cells were then seeded onto the cell culture vessel containing iMEFs. In this example, the cells were cultured under a 5% CO2 concentration.

[0177] Step (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 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) was the same as that used in Example 2 to produce naive iPS cells from human dermal fibroblasts.

[0178] Step (3): From around day 10, layered, dome-shaped colonies (naive iPS cell colonies) appeared. After day 10, when the colonies reached a sufficient size (approximately 50-200 μm in diameter), they were passaged, and the following day, high-temperature culture (typically 38°C) began. The maintenance culture and passaging methods were the same as those used in Example 2 to produce naive iPS cells from human dermal fibroblasts.

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

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

[0181] Therefore, it has been demonstrated that the method of the present application enables rapid production of naive induced pluripotent stem cells (more specifically, naive iPS cells that are substantially free of reprogramming factors) from various human somatic cells.

[0182] Example 4: Phenotypic Stability of Naive-Type Induced Pluripotent Stem Cells. The phenotypic stability of the naive-type induced pluripotent stem cells obtained in Examples 2 and 3 after long-term passage was analyzed. Principal component analysis was performed on the single-cell data of reset naive ES cells (in-house reset naive ESCs), the standard for naive-type induced pluripotent stem cells; eight clones of HDF-derived primed iPS cells established by the present 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; previously reported naive ES cells (Reset Naive ESCs, 6 / 5 / 4iLA Naive ESCs); and human embryos (Figure 7). The red-framed area indicates the naive iPS cells established by the present inventors. Reset Naive ESCs (in-house reset naive ESCs) were also present in the same group, demonstrating that their global gene expression was comparable to that of Reset Naive ESCs.

[0183] Using the same RNA-seq data as in Figure 7, we compared the expression patterns of imprinted genes and X-chromosome-linked genes in each cell type using SNP analysis (Figure 8). HDFs, primed iPSCs, and naive iPSCs were all derived from the HDFs of the same woman. In HDFs and primed iPSCs, imprinted genes, such as MEG3, generally showed expression patterns from only one allele, whereas in naive iPSCs, imprinting was lost and expression patterns from both alleles were observed. Furthermore, in the female-derived HDFs and primed iPSCs used in this study, X-chromosome-linked genes showed expression patterns from only one allele due to inactivation of one X chromosome, whereas in naive iPSCs, expression patterns from both alleles were observed due to X chromosome reactivation.

[0184] Using DNA obtained simultaneously with the RNA from the same cells used in the RNA-seq analysis in Figure 7, global methylation in HDF-derived and PBMC-derived naive iPS cells was analyzed using a methylation array, with density bean plots (Figure 9A) and principal component analysis (Figure 9B). Primed refers to primed iPS cells established by the applicant, and naive refers to naive iPS cells. In Figure 9A, the naive iPS cells obtained in this study tended to have lower beta values ​​(i.e., hypomethylated) compared to the primed iPS cells, and showed hypomethylation comparable to that of reset naive ESCs (in-house). In Figure 9B, the primed and naive iPS cells were clearly separated, demonstrating distinct methylation patterns. Furthermore, the proportion of 5-methylcytosine in total cytosines in the HDF-derived naive iPS cells obtained in this example was analyzed using LC-MS / MS (Figure 9C). Naive iPS cells showed a low ratio similar to that of reset naive ESCs, suggesting that the ratio of methylated cytosines in the genome was reduced.

[0185] The early differentiation potential of the three germ layers was compared and analyzed for two clones of naive iPS cells derived from HDFs after long-term passage (P16-45) used in the RNA-seq and methylation array, two clones of naive iPS cells derived from PBMCs, and reset naive ESCs (in-house) as a control (Figure 10). ES cells / iPS cells were plated in 96-well plates at 10 × 10 6The embryoid bodies were then harvested using differentiation medium (DMEM + 20% FBS) in a hypoxic (5% O2) environment for 24 days. RNA was extracted from the harvested samples and reverse-transcribed to obtain cDNA. The TaqMan hPSC Scorecard Panel (ThermoFisher Scientific) was used to semi-comprehensively assess the early differentiation potential of the three germ layers. The expression levels of pluripotency markers, ectodermal markers, mesodermal markers, and endodermal markers are shown from the left to the right of the figure. The gray plots represent reset naive ESCs, and the red plots represent naive iPS cells obtained using the present invention. All clones exhibited pluripotency comparable to reset naive ESCs or even better, particularly in the mesoderm.

[0186] Thus, it was confirmed that the naive induced pluripotent stem cells obtained by the method of the present application exhibited the same global gene expression pattern, extensive genome-wide DNA demethylation, X chromosome reactivation, and early differentiation ability of three germ layer components as the standard for naive induced pluripotent stem cells, even after long-term passaging of more than 180 days. Therefore, it was demonstrated that the method of the present application can rapidly produce true naive induced pluripotent stem cells.

[0187] Example 5: Development of a Modified OSKL-SEV Vector Reprogramming System. Naive human iPS cells (nCT2.0L_1-4) were established from human dermal fibroblasts (HDFs) using the 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). On day 94 after SeV transduction, the presence of residual SeV in the established naive human iPSCs was verified by immunostaining (Figure 11A). As shown in the upper right panel of Figure 11A, most cells were strongly positive, indicating high levels of SeV persistence. Cells were then harvested 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), SeV levels initially decreased with each passage in HDFs or human peripheral blood mononuclear cells (PBMCs), but then tended to increase again, so SeV did not disappear even with repeated passaging (Fig. 11B). Furthermore, SeV genome expression remained in all clones, and in some cell lines, expression levels were equal to or higher than those of the housekeeping gene GAPDH. Quantification of the expression levels of the three remaining SeV vector genomes revealed that the SeV-KOS and SeV-CMYC / LMYC vectors remained at particularly high levels in all clones, and the SeV-KLF4 vector remained at particularly high levels in more than half of the clones (Fig. 11C).

[0188] We investigated the reasons for the persistence of these vectors. Although SeV vectors share viral proteins during mixed infection, the SeV-TSΔF vector, which forms the backbone of the SeV-KLF4 / TS vector, is known to be highly temperature-insensitive, unlike the SeV-TS12ΔF or SeV-TS15ΔF vectors, and therefore 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. Therefore, we hypothesized that the presence of the SeV18+KLF4 / TSΔF (KLF4 / TS) vector in the reprogramming cocktail was the primary cause of persistence of all SeV vector genomes.

[0189] To test this hypothesis, we applied three modified SeV-KLF4 vectors as alternatives to the SeV-KLF4 / TS vector (Fig. 11D): SeV18+KLF4 / TS12ΔF (SeV-KLF4 / TS12), SeV18+KLF4 / PmiR367T2 / TSΔF (SeV-KLF4 / miR / TS), and SeV18+KLF4 / PmiR367T2 / TS12 (SeV-KLF4 / miR / TS12). The SeV-KLF4 / TS12 vector was constructed based on the SeV-TS12ΔF vector, which harbors multiple point mutations in the P gene to enhance temperature sensitivity (Fig. 11D). The SeV-KLF4 / miR / TS vector contains hsa-microRNA-367 (miR-367) target sequences (i.e., the miR-367 target sequence, not miR-367 itself) inserted in tandem at the 5' end of the P gene of the SeV vector genome (Fig. 11D). miR-367 has been shown to be specifically and abundantly expressed in primed human pluripotent stem cells (PSCs). We also confirmed that miR-367 is highly expressed in naive human PSCs compared with HDFs (Fig. 11E). Therefore, miR-367 acceptance is considered useful for rapid SeV vector clearance 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 clearance (Fig. 11D).

[0190] We compared the reprogramming efficiency of these vector combinations. It has been reported that when a temperature-sensitive SeV vector carrying the KLF4 gene was used to generate iPSCs from human fibroblasts, iPSC generation was not possible at 37°C except at high multiplicities of infection (presumably due to the loss of most of the vector before reprogramming was complete). Therefore, cells were cultured at 35°C from the time of infection until day 14, when sufficient naive iPSC colonies appeared, and then passaged for the first time. After the first passage, the culture temperature was raised to 38°C, and the SeV vector was removed (Figure 11F). Reprogramming of HDFs with SeV-KOS, SeV-LMYC, and modified SeV-KLF4 vectors resulted in the emergence of naive iPSC colonies at approximately day 10, consistent with the conventional method using the SeV-KLF4 / TS vector.

[0191] There have been no reports of the establishment of naive human iPSCs from human peripheral blood mononuclear cells (PBMCs), and in fact, the present inventors were unable to establish naive human iPSCs from PBMCs using CytoTune-iPS 2.0 (Figure 11G).

[0192] Example 6: Verification of SeV clearance rate and application to feeder-free naive iPSCs. The establishment efficiency of naive human iPS cells established from HDFs using each KLF4 vector was compared (Fig. 12A). None of the vectors showed a decrease in establishment efficiency compared to the conventional vector (SeV-KLF4 / TS). However, the establishment efficiency of the SeV-KLF4 / TS12 or SeV-KLF4 / miR / TS12-containing OSKL (OSKM with CMYC substituted with LMYC) cocktail was significantly improved compared to the conventional vector.

[0193] Next, we detected residual SeV vectors in naive iPSCs generated with these vectors. For the application of iPSC-derived differentiated cells to regenerative medicine, at least 1x10 5 The sensitivity required to detect a single undifferentiated cell resulting from the residual exogenous pluripotency gene from 1 x 10 cells is required. 6We confirmed that the method was sensitive enough to detect a single SeV-positive cell among 1,638,400 SeV-negative cells (Fig. 12B). This theoretically indicates that even a single SeV-positive cell can be detected among 1,638,400 SeV-negative cells, which is far more sensitive than methods such as immunostaining. This qRT-PCR analysis quantitatively assessed the residual SeV genome levels. After the temperature shift 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 decline in SeV-KLF4 / TS levels was very slow. In particular, SeV-KLF4 / miR / TS12 reached the detection limit of qRT-PCR on day 34, the fastest among the SeV-KLF4 vectors (Fig. 12C). These results suggest that the SeV-KLF4 / miR / TS12 vector is the most effective SeV-KLF4 vector in terms of reprogramming efficiency and vector clearance rate.

[0194] We further verified the reprogramming performance of the SeV-KLF4 / miR / TS12 vector. Using the SeV-KOS, SeV-LMYC, and SeV-KLF4 / miR / TS12 vectors, we successfully generated naive iPSC clones from HDFs (nOSKL_1, 2) as well as PBMCs (nOSKL_3, 4). By day 34, SeV expression was confirmed to have disappeared in all nOSKL-iPSC clones (Figure 12D). Interestingly, we successfully established and maintained feeder-free naive iPSCs from HDFs by using conditioned medium prepared by incubating t2iLGo medium with irradiated mouse embryonic fibroblasts (iMEFs) from the beginning of the reprogramming process (nOSKL_FF_1, 2) (Figure 12E). We then measured the cell proliferation rate of nOSKL_1-4. No significant decline in cell proliferation was observed in any of the clones even after repeated passages (Figure 12F).

[0195] Example 7: Gene Expression of Established Naive iPSCs Next, we examined the naive-specific pluripotency characteristics of the generated nOSKL-iPSCs. For comparison, we collected primed human iPSCs (OSKL_1-4), primed WA09 H9 embryonic stem cells (ESCs; H9), and primed 201B7 iPSCs (201B7), all derived from the same somatic cells as the nOSKL-iPSCs. We also collected reset naive H9 ESCs (nH9), which were reset from primed to naive by overexpressing KLF2 and NANOG and maintained under the same conditions as the nOSKL-iPSCs. The expression of naive iPSC-specific markers was examined by qRT-PCR (Figure 13A). While NANOG is expressed in primed iPSCs, it is known to be significantly increased in naive iPSCs. Both naive iPSC lines expressed significantly higher levels of naive iPSC-specific markers than primed iPSC lines. Similarly, the expression of primed iPSC-specific markers was verified by qRT-PCR (Figure 13B). Both naive iPSC lines expressed significantly lower levels of primed iPSC-specific markers than primed iPSC lines. RNA-seq was performed on these cell lines to compare the expression of representative pluripotency markers (Figure 13C). nOSKL-iPSCs showed nearly identical pluripotency marker expression to nH9.Next, we compared the transcriptome of nOSKL-iPSCs with those of primed PSCs and previously published datasets of reset naive PSCs and human inner cell mass (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) revealed that nOSKL-iPSCs were clearly distinct from primed PSCs and shared almost identical characteristics with nH9 (Figure 13D). The naive human iPSCs established by the present inventors were most similar to epiblasts.

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

[0197] X chromosome reactivation was verified by RNA-FISH (Figure 14C). HUWE1 is a gene expressed in the activated X chromosome. XIST is a lncRNA that normally acts to inactivate the X chromosome. However, in preimplantation epiblasts, which have characteristics similar to those of naive iPSCs, XIST is expressed in both activated X chromosomes, a seemingly contradictory finding (Figure 14C, leftmost panel). The gene expression pattern shifted toward primed iPSCs, as shown in the right panel of Figure 14C. Most primed iPSCs essentially exhibited the gene expression pattern shown in the rightmost panel of Figure 14C (HUWEI+ / -, XIST- / -). Furthermore, RNA-FISH was performed on established clones (Figure 14D). Although there was some variability between clones, nOSKL-iPSCs clearly exhibited a pattern similar to that of preimplantation epiblasts compared to primed PSCs. These observations confirmed the naive state of nOSKL-iPSCs.

[0198] Example 9: Evaluation of Mitochondrial Function of Established Naive iPSCs Based on the RNA-seq data from Example 7, we compared the expression of mitochondrial genes related to oxidative phosphorylation (Figure 15A). Naive iPSCs showed higher expression levels of mitochondrial genes than primed iPSCs, suggesting activated mitochondrial functions such as aerobic metabolism. Next, we compared the metabolic capacity of naive and primed iPSCs using an extracellular flux analyzer (Figure 15B). Naive iPSCs showed significantly elevated oxygen consumption rates (OCRs) after FCCP administration compared with primed iPSCs, suggesting superior spare respiratory capacity for naive iPSCs. The spare respiratory capacity, an index of aerobic metabolic capacity, was quantitatively compared between naive and primed iPSCs (Figure 15C). Similar to nH9, nOSKL-iPSCs showed higher values ​​compared with primed iPSCs. Furthermore, we examined the extracellular acidification rate (ECAR), an index of glycolytic metabolic function, and the OCR, an index of aerobic metabolic function (Fig. 15D). Naive iPSCs had higher ECAR and OCR than primed iPSCs, suggesting a high-energy metabolic state.

[0199] Example 10: Verification of the differentiation potential of established naive iPSCs into primitive endoderm. Naive PSCs resemble preimplantation epiblasts and have the potential to differentiate into extraembryonic tissues. This characteristic differs from primed PSCs. Primitive endoderm differentiation is rarely possible from primed iPSCs, but it is possible from naive iPSCs. Therefore, we investigated the ability of nOSKL-iPSCs to differentiate into naive PSC-derived primitive endoderm in a defined differentiation medium (Figure 16A). Light microscopic images 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 exhibited endodermal cell-like morphology. Next, we used flow cytometry to compare and verify the expression levels of primitive endoderm-specific markers, PDGFRA and ANPEP (Figure 16C). n9 and nOSKL-iPSCs highly expressed the primitive endoderm-specific markers PDGFRA and ANPEP, indicating good differentiation potential toward primitive endoderm. Primed PSCs and naive iPSCs established using the conventional SeV-KLF4 / TS vector (CytoTune-iPS 2.0L) with residual SeV expressed very low levels of PDGFRA and ANPEP, indicating significantly reduced differentiation potential toward primitive endoderm. The median signal intensity of PDGFRA / ANPEP co-positive cells was examined (Figure 16D). Primed iPSCs and naive iPSCs with residual SeV were located in the lower left corner of Figure 16D, whereas nH9 and naive iPSCs established using this method were located in the upper right corner, suggesting that naive iPSCs established using this method have excellent differentiation potential similar to nH9.

[0200] Example 11: Verification of the Differentiation Potential of Established Naive iPSCs into Trophectoderm. Trophectoderm (TE) differentiation from primed iPSCs is also largely impossible, but it is possible from naive iPSCs. We investigated the ability of nOSKL-iPSCs to differentiate into trophectoderm (TE) derived from naive PSCs in a defined differentiation medium (Figure 17A). Light microscopic images of primed ESCs and nOSKL-iPSCs on day 3 after the start of differentiation induction are shown in Figure 17B. The morphology of the two trophectoderm-specific markers on day 3 after the start of differentiation induction was clearly different. Next, we used flow cytometry to compare the expression levels of trophectoderm-specific markers TACSTD2, ENPEP, and HAVCR1, as well as the negative marker HLA-ABC (Figure 17C). n9 and nOSKL-iPSCs exhibited high expression of the TE-specific marker HAVCR1 and low expression of HLA-ABC, demonstrating good differentiation potential into TE. Primed PSCs did not express HAVCR1 at all, and nearly half were HLA-ABC positive, suggesting that they rarely differentiated into TEs. Naive iPSCs with residual SeV, established using the conventional SeV-KLF4 / TS vector (CytoTune-iPS 2.0L), expressed very low levels of TACSTD2, ENPEP, and HAVCR1, indicating their significantly reduced TE differentiation potential, similar to that of primed PSCs. The percentages of TACSTD2 / ENPEP co-positive and HAVCR1-positive cells were examined by flow cytometry (Fig. 17D). Primed iPSCs and naive iPSCs with residual SeV were distributed on the left side of Fig. 17D, whereas nH9 and naive iPSCs established by this method were distributed on the right side, suggesting that naive iPSCs established by this method have excellent differentiation potential similar to nH9. Interestingly, some nOSKL-iPSC clones expressed TACSTD2 and ENPEP at higher levels than nH9.

[0201] In summary, we developed a significantly faster SeV genome removal system for generating naive human iPSCs from somatic cells by improving the SeV-KLF4 vector configuration. As a result, we were able to obtain naive human iPSCs that exhibited robust transgene independence and superior naive-specific differentiation potential at early passages compared to naive iPSCs generated by conventional methods. Furthermore, we successfully reprogrammed PBMCs to naive human iPSCs by changing the combination of reprogramming factors to SeV-OSKL. We also established naive human iPSCs from skin fibroblasts in a feeder-free environment using iMEF-conditioned medium. These technological innovations will greatly advance early developmental research and clinical applications using naive human iPSCs.

Claims

1. A method for producing naive induced pluripotent stem cells from human somatic cells, comprising: (1) introducing into a human somatic cell one or more vectors containing reprogramming factors; (2) culturing the somatic cells in the presence of naive medium; and (3) After step (2), culturing the cells obtained 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 amount at the start of step (3). wherein the one or more vectors are temperature-sensitive vectors in a cell culture environment for establishing naive pluripotent stem cells.

2. The method of claim 1 , wherein the one or more vectors are negative-strand RNA viral vectors.

3. The method of claim 2 , wherein the one or more vectors are paramyxovirus vectors.

4. The method of claim 3 , wherein the one or more vectors are Sendai virus vectors.

5. The method according to any one of claims 1 to 4, wherein step (2) is initiated 1 to 10 days after step (1).

6. The method according to any one of claims 1 to 5, wherein step (2) is carried out for 1 to 20 days.

7. The method of any one of claims 1 to 6, wherein the one or more vectors are selected from Sendai virus vectors comprising TS12 mutations (TS 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) as well as D433A / R434A / K437A mutations in the P protein), TS7 mutations (Y942H / L1361C / L1558I mutations in the L protein as well as TS mutations), or TS15 mutations (D433A / R434A / K437A mutations in the P protein and L1361C / L1558I mutations in the L protein as well as TS mutations).

8. The method according to any one of claims 1 to 7, wherein the step (3) comprises culturing the obtained cells at 38°C or higher.

9. The method according to any one of claims 1 to 8, wherein the one or more vectors include a vector comprising a target sequence of a microRNA specific to an induced pluripotent stem cell.

10. The method of claim 9, wherein the target sequence of the microRNA is present in the translated region, 5'UTR, or 3'UTR of the NP gene or the P gene.

11. The method of claim 10, wherein the microRNA is miR-367.

12. The method according to any one of claims 1 to 11, wherein the reprogramming factors include an OCT gene, a SOX gene, a MYC gene and / or a KLF gene.

13. The method of claim 12, wherein the one or more vectors include a vector containing an OCT gene, a SOX gene and a KLF gene, a vector containing a MYC gene, and a vector containing a KLF gene.

14. The method according to claim 13, wherein the vector containing the MYC gene is a Sendai virus vector containing a TS15 mutation.

15. The method according to claim 13 or 14, 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 a TS12 mutation.

16. The method according to any one of claims 1 to 15, wherein in the step (3), the amount of the vector per somatic cell is reduced to 30% or less within 12 days from the start of step 3 compared to the amount at the start of step 3.

17. The method according to any one of claims 1 to 16, wherein the naive culture medium contains one or more compounds selected from LIF, a MEK inhibitor, a GSK3 inhibitor, a cAMP production promoter, a TGF-β inhibitor, and a PKC inhibitor.

18. 18. The method of claim 17, wherein the naive medium is a medium selected from the group consisting of t2iLGo, 5iLAF, and tt2iLGo.

19. The method according to any one of claims 1 to 18, wherein steps (2) and (3) are carried out in the absence of feeder cells.

20. The method according to any one of claims 1 to 19, wherein the human somatic cells are mononuclear cells or fibroblasts.

21. A kit for producing naive induced pluripotent stem cells from human somatic cells, comprising: One or more vectors containing reprogramming factors, wherein the one or more vectors are temperature-sensitive in a cell culture environment for establishing naive pluripotent stem cells; and Naïve medium.

22. The kit of claim 21 , wherein the one or more vectors are negative-strand RNA viral vectors.

23. The kit of claim 22, wherein the one or more vectors are paramyxovirus vectors.

24. 24. The kit of claim 23, wherein the one or more vectors are Sendai virus vectors.

25. The kit of claim 24, wherein the one or more vectors are selected from Sendai virus vectors comprising TS12 mutations (TS 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) plus D433A / R434A / K437A mutations in the P protein), TS7 mutations (Y942H / L1361C / L1558I mutations in the L protein in addition to TS mutations), or TS15 mutations (D433A / R434A / K437A mutations in the P protein and L1361C / L1558I mutations in the L protein in addition to TS mutations).

26. The kit of claim 25, wherein the one or more vectors include a vector comprising a target sequence of a microRNA specific for induced pluripotent stem cells.

27. The kit of claim 26, wherein the target sequence of the microRNA is present in the translated region, 5'UTR, or 3'UTR of the NP gene or the P gene.

28. The kit of claim 27, wherein the microRNA is miR-367.

29. The kit according to any one of claims 21 to 28, wherein the reprogramming factors include an OCT gene, a SOX gene, a MYC gene and / or a KLF gene.

30. 30. The kit of claim 29, wherein the one or more vectors comprise a vector comprising an OCT gene, a SOX gene and a KLF gene, a vector comprising a MYC gene, and a vector comprising a KLF gene.

31. The kit of claim 30, wherein the vector containing the MYC gene is a Sendai virus vector containing a TS15 mutation.

32. The kit according to claim 30 or 31, 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 a TS12 mutation.

33. The kit according to any one of claims 21 to 32, wherein the naive medium contains one or more compounds selected from LIF, a MEK inhibitor, a GSK3 inhibitor, a cAMP production promoter, a TGF-β inhibitor, and a PKC inhibitor.

34. The kit of claim 33, wherein the naive medium is a medium selected from the group consisting of t2iLGo, 5iLAF, and tt2iLGo.

35. The kit according to any one of claims 21 to 34, wherein the human somatic cells are mononuclear cells or fibroblasts.

36. A culture supernatant of naive induced pluripotent stem cells produced by the method according to any one of claims 1 to 20.

37. A cosmetic comprising as an ingredient a culture supernatant of naive induced pluripotent stem cells produced by the method according to any one of claims 1 to 20.