WO2010008054A1 - 染色体非組み込み型ウイルスベクターを用いてリプログラムされた細胞を製造する方法 - Google Patents
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Definitions
- the present invention relates to a method for producing a reprogrammed cell, a cell produced by the method, a composition used in the method, and the like.
- the present invention relates to a method for producing pluripotent stem cells from differentiated somatic cells and pluripotent stem cells prepared by the method.
- Embryonic stem cells are stem cells established from the inner cell mass of mammalian blastocysts and can be expanded indefinitely while maintaining the ability to differentiate into all cells (differentiation pluripotency). . Because of this characteristic, stem cell therapy is expected in which cardiomyocytes and nerve cells derived and prepared in large quantities from ES cells are transplanted and treated in patients with myocardial infarction or Parkinson's disease. It is also expected to be used as a development tool in basic research on pathology and pharmacology and drug discovery. However, this ES cell has an ethical problem of using and sacrificing human fertilized eggs. There is also the problem of immune rejection where the limited histocompatibility antigens of fertilized eggs do not match the patient.
- tissue stem cells such as neural stem cells, hematopoietic stem cells, and mesenchymal stem cells exist in each tissue of a living body. Since tissue stem cells do not use fertilized eggs, there are few or no ethical problems, and immune rejection can be avoided because the patient's own cells can be used. However, tissue stem cells are difficult to isolate because their properties are not always clear, and the number is very small. Proliferative ability and differentiation ability are also limited so that they cannot be compared with ES cells. If somatic cells such as tissue stem cells and differentiated cells can be converted into cells similar to ES cells having high proliferation ability and differentiation pluripotency (called ES-like cells) by some means, this ES-like cell is It is an ideal stem cell for clinical applications.
- somatic cells such as tissue stem cells and differentiated cells can be converted into cells similar to ES cells having high proliferation ability and differentiation pluripotency (called ES-like cells) by some means, this ES-like cell is It is an ideal stem cell for clinical applications.
- mammalian cells especially patient somatic cells (skin, stomach and lung tissues, blood cells, etc.) are collected and cultured, and these cells are used as nuclear reprogramming factors (nuclear early stage). Stimulated with a factor that induces nuclear reprogramming), sometimes called ES-like cells (“artificial pluripotent stem cells”, “induced pluripotent stem cells (iPS cells)” or “embryonic stem cell-like cells”) ).
- ES-like cells artificial pluripotent stem cells”, “induced pluripotent stem cells (iPS cells)” or “embryonic stem cell-like cells”.
- ES-like cells artificial pluripotent stem cells”, “induced pluripotent stem cells (iPS cells)” or “embryonic stem cell-like cells”. It is expected that the prepared product is stored as it is or as a cell bank and applied clinically as a stem cell, or used for basic research including pharmacology and pathology (Patent Document 1). It is also possible to conduct a drug eff
- nuclear reprogramming factors examples include Oct gene, Klf gene, Myc gene, Sox gene, Nanog gene, Lin28 gene, TERT gene, and SV40 LargeT gene (Patent Document 2, Non-Patent Document). References 1-7).
- Non-Patent Documents 1 to 7 Gamma retroviral vector or lentiviral vector containing Oct3 / 4 gene (hereinafter collectively referred to as “retroviral vector”) (2) Retroviral vector containing Klf4 gene (3) Retroviral vector containing c-Myc gene (4) Retroviral vector containing Sox2 gene
- the ES-like cells prepared using the retroviral vector have structurally modified chromosomes due to vector integration into the host chromosome.
- the cause is the use of retroviral vectors.
- the vector When using a retroviral vector, the vector is randomly integrated into the chromosome of the introduced cell, inactivating the tumor suppressor gene in the chromosome or activating the gene involved in canceration adjacent to the insertion site. (Experimental Medicine Vol.26 No.5 (extra number): pp.35-40, 2008).
- it when it is incorporated into another gene or a gene that modifies the expression of the gene, it may change into a cell having unexpected properties.
- non-encrypted regions of chromosomes have recently been assumed to have a certain chromosomal function, it is also necessary to consider undesirable results brought about by the incorporation of retroviral vectors into the non-encrypted regions.
- retroviral vectors when a vector is inserted into a gene that is involved in the differentiation of pluripotent stem cells, treatment and research using cells obtained by differentiating the stem cells will result in cells not being differentiated. There is also a possibility that it cannot be implemented because it is not possible.
- the cell reprogramming by the conventional method has a problem of safety in the treatment using the obtained ES-like cell, and also in the pharmacological effect and pathological analysis using the ES-like cell established from the patient, Consideration must be given to the inactivation and activation of genes originally functioning in cells due to the insertion of foreign genes into the cell, and the analysis is extremely difficult.
- retroviral vectors when retroviral vectors are used, even if they are the same researcher, for each lot, or even if different producers make the same protocol, the established ES-like cells are located at different locations in the chromosome. Since the vector is inserted, there is a problem that the uniformity of induced pluripotent stem cells cannot be guaranteed.
- the present invention has been made with the intention of fundamentally solving such a situation.
- An ES-like cell in which a foreign gene is not integrated into a chromosome, that is, a non-chromosomal non-recombinant ES cell can be easily and efficiently obtained.
- a method of manufacturing is provided.
- the present invention also provides a gene transfer composition useful for inducing reprogramming in the method.
- the present invention also provides pluripotent stem cells obtained by the method of the present invention.
- the present inventors have found that pluripotent stem cells in which a foreign gene is not integrated into a chromosome can be produced by using a non-chromosomal integration vector. That is, the present invention relates to a method for producing pluripotent stem cells using a non-chromosomal integration vector, and ES-like cells prepared by the method of the present invention, and more specifically, the inventions described in the respective claims.
- An invention composed of any combination of two or more of the inventions recited in the claims that refer to the same claim is also an invention intended in the present specification.
- the present invention [1] A method for introducing a gene in cell reprogramming, which comprises introducing the gene into a cell using a non-chromosomal viral vector, [2] The method according to [1], wherein the reprogramming is induction of pluripotent stem cells, [3] The method according to [1] or [2], wherein the non-chromosomal viral vector is an RNA viral vector, [4] The method according to [3], wherein the RNA viral vector is a minus-strand RNA viral vector, [5] The method according to [4], wherein the minus-strand RNA viral vector is a paramyxovirus vector, [6] The method according to [5], wherein the paramyxovirus vector is a Sendai virus vector, [7] The method according to any one of [1] to [6], wherein the gene is selected from the group consisting of the following (1) to (8): (1) Oct gene (2) Klf gene (3) Myc gene (4) Sox gene (5) Nanog gene (6) Lin,
- the present invention also provides [1] A method for producing a reprogrammed cell, comprising the step of bringing a differentiated cell into contact with at least one non-chromosomal viral vector; [2] The method according to [1], wherein the reprogrammed cell is an induced pluripotent stem cell, [3] The method according to [1] or [2], wherein the vector is at least one non-chromosomal viral vector carrying at least one gene encoding a nuclear reprogramming factor, [4] The method according to [3], wherein the gene is selected from the group consisting of the following (1) to (8): (1) Oct gene (2) Klf gene (3) Myc gene (4) Sox gene (5) Nanog gene (6) Lin28 gene (7) SV40 LargeT antigen gene (8) TERT gene [5] At least in the cell The vectors are combined so that three types of Oct gene, Klf gene and Sox gene, or at least four types of Oct gene, Sox gene, Nanog gene and Lin28 gene are expressed endogenously or exogen
- the present invention also provides [1] A non-chromosomal viral vector carrying a gene selected from the group consisting of the following (1) to (8): (1) Oct gene (2) Klf gene (3) Myc gene (4) Sox gene (5) Nanog gene (6) Lin28 gene (7) SV40 LargeT antigen gene (8) TERT gene [2]
- Non-chromosomal virus The vector according to [1], wherein the vector is an RNA virus vector, [3] The vector according to [2], wherein the RNA virus vector is a minus-strand RNA virus vector, [4] The vector according to [3], wherein the minus-strand RNA viral vector is a paramyxovirus vector, [5] The vector according to [4], wherein the paramyxovirus vector is a Sendai virus vector.
- the cells produced by the method according to the present invention are not only useful for testing and research using the cells, but also in the treatment of diseases, because the foreign gene is not integrated into the chromosome. It is expected to be able to avoid problems of immune rejection and ethical problems, and to avoid the risk of canceration based on genotoxicity, unexpected side effects due to changes in chromosomal function, and changes in cell properties. Furthermore, according to the method of the present invention, pluripotent stem cells can be induced from a desired cell type including adult skin cells with a significantly higher efficiency (for example, about 10 times) than conventional methods using retroviruses. Is possible.
- retroviruses are generally highly directional.
- the method of the present invention can be applied to a wide range of animal species (mammals in general). For example, it can also be applied to species having great demand as disease model animals such as monkeys and pigs.
- the control is a negative control without template DNA. It is the result of having detected the telomerase activity of the cell obtained by the method which concerns on this invention. It is the result which showed the pluripotency of the cell obtained by the method which concerns on this invention. The result of the embryoid body formation experiment was shown. It is the result which showed the pluripotency (invitro) of the cell obtained by the method which concerns on this invention.
- FIG. 5 shows the results of the pluripotency of cells obtained by the method according to the present invention.
- a various differentiated tissues
- b costal cartilage and secretory cells (black arrow)
- c rib tissue
- d costal secretory tissue (black arrow) and retina-like tissue differentiated from neuroepithelium (white arrow)
- e salmon transitional epithelial tissue (center)
- f Bone bone and myeloid tissue (white arrow)
- g Gastrointestinal tract-like tissue
- i Acupuncture myocardium-like tissue It is the result which showed the epigenetics of the cell obtained by the method which concerns on this invention.
- A The activation state of a human ES cell-specific promoter region was analyzed by the bisulfite sequencing method for Oct3 / 4 and Nanog, respectively.
- the activated demethylated region is indicated by a white circle, and the methylated region is indicated by a black circle.
- SeV-iPS clones HNL1 and HNLs derived from parental human neonatal foreskin cells BJ and SeV-iPS clone 7H5 derived from human adult skin cell HDF were analyzed. In both SeV-iPS cells, activation of the relevant promoter was observed in both regions.
- BJ-derived clones: HNLs, HNL1-6, HNLp Decreased expression of the introduced foreign gene in was measured over time by RT-PCR using primers recognizing the sequence of the vector part (P represents the number of passages) ). As passage progressed, the phenomenon of 4 introduction initialization factors decreasing to 3 and 2 was recognized.
- Alkaline phosphatase (ALP) positive ES-like cell colonies were obtained.
- B iPS cell induction by 4 factors of Thomson (Oct3 / 4, Sox2, Nanog, Lin28 ⁇ F / TS / SeV). Other than Yamanaka's 4 factors (Oct3 / 4, Sox2, Klf4, c-Myc) (left panel), Thomson's 4 factors (Oct3 / 4, Sox2, Nanog, Lin28 ⁇ F / TS / SeV) (right panel) Cells were induced.
- the present invention provides a method for inducing reprogramming of differentiated cells using a non-chromosomal viral vector, particularly a method for producing pluripotent stem cells from somatic cells.
- This method includes, for example, a step of bringing a non-chromosomal viral vector carrying a gene encoding a nuclear reprogramming factor (s) to be introduced into contact with a differentiated cell such as a somatic cell.
- the present invention relates to a method for introducing a gene in reprogramming of a cell, a method for introducing the gene into a cell in need thereof using a non-chromosomal viral vector, and therefore A non-chromosomal non-integrating viral vector is provided.
- the pluripotent stem cell refers to a stem cell made from an inner cell mass of an embryo at the blastocyst stage of an animal or a cell having a phenotype similar to that.
- the pluripotent stem cell induced in the present invention is a cell that expresses alkaline phosphatase, which is an indicator of ES-like cells.
- the pluripotent stem cell forms a flat colony composed of cells having a higher nucleus ratio than the cytoplasm by culturing. The culture may be performed with a feeder as appropriate.
- pluripotent stem cells can be passaged for a long time, for example, 15 times or more, preferably 20 times or more every 3 days. It can be confirmed that the growth is not lost even after passage 25 times or more, 30 times or more, 35 times or more, or 40 times or more.
- the pluripotent stem cells preferably express endogenous Oct3 / 4 or Nanog, more preferably both.
- the pluripotent stem cell preferably expresses TERT and exhibits telomerase activity (activity for synthesizing telomeric repeat sequences).
- the pluripotent stem cells preferably have the ability to differentiate into three germ layers (endoderm, mesoderm, ectoderm) (for example in teratoma formation and / or embryoid body formation). More preferably, pluripotent stem cells generate germline chimeras by transplanting into blastocysts. A pluripotent stem cell capable of Germline transmission is called a germline-competent pluripotent stem cell. Confirmation of these phenotypes can be performed by a well-known method (WO2007 / 69666; Ichisaka T et al., Nature 448 (7151): 313-7,) 2007).
- the term “differentiated” means, for example, that the cells are differentiated more than pluripotent stem cells, the state that still has the ability to differentiate into a plurality of cell lineages (eg, somatic stem cells), and the terminally differentiated cells. Includes state.
- Differentiated cells are cells derived from pluripotent stem cells (other than pluripotent stem cells). Differentiated cells may have no ability to differentiate into, for example, three germ layers (endoderm, mesoderm, ectoderm). Such cells do not have the ability to form three germ layers unless reprogrammed.
- the differentiated cell may be a cell that cannot generate cells other than the germ layer type to which it belongs, for example. Differentiated cells may be somatic cells, for example, cells other than germ cells.
- reprogramming means that a differentiated state of a cell is changed to an undifferentiated state, for example, that a differentiated cell is dedifferentiated, for example, from a cell having no differentiation pluripotency. Inducing cells possessed, such as pluripotent stem cells.
- dedifferentiation refers to making a certain cell immature (for example, undifferentiated). Dedifferentiation may mean returning a cell to its initial or developing state. In addition, dedifferentiation may mean that a cell that cannot generate cells other than the germ layer type to which it belongs can be differentiated into cells of other germ layers. Dedifferentiation includes, for example, that cells that do not have the ability to differentiate from three germ layers acquire the ability to differentiate from three germ layers. Dedifferentiation also includes the generation of pluripotent stem cells.
- somatic cells are cells other than pluripotent stem cells, for example.
- Somatic cells include, for example, cells other than pluripotent stem cells among cells constituting multicellular organisms, and cultured cells thereof.
- Somatic cells include, for example, somatic stem cells and terminally differentiated cells.
- a viral vector is a vector that has a genomic nucleic acid derived from the virus and can express the gene by incorporating a transgene into the nucleic acid.
- a non-chromosomal viral vector for producing pluripotent stem cells is a viral vector derived from a virus and capable of introducing a gene into a target cell, wherein the introduced gene is a host.
- a carrier that has no danger of being incorporated into the chromosome nuclear-derived chromosome.
- the viral vector is a complex composed of a virus core, a complex of a virus genome and a virus protein, or a non-infectious virus particle in addition to an infectious virus particle, and is loaded by introduction into a cell.
- a complex capable of expressing the gene to be expressed for example, in an RNA virus, a ribonucleoprotein (virus core portion) comprising a viral genome and a viral protein that binds to it can be introduced into the cell to express the transgene in the cell (WO00 / 70055). The introduction into the cells may be appropriately performed using a transfection reagent or the like.
- ribonucleoprotein (RNP) is also included in the viral vector in the present invention.
- “there is no risk of integration into the host chromosome” means that the frequency of integration into the host chromosome is sufficiently low when a viral vector is introduced.
- the frequency of integration into the chromosome of the host is, for example, 5 ⁇ 10 ⁇ 4 or less when the human fibrosarcoma-derived cell line HT1080 (ATCC CCL121) is infected at 10 PFU / cell, more preferably 10 ⁇ 4. Or less, more preferably 10 ⁇ 5 or less, more preferably 10 ⁇ 6 or less, and even more preferably 10 ⁇ 7 or less.
- the non-integrating viral vector used in the present invention is particularly preferably an RNA virus.
- RNA virus refers to a virus having an RNA genome and having no DNA phase in its life cycle.
- RNA viruses do not have reverse transcriptase (ie, do not include retroviruses). That is, in virus propagation, the viral genome is replicated by RNA-dependent RNA polymerase without DNA. Since RNA viruses do not have a DNA phase, the risk of integration into the host chromosome can be minimized by using RNA virus vectors.
- RNA viruses include single stranded RNA viruses (including positive and negative stranded RNA viruses) and double stranded RNA viruses.
- RNA viruses specifically include viruses belonging to the following families.
- Arenaviridae such as Lassa virus Orthomyxoviridae, such as influenza virus Coronaviridae such as SARS virus Togaviridae such as rubella virus Paramyxoviridae such as mumps virus, measles virus, Sendai virus, RS virus Picornaviridae such as Poliovirus, Coxsackie virus, Echovirus Filoviridae, such as Marburg virus and Ebola virus Flaviviridae such as yellow fever virus, dengue virus, hepatitis C virus, hepatitis G virus Bunyaviridae (including Bunyaviridae; Bunyavirus, Hantavirus, Nairovirus, and Phlebovirus genera, etc.) Rhabdoviridae such as rabies virus Reoviridae
- the non-chromosomal non-integrated virus vector used in the present invention is, for example, a minus-strand RNA virus vector.
- a minus-strand RNA viral vector is a vector consisting of a virus containing, as a genome, a minus-strand (antisense strand against a sense strand encoding a viral protein) RNA. Negative strand RNA is also called negative strand RNA. Examples of minus-strand RNA viruses listed as examples in the present invention include single-strand minus-strand RNA viruses (also referred to as non-segmented minus-strand RNA viruses).
- Single-stranded negative strand RNA virus refers to a virus having a single-stranded negative strand [ie, minus strand] RNA in the genome.
- viruses include paramyxovirus (including Paramyxoviridae; Paramyxovirus, Morbillivirus, Rubulavirus, and Pneumovirus), rhabdoviridae; Vesiculovirus, Lyssavirus, Lyssavirus, and Ephemerovirus etc.
- minus-strand RNA viral vectors listed as examples in the present invention include paramyxovirus vectors.
- Paramyxovirus vectors are viral vectors derived from the Paramyxoviridae virus.
- the Sendai virus of Paramyxoviridae virus can be mentioned.
- Newcastle disease virus (Newcastle disease virus), mumps virus (Mumps virus), measles virus (Measles virus), RS virus (Respiratory syncytial virus), rinderpest virus, distemper virus (distemper virus) , Simian parainfluenza virus (SV5), human parainfluenza virus types 1,2,3, orthomyxoviridae influenza virus (Influenza virus), rhabdoviridae vesicular stomatitis virus (Vesicular stomatitis virus) ), And rabies virus (Rabies virus).
- Sendai virus SeV
- HPIV-1 human parainfluenza virus-1
- HPIV-3 human parainfluenza virus-3
- PDV canine
- 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
- melesles virus MV
- rinderpest virus RSV
- Hendra virus Hendra
- the vector used in the present invention is, for example, a virus belonging to the Paramyxovirus subfamily (including the Respirovirus genus, Rubravirus genus, and Morbillivirus genus) or a derivative thereof, such as the Respirovirus genus (genus Respirovirus). ) (Also referred to as Paramyxovirus) or a derivative thereof.
- Derivatives include viruses in which viral genes have been modified, chemically modified viruses, and the like so as not to impair the ability to introduce genes by viruses.
- respirovirus viruses to which the present invention can be applied examples include human parainfluenza virus type 1 (HPIV-1), human parainfluenza virus type 3 (HPIV-3), and bovine parainfluenza virus type 3 (BPIV-3).
- HPIV-1 human parainfluenza virus type 1
- HPIV-3 human parainfluenza virus type 3
- BPIV-3 bovine parainfluenza virus type 3
- Sendai virus also referred to as mouse murine parainfluenza virus type 1
- SPIV-10 simian parainfluenza virus type 10
- the minus-strand RNA virus listed as an example in the present invention includes Sendai virus.
- the genome of the wild type Sendai virus follows the 3 'short leader region, followed by the nucleocapsid (N) gene, phospho (P) gene, matrix (M) gene, fusion (F) gene, hemagglutinin-neuraminidase (HN) gene, And the large (L) gene and the short 5 'trailer region in this order.
- N nucleocapsid
- P phospho
- M matrix
- F fusion
- HN hemagglutinin-neuraminidase
- L large gene and the short 5 'trailer region in this order.
- Production of recombinant vectors corresponding to wild-type viruses and various mutant vectors is already known.
- the non-chromosomal integration virus in the present invention may be derived from natural strains, wild strains, mutant strains, laboratory passage strains, artificially constructed strains, and the like. That is, as long as the target reprogramming can be induced, the virus may be a virus vector having the same structure as a virus isolated from nature, or a virus artificially modified by genetic recombination. . For example, any gene possessed by the wild-type virus may be mutated or defective. It is also possible to use incomplete viruses such as DI particles (J. Virol. 68: 8413-8417, 1994). For example, a virus having a mutation or deletion in at least one gene encoding a viral envelope protein or outer shell protein can be preferably used.
- Such a viral vector is, for example, a viral vector that can replicate the genome in infected cells but cannot form infectious viral particles.
- a replication-defective virus vector is highly safe because there is no concern of spreading infection around it.
- minus-strand RNA viruses that do not contain at least one gene encoding an envelope protein or spike protein such as F, H, HN, or G, or a combination thereof can be used (WO00 / 70055 and WO00 / 70070; Li, H.-O. et al., J. Virol. 74 (14) 6564-65692000 (2000)).
- proteins necessary for genome replication for example, N, P, and L proteins
- the genome can be amplified in infected cells.
- a defective gene product or a protein capable of complementing it is supplied exogenously in virus-producing cells (WO00 / 70055 and WO00 / 70070; Li, H.-O. et al., J. Virol. 74 (14) 6654-6569 (2000)).
- a method of recovering a viral vector as a non-infectious viral particle (VLP) without completely complementing a defective viral protein is also known (WO00 / 70070).
- VLP non-infectious viral particle
- the vector can be produced without complementing the envelope protein.
- the present invention provides a method for gene transfer in reprogramming and a method for producing reprogrammed cells, particularly using an RNA viral vector having a mutation and / or deletion in a viral gene.
- an RNA viral vector having a mutation and / or deletion in a viral gene For example, many mutations including an attenuation mutation and a temperature-sensitive mutation are known in envelope proteins and outer shell proteins.
- An RNA virus having these mutant protein genes can be preferably used in the present invention.
- LDH lactate dehydrogenase
- a vector whose cytotoxicity is significantly attenuated compared to the wild type can be used.
- the degree of attenuation of cytotoxicity is, for example, the amount of LDH released in the culture medium in which HeLa (ATCC CCL-2) or monkey CV-1 (ATCC CCL 70) is infected with MOI 3 and cultured for 3 days is wild type.
- a significantly reduced vector such as 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 50% or more can be used.
- mutations that reduce cytotoxicity include temperature-sensitive mutations.
- a temperature-sensitive mutation is a mutation whose activity is significantly reduced at a normal temperature (eg, 37 ° C.
- a virus vector having a temperature-sensitive mutation useful in the present invention has a growth rate or gene expression level of at least 1/2 or less when infected at 37 ° C. compared to, for example, infection at 30 ° C. in cultured cells. It is preferably 1/3 or less, more preferably 1/5 or less, more preferably 1/10 or less, and more preferably 1/20 or less.
- the non-chromosomal viral vector used in the present invention may be wild-type as long as it does not inhibit reprogramming and can induce reprogramming by a reprogramming factor, and is preferably at least 1, more preferably at least 2, 3 Has deletions or mutations in 4, 5, or more viral genes. Deletions and mutations may be introduced in any combination for each gene.
- the mutation may be a reduced function mutation or a temperature-sensitive mutation, and at least at 37 ° C., the virus growth rate or the expression level of the loaded gene is preferably 1/2 or less, more preferably, compared to the wild type.
- the mutation is reduced to 1/3 or less, more preferably 1/5 or less, more preferably 1/10 or less, more preferably 1/20 or less.
- modified viral vectors can be particularly important for the induction of pluripotent stem cells.
- at least two viral genes are deleted or mutated.
- Such viruses have at least two viral genes deleted, at least two viral genes mutated, at least one viral gene mutated and at least one viral gene deleted Is included.
- the at least two viral genes that are mutated or deleted are preferably genes that encode envelope-constituting proteins.
- a vector having the F gene deleted, the M or HN (or H) gene further deleted, and the remaining M and / or HN (or H) gene further having a mutation (for example, a temperature-sensitive mutation) It is suitably used in the invention.
- at least three viral genes are deleted or mutated.
- Such viral vectors have at least 3 genes deleted, at least 3 genes mutated, at least 1 gene mutated and at least 2 genes deleted And those in which at least two genes are mutated and at least one gene is deleted.
- a vector that lacks the F gene and further deletes the M and HN (or H) genes, or further has a mutation (for example, a temperature-sensitive mutation) in the M and HN (or H) genes. is preferably used in the present invention.
- a vector having the F gene deleted, the M or HN (or H) gene further deleted, and the remaining M or HN (or H) gene further having a mutation (for example, a temperature sensitive mutation) is suitable in the present invention. Used for.
- Such a mutant virus can be prepared according to a known method.
- the temperature-sensitive mutation of the M gene of the minus-strand RNA virus is a site arbitrarily selected from the group consisting of positions 69 (G69), 116 (T116), and 183 (A183) in the Sendai virus M protein.
- amino acid substitutions at homologous sites of other minus-strand RNA virus M proteins can be mentioned (Inoue, M. et al., J.Virol. 2003, 77: 3238-3246).
- the amino acid at the homologous site of other minus-strand RNA virus M protein can be easily identified.
- human parainfluenza virus-1 HPIV-1 (the parentheses are abbreviations) G69, human parainfluenza virus-3 (HPIV-3) G73, phocine distemper virus (PDV) and canine distemper virus (CDV) G70, dolphin molbillivirus ( DM71), G71 for peste-des-petits-ruminants virus (PDPR), melesles virus (MV), and rinderpest virus (RPV), G70 for Hendra virus (Hendra) and Nipah virus (Nipah) G81, human parainfluenza virus-2 (HPIV-2) G70, human parainfluenza virus-4a (HPIV-4a) and human parainfluenza virus-4b (HPIV-4b) E47, mumps virus (Mumps) E72 (letters
- homologous sites of each M protein corresponding to T116 of SeV M protein are T116 for human parainfluenza virus-1 (HPIV-1), T120 for humanphoparainfluenza virus-3 (HPIV-3), phocine T104 for distemper virus (PDV) and canine distemper virus (CDV), T105 for dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MV) and rinderpest virus (RPV) T104, Hendra virus (Hendra) and Nipah virus (Nipah) T120, human parainfluenza virus-2 (HPIV-2) and siman parainfluenza virus 5 (SV5) T117, human parainfluenza virus-4a ( T121 for HPIV-4a) and human parainfluenza virus-4b (HPIV-4b), T119 for mumps virus (Mumps), and S120 for Newcastle disease virus (NDV).
- HPIV-1 human parainfluenza virus-1
- HPIV-3
- the homologous sites of each M protein corresponding to A183 of SeV M protein are A183 for human parainfluenza virus-1 (HPIV-1), F187 for human parainfluenza virus-3 (HPIV-3), phocine distemper virus (PDV) and canine distemper virus (CDV) Y171, dolphin molbillivirus (DMV) Y172, peste-des-petits-ruminants virus (PDPR), measles virus (MV) and rinderpest virus (RPV) Y171, Y187 for Hendra virus (Hendra) and Nipah virus (Nipah), Y184 for human parainfluenza virus-2 (HPIV-2), F184 for siman parainfluenza virus 5 (SV5), human parainfluenza virus- F188 for 4a 4 (HPIV-4a) and human parainfluenza virus-4b (HPIV-4b), F186 for mumps virus (Mumps), and Y187 for Newcastle disease virus (NDV).
- HPIV-1 human para
- each M protein is a mutant M protein in which any one of the above three sites, preferably a combination of any two sites, and more preferably all amino acids in all three sites are replaced with other amino acids.
- a virus having a genome encoding is preferably used in the present invention.
- the amino acid mutation is preferably substitution with another amino acid having a different side chain chemistry, for example, BLOSUM62 matrix (Henikoff, S. and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) is substituted with an amino acid having a value of 3 or less, preferably 2 or less, more preferably 1 or less, more preferably 0 or less.
- BLOSUM62 matrix Henikoff, S. and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919
- an amino acid having a value of 3 or less preferably 2 or less, more preferably 1 or less, more preferably 0 or less.
- the homologous sites of Sendai virus M protein G69, T116, and A183 or other viral M proteins can be replaced with Glu (E), Ala (A), and Ser (S), respectively.
- HN a site arbitrarily selected from the group consisting of positions 262 (A262), 264 (G264), and 461 (K461) of the HN protein of Sendai virus
- a virus having a genome encoding a mutant HN protein in which any one of the three sites, preferably a combination of any two sites, more preferably amino acids in all three sites are substituted with other amino acids, is used in the present invention. Preferably used.
- the substitution of amino acids is preferably substitution with other amino acids having different side chain chemical properties.
- the homologous sites of Sendai virus ⁇ HN protein A262, G264, and K461 or other viral HN proteins are replaced with Thr (T), Arg (R), and Gly (G) ⁇ ⁇ ⁇ ⁇ , respectively.
- mutations can be introduced into amino acids 464 and 468 of the HN protein with reference to the mumps virus temperature-sensitive vaccine strain Urabe AM9 (Wright, K. E. et al., Virus Res. 2000: 67). ; 49-57).
- the minus-strand RNA virus may have a mutation in the P gene and / or L gene.
- mutations include the 86th Glu (E86) mutation of the SeV P protein, the substitution of the 511st Leu (L511) of the SeV P protein with other amino acids, or other negative strands.
- Examples include substitution of homologous sites of RNA virus P protein.
- the substitution of amino acids is preferably substitution with other amino acids having different side chain chemical properties. Specific examples include substitution of the 86th amino acid with Lys and substitution of the 511st amino acid with Phe.
- the substitution of an amino acid is preferably a substitution with another amino acid having a different side chain chemical property.
- Specific examples include substitution of the 1197th amino acid with Ser and substitution of the 1795th amino acid with Glu.
- 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, by combining mutations and / or deletions in the envelope protein gene, these effects can be dramatically increased.
- the L gene includes substitution of the 1214th Tyr (Y1214) and / or 1602 Met (M1602) of the SeV L protein with other amino acids, or substitution of homologous sites of other minus-strand RNA virus L proteins.
- the substitution of an amino acid is preferably a substitution with another amino acid having a different side chain chemical property. Specific examples include substitution of amino acid 1214 with Phe, substitution of amino acid 1602 with Leu, and the like. The mutations exemplified above can be arbitrarily combined.
- At least 69-position G of SeV ⁇ M protein, 116-position T, 183-position A, SeV HN protein at least 262-position A, 264-position G, 461-position K, and SeV P protein at least 511.
- Each homologous protein has a substitution mutation at the homologous site, an F gene deletion or deletion vector in which the F gene is deleted or deleted, and cytotoxicity is the same as or less than these, and / or temperature sensitivity.
- F gene-deficient or deleted minus-strand RNA viral vectors similar to or more than these are particularly suitable for expressing nuclear reprogramming factors in the present invention.
- specific substitutions include, for example, substitution of G69E, T116A, and A183S for the M protein, substitution of A262T, G264, and K461G for the HN protein, substitution of L511F for the P protein, and L As for proteins, N1197S and K1795E substitutions can be mentioned.
- Genes encoding nuclear reprogramming factors can be arranged, for example, at the most upstream (3 ′ side) of the minus-strand RNA genome (for example, 3 ′ side of N gene).
- the Myc gene may be arranged at other positions, for example, behind the minus-strand RNA genome, that is, on the 5 ′ side. For example, it can be inserted between the HN gene and the L gene.
- the substitution of the amino acid at the site arbitrarily selected from positions 942 (Y942), 1361 (L1361), and 1558 (L1558) of the SeV L protein, or other The substitution of the homologous site of the minus-strand RNA virus L protein is also included.
- the substitution of amino acids is preferably substitution with other amino acids having different side chain chemical properties. Specific examples include substitution of the 942nd amino acid with His, substitution of the 1361st amino acid with Cys, substitution of the 1558th amino acid with Ile, and the like.
- L protein substituted at least at positions 942 or 1558 can be used preferably.
- a mutant L protein in which the 1361 position is substituted with another amino acid in addition to the 1558 position is also suitable.
- a mutant L protein in which positions 1558 and / or 1361 are substituted with other amino acids is also suitable.
- These mutations can increase the temperature sensitivity of the L protein.
- part arbitrarily chosen from 433 position (D433), 434 position (R434), and 437 position (K437) of SeV P protein, or other Examples include substitution of a homologous site of a minus-strand RNA virus P protein.
- the substitution of amino acids is preferably substitution with other amino acids having different side chain chemical properties.
- a P protein in which all of these three sites are substituted can be preferably used. These mutations can increase the temperature sensitivity of P protein.
- Mutant P protein in which at least 433-position D, 434-position R, and 437-position K in SeV P protein were substituted with other amino acids, and L in at least 1558-position in SeV L protein were substituted A homologous site in a Sendai virus vector that lacks or deletes the F gene, and that encodes a mutant L protein (preferably a mutant L protein in which at least L at position 1361 is also substituted with another amino acid), and other minus-strand RNA viruses Mutated F gene-deficient or deleted vectors, and minus-strand RNA viruses that lack or delete F genes with similar or less cytotoxicity and / or temperature sensitivity Vectors are also preferably used in the present invention.
- Each viral protein may have a mutation in other amino acids (for example, within 10, within 5, within 4, within 3, within 2, or 1 amino acid) in addition to the above mutation.
- the vectors having the mutations shown above are highly temperature sensitive, after reprogramming is completed, the cells are cultured at a slightly high temperature (eg, 37.5 to 39 ° C, preferably 38 to 39 ° C, or 38.5 to 39 ° C). Thus, the vector can be easily removed.
- Nuclear reprogramming factors can be appropriately inserted at an appropriate position in the genome. For example, the nuclear reprogramming factors are inserted into the uppermost stream (3 ′ side) of the genome (for example, 3 ′ side of the NP gene).
- the Myc gene is, for example, 5 ′ end from the center of the genome of the minus-strand RNA virus (5 ′ end from the middle gene), for example, 5 ′ side or 3 ′ side of the L gene, especially 3 ′ side of the L gene (For example, between HN and L) may be inserted.
- the cytotoxicity of the vector can be measured, for example, by quantifying the release of lactate dehydrogenase (LDH) from the cells. Specifically, for example, the amount of LDH released in a culture solution obtained by infecting HeLa (ATCC CCL-2) or monkey CV-1 (ATCC CCL 70) with MOI 3 and culturing for 3 days is measured. The smaller the amount of LDH released, the lower the cytotoxicity.
- the temperature sensitivity can be determined by measuring the virus growth rate or the expression level of the loaded gene at the normal temperature of the virus host (eg, 37 ° C. to 38 ° C.). It is judged that the temperature sensitivity is higher as the growth rate of the virus and / or the expression level of the loaded gene are lower than those without the mutation.
- a virus containing a protein different from the envelope protein inherent in the virus may be used.
- a virus containing this can be produced by expressing a desired foreign envelope protein in a virus-producing cell during virus production.
- Proteins such as a desired adhesion factor, a ligand, and a receptor which provide the infectious ability to a mammalian cell, are used.
- Specific examples include G protein (VSV-G) of vesicular stomatitis virus (VSV).
- VSV-G protein may be derived from any VSV strain.
- a VSV-G protein derived from a Indiana serotype strain J. Virology 39: 519-528 (1981)
- the minus-strand RNA viruses listed as examples in the present invention can contain any combination of envelope proteins derived from other viruses.
- RNA viruses having nuclear reprogramming factors may be performed using known methods.
- the minus-strand RNA virus listed as an example of the present invention typically, (a) a cDNA encoding a minus-strand RNA virus genomic RNA (minus strand) or its complementary strand (plus strand) is used. , A step of transcription in cells expressing viral proteins (N, P, and L) necessary for virus particle formation, and (b) a step of recovering the culture supernatant containing the produced virus.
- Viral proteins necessary for particle formation may be expressed from transcribed viral genomic RNA or supplied to trans from other than genomic RNA.
- expression plasmids encoding N, P, and L proteins can be introduced into cells and supplied. If the genomic RNA lacks a viral gene necessary for particle formation, the viral gene is separately expressed in virus-producing cells to complement particle formation.
- a vector in which DNA encoding the protein or genomic RNA is linked downstream of an appropriate promoter that functions in the host cell is introduced into the host cell. Transcribed genomic RNA is replicated in the presence of viral proteins to form infectious viral particles.
- the defective virus or other viral protein capable of complementing its function is expressed in the virus-producing cell.
- production of the minus-strand RNA virus as an example of the present invention can be carried out using the following known methods (WO97 / 16539; WO97 / 16538; WO00 / 70055; WO00 / 70070; WO01 / 18223 ; WO03 / 025570; WO2005 / 071092; WO2006 / 137517; WO2007 / 083644; WO2008 / 007581; Hasan, M. K. et al., J. Gen. Virol. 78: 2813-2820, 1997, Kato, A. et al., 1997, EMBO J. 16: 578-587 and Yu, D.
- RNA viruses including parainfluenza, vesicular stomatitis virus, rabies virus, measles virus, Linder pest virus, Sendai virus and the like can be reconstituted from DNA.
- Examples of the method for producing a plus (+) strand RNA virus include the following examples. 1) Coronavirus Enjuanes L, Sola I, Alonso S, Escors D, Zuniga S. Coronavirus reverse genetics and development of vectors for gene expression. Curr Top Microbiol Immunol. 2005; 287: 161-97. Review. 2) Toga virus Yamanaka R, Zullo SA, Ramsey J, Onodera M, Tanaka R, Blaese M, Xanthopoulos KG. Induction of therapeutic antitumor antiangiogenesis by intratumoral injection of genetically engineered endostatin-producing Semliki Forest virus. Cancer Gene Ther. 2001 Oct; 8 (10): 796-802.
- RNA virus propagation methods and recombinant virus production methods refer to Virology Experimental Studies, 2nd revised edition (edited by National Institute of Preventive Health, Alumni Association, Maruzen, 1982).
- the above non-chromosomal viral vector can be appropriately loaded with a gene for cell reprogramming.
- the gene to be mounted may be a desired gene involved in induction of various stem cells such as pluripotent stem cells from differentiated cells.
- genes that are essential for reprogramming or genes that increase the efficiency of reprogramming can be mounted. That is, the present invention uses the non-chromosomal viral vector of the present invention for introducing a gene in cell reprogramming, and for expressing a reprogramming factor in a cell and inducing reprogramming of the cell. Provide use.
- the present invention also includes an agent for introducing a gene in cell reprogramming (introduction agent, gene introduction agent) and an agent for expressing a reprogramming factor in a cell, including the non-chromosomal viral vector of the present invention.
- the present invention also relates to an agent for expressing a reprogramming factor in a cell and inducing reprogramming of the cell, comprising the non-chromosomal viral vector of the present invention.
- the vector of the present invention is also useful for expressing a desired gene in a cell when performing nuclear reprogramming of the cell.
- Non-integrating viral vectors carrying a gene encoding one or more nuclear reprogramming factors can be used for cell reprogramming in accordance with the present invention.
- the present invention can be used for medical and non-medical applications and is useful in medical and non-medical embodiments.
- the present invention can be used for therapeutic, surgical, and / or diagnostic, or non-therapeutic, non-surgical, and / or non-diagnostic purposes.
- a nuclear reprogramming factor is a gene used to induce a differentiation state of a cell to a more undifferentiated state, or a product thereof, alone or in combination with a plurality of factors.
- a gene or product thereof used to induce dedifferentiation of differentiated cells the nuclear reprogramming factor includes a factor essential for nuclear reprogramming and an auxiliary factor (cofactor) that increases the efficiency of nuclear reprogramming.
- a desired gene for use in nuclear reprogramming may be mounted on a vector.
- a gene for use in the production of pluripotent stem cells can be mounted.
- nuclear reprogramming factors for inducing pluripotent stem cells are expressed in, for example, ES cells and early embryos, but not expressed in many differentiated somatic cells or expressed. Can be used, such as ES cell-specific genes. Such a gene is preferably a gene encoding a transcription factor, a nuclear protein or the like. Methods for identifying nuclear reprogramming genes (nuclear reprogramming factors) are already known (WO2005 / 80598). In fact, genes identified using this method are useful for reprogramming into pluripotent stem cells Is shown (WO2007 / 69666).
- genes include DPPA5 (developmental pluripotency associated 5, ES cell specific gene 1 (ESG1); accession numbers NM_001025290, NM_025274, XM_236761), F-box protein 15 (Fbx15, NM_152676, NM_798 NM_024865, AB093574), ECAT1 (ES cell associated transcript 1; AB211062, AB211060), ERAS (ES cell expressed Ras; NM_181532, NM_181548), DNMT3L (DNA (cytosine-5-)-methyltransferase 3-like; NM_013369, NM_194348, 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 pl4 (NM_)-
- a non-chromosomal viral vector carrying any of these genes is useful for use in the induction of cell dedifferentiation in the present invention, and is particularly suitable for the induction of pluripotent stem cells.
- RNA viral vector carrying any of these genes, such as an RNA viral vector
- Each of these genes may be incorporated into another vector, or a plurality of genes may be integrated into one vector.
- each gene may be incorporated into one type of vector, or different types of vectors (including chromosomally integrated viral vectors and / or non-viral vectors) may be used in combination with non-chromosomally integrated viral vectors. .
- kits or compositions containing the vectors also can be used for cell reprogramming, particularly for pluripotent stem cells. It can use suitably in manufacture.
- the vector may be appropriately mixed in sterilized water, pH buffer solution, physiological saline, culture solution or the like. In these systems, a part or most of the nuclear reprogramming gene can be replaced with a protein that is an expression product thereof.
- composition and kit of the present invention include at least one non-chromosomal viral vector, another vector (chromosomal viral vector and / or non-viral vector) that expresses the reprogramming factor and / or reprogramming. It may contain compounds, proteins, etc. Factors necessary for reprogramming may be expressed from all non-chromosomal viral vectors, or only a part may be expressed from non-chromosomal viral vectors, and others may be expressed by other vectors and / or compounds (for example, You may supply by protein and a low molecular weight compound).
- the method for producing a reprogrammed cell of the present invention is not limited to a method in which all gene transfer is performed using a non-chromosomal viral vector. That is, the method of the present invention may use at least one non-chromosomal viral vector, and induces other vectors (chromosomal viral vectors and / or non-viral vectors) that express reprogramming factors and / or reprogramming. It is included to use a compound to be used in combination.
- the present invention relates to a composition for use in cell reprogramming, comprising a non-chromosomal viral vector as an expression vector.
- the present invention also relates to the use of a non-chromosomal viral vector for reprogramming differentiated cells.
- the present invention provides the use of a non-chromosomal viral vector for introducing a gene into a cell in need of cell reprogramming.
- the present invention also relates to a method for introducing a gene in reprogramming of a cell, wherein the gene is introduced into a cell in need thereof using a non-chromosomal viral vector.
- the present invention also includes a composition for use in gene transfer in cell reprogramming, including a non-chromosomal viral vector, and an agent for use in gene transfer in cell reprogramming (used for gene transfer in cell reprogramming). And a gene transfer agent for cell reprogramming).
- the present invention also relates to the use of a non-chromosomal viral vector for use in the manufacture of a medicament for introducing a gene into a cell in need of cell reprogramming.
- the present invention also provides a gene introduction agent (gene expression agent or expression vector) for use in cell reprogramming, including a non-chromosomal viral vector.
- the present invention also provides a reprogramming gene introduction agent (gene expression agent or expression vector) containing a non-chromosomal viral vector.
- the present invention also includes a nuclear reprogramming factor expression agent (a nuclear reprogramming gene, an a nuclear nuclear reprogramming gene, a nuclear nuclear reprogramming gene, and a non-chromosomal viral vector. Expression vector).
- the present invention also provides a pluripotent stem cell inducing agent and a pluripotent stem cell inducing agent comprising a non-chromosomal viral vector encoding a nuclear reprogramming factor.
- the present invention also provides the use of a non-chromosomal viral vector for reprogramming of differentiated cells.
- the invention also provides the use of a non-chromosomally integrated viral vector in the manufacture of a medicament, reagent and / or medicament for reprogramming differentiated cells.
- the present invention also relates to the use of a non-chromosomal integration-type viral vector in the production of a nuclear reprogramming factor introduction agent for differentiated cells.
- reprogramming may be, for example, induction of pluripotent stem cells from differentiated cells.
- the vector is used by incorporating a gene encoding a factor for reprogramming.
- Examples of the gene encoding the reprogramming factor include a gene encoding any of the factors described above and below.
- the factor to be introduced may be appropriately selected according to the origin of the cell to be reprogrammed, and may be derived from other mammals, for example, from primates such as mice, rats, rabbits, pigs, monkeys, etc. It may be.
- the gene and protein sequences do not necessarily have to be wild-type sequences, and may have any mutation as long as reprogramming can be induced.
- an example of producing pluripotent stem cells using a mutant gene is known (WO2007 / 69666).
- amino acids eg, several, within 3, within 5, within 10, within 15, within 20, within 25
- a gene encoding an amino acid sequence and capable of inducing reprogramming can be used in the present invention.
- the biological activity for example, 1 to several residues (for example, 2, 3, 4, 5, 6, 10, 15 or 20 at the N-terminus and / or C-terminus)
- a polypeptide in which one or more residues (for example, 2, 3, 4, 5, 6, 10, 15 or 20 residues) are substituted, etc. Can also be used.
- Variants that can be used include, for example, fragments of natural proteins, analogs, derivatives, and fusion proteins with other polypeptides (eg, those added with heterologous signal peptides or antibody fragments). Specifically, it includes a sequence in which one or more amino acids of the wild-type amino acid sequence are substituted, deleted, and / or added, and has a biological activity equivalent to that of the wild-type protein (eg, activity that induces reprogramming). ). When a wild-type protein fragment is used, it is usually 70% or more, preferably 80% or more, 85% or more, more preferably 90% or more, 95% of the wild-type polypeptide (in the case of a secreted protein, the mature form). % Or more than 98% continuous area.
- Amino acid sequence variants can be prepared, for example, by introducing mutations into DNA encoding the natural polypeptide (Walker and Gaastra, eds. Techniques in Molecular Biology (MacMillan Publishing Company, New York, k1983); 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 Press, Plainview, NY), 1989; US Pat. No. 4,873,192).
- Guidance for substitution of amino acids so as not to affect biological activity includes, for example, Dayhoff et al. (Dayhoffhet al., In Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,. Washington, ingtonDC ), 1978).
- the number of amino acids to be modified is not particularly limited, but for example, within 30%, preferably within 25%, more preferably within 20%, more preferably within 15%, more preferably within the total amino acids of a natural mature polypeptide. It is within 10%, within 5%, or within 3%, for example within 15 amino acids, preferably within 10 amino acids, more preferably within 8 amino acids, more preferably within 5 amino acids, more preferably within 3 amino acids.
- substituting an amino acid it can be expected to maintain the activity of the protein by substituting an amino acid having a similar side chain property. Such substitution is referred to as conservative substitution in the present invention.
- Conservative substitutions include, for example, basic amino acids (eg, lysine, arginine, histidine), acidic amino acids (eg, aspartic acid, glutamic acid), uncharged polar amino acids (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non- Polar amino acids (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched amino acids (eg threonine, valine, isoleucine), and aromatic amino acids (eg tyrosine, phenylalanine, tryptophan, histidine) Examples include substitution between amino acids in the group.
- basic amino acids eg, lysine, arginine, histidine
- acidic amino acids eg, aspartic acid, glutamic acid
- uncharged polar amino acids eg,
- the modified protein shows high homology with the amino acid sequence of the wild type protein.
- High homology is, for example, an amino acid sequence having 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, S. F. et al., J. Mol. Biol. 215: 403-410, 1990). For example, on the BLAST web page of NCBI (National Center ch Biothchnology Information), search can be performed using default parameters (Altschul SF et al., Nature Genet.
- blast2sequences program (Tatiana A et al., FEMS Microbiol Lett. 174: 247-250, 1999) that compares two sequences can create an alignment of the two sequences and determine the identity of the sequences. Gaps are treated in the same way as mismatches, and for example, the identity value for the entire amino acid sequence of a natural cytokine (the mature form after secretion) is calculated. Specifically, the ratio of the number of matching amino acids in the total number of amino acids of wild type protein cocoon (or mature type in the case of secreted protein) cocoon is calculated.
- silent mutations can be introduced into genes so as not to change the encoded amino acid sequence.
- AT ⁇ ⁇ ⁇ rich genes by substituting 5 or more consecutive A or T bases with G or C so as not to change the encoded amino acid sequence, stable high expression of the gene can be obtained. Can do.
- the modified protein or the protein used for reprogramming is a protein encoded by a nucleic acid that hybridizes under stringent conditions with part or all of the coding region of the gene encoding the wild type protein, and is equivalent to the wild type protein.
- examples thereof include proteins having activity (activity for inducing reprogramming).
- a probe is prepared from either a nucleic acid containing a sequence of the coding region of a wild-type protein gene or a complementary sequence thereof, or a nucleic acid to be hybridized, and whether it hybridizes to the other nucleic acid. Can be identified by detecting.
- stringent hybridization conditions are: 5xSSC, 7% (W / V) SDS, 100 micro-g / ml denatured salmon sperm DNA, 5x Denhardt solution (1x Denhardt solution is 0.2% polyvinylpyrrolidone, 0.2% bovine serum In a solution containing albumin and 0.2% Ficoll), preferably at 60 ° C., preferably at 65 ° C., more preferably at 68 ° C., and then in 2 ⁇ SSC, preferably in 1 ⁇ SSC at the same temperature as the hybridization. Is a condition of washing in 0.5xSSC, more preferably 0.1xSSC for 2 hours with shaking.
- Examples of particularly preferred genes 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 called POU5F1; NM_002701, NM_203289, NM_013633, NM_001009178), Sox2 (NM_003106, NM_011443, XM_574919), TCL1A (T-cell leukemia / lyNM 1966 -like factor 4; NM_004235, NM_010637), catenin ⁇ 1 (cadherin-associated protein beta 1; NM_001904, NM_007614; S33Y mutant included), and c-Myc (NM_002467, NM_010849; T58
- the non-chromosomal viral vector carrying any of these is useful for use in the induction of cell reprogramming in the present invention, and can be particularly suitably used for the induction of pluripotent stem cells.
- Individual viral vectors can be used in combination at the time of use. Moreover, it is good also as a kit collectively, or it is good also as a composition by mixing. Also included in the present invention are one or more non-chromosomal viral vectors containing any combination (or all) of these genes, and kits or compositions containing the vectors.
- one of the particularly preferred gene combinations for the induction of pluripotent stem cells is a combination comprising at least four genes, Sox gene, KLF gene, Myc gene, and Oct gene (Takahashi, K. and Yamanaka S ., Cell 126, 663-676, 2006; Lowry WE et al., Proc Natl Acad Sci U S A, 105 (8): 2883-8, 2008; Masaki, H. et al., Stem Cell Res. 1: 105-115, 2008; WO2007 / 69666).
- the Sox protein, the KLF protein, the Myc protein, and the Oct protein and their genes refer to member proteins and genes belonging to the Sox family, the KLF family, the Myc family, and the Oct family, respectively. It has been reported that pluripotent stem cells can be induced from various differentiated cells by adjusting one or more of these four family members to be expressed. For example, with regard to the Sox family of genes, it has been reported that pluripotent stem cells can be induced using any of the genes Sox1, Sox2, Sox3, Sox15, and Sox17 (WO2007 / 69666). As for the KLF family, pluripotent stem cells could be induced by either KLF4 or KLF2 (WO2007 / 69666).
- pluripotent stem cells could be induced not only by wild type c-Myc but also by T58A mutant, N-Myc, and L-Myc (WO2007 / 69666; Blelloch R. et al., Cell Stem Cell , 1: 245-247, 2007).
- T58A mutant N-Myc
- L-Myc L-Myc
- wild-type c-Myc was found to have less expression from RNA virus vectors such as Sendai virus vectors.
- RNA virus vectors such as Sendai virus vectors.
- the gene can be stably expressed at a high level.
- the modified c-Myc gene shown in SEQ ID NO: 45 can be preferably used.
- a desired site can be selected for the insertion position of the gene in the vector.
- the Myc gene is located behind the minus-strand RNA genome (5 'side), that is, in the multiple protein coding sequences placed on the genome, at a position earlier counted from the 5' side than from the 3 'side. You may do (refer an Example).
- the Myc gene can be arranged, for example, on the most 5 ′ side (that is, the first from the 5 ′ side) or the second or third from the 5 ′ side.
- the Myc gene may be arranged between the second gene from the 5 ′ side of the genome, specifically, the L gene at the 5 ′ most side of the genome and the HN gene next to it.
- the Myc gene can be substituted with a continuous A or T base sequence by appropriately introducing a silent mutation so as not to change the encoded amino acid sequence.
- the minus-strand RNA viral vector in which the Myc gene is arranged at the rear (5 ′ side) of the minus-strand RNA genome can be used in combination with a minus-strand RNA viral vector encoding another nuclear reprogramming factor.
- these nuclear reprogramming factors are placed forward (3 ′ side), ie, on the genome, in the minus-strand RNA genome of each vector.
- it can be arranged at a position earlier when counted from the 3 ′ side than from the 5 ′ side.
- a gene encoding a nuclear reprogramming factor other than Myc is the first or second from the 5 ′ side of the genome in each minus-strand RNA viral vector, more preferably Arranged first.
- a gene encoding a nuclear reprogramming factor can be arranged at the 3 ′ end of the 3 ′ end of the NP gene in the genome.
- cells from which the vector has been removed can be selected as appropriate.
- a cell from which the vector has been naturally removed may be selected.
- negative selection can be performed with an antibody specific to a viral vector (for example, an anti-HN antibody).
- a temperature sensitive vector the vector can be easily removed by culturing at a high temperature (eg, 37.5 to 39 ° C., preferably 38 to 39 ° C., or 38.5 to 39 ° C.).
- the KLF family includes Klf1 (NM_006563, NM_010635), Klf2 (NM_016270, NM_008452), Klf4 (NM_004235, NM_010637), Klf5 (NM_001730, NM_009769), and the Myc family includes c-Myc ( NM_002467, NM_010849, T58A mutant included), N-Myc (NM_005378, NM_008709), L-Myc (NM_005376, NM_005806) are included, and Oct1A (NM_002697, NM_198934), Oct3 / 4 (NM_002701, NM_203289, NM_013633, _NM_001009178), Oct6 (NM_002699, NM_011141), and the Sox family includes Sox1 (NM_005986, NM_009233), Sox2 (NM_003106, NM_01
- Myc family genes are not essential for induction of pluripotent stem cells, and pluripotent stem cells can be induced by only three family genes except Myc family genes (Nakagawa M. et al., Nat Biotechnol. 26 ( 1): 101-6, 2008; Wering M. et al., Cell Stem Cell 2 (1): 10-2, 2008; Example 5).
- Myc gene is not expressed, for example, p53 ⁇ siRNA and UTF1 can significantly increase the induction efficiency of pluripotent stem cells (Y. Zhao et al., Cell Stem Cell, 3 (5): 475-479, 2008; N. Maherali, and K. Hochedlinger, Cell Stem Cell, 3 (6): 595-605, 2008).
- pluripotent stem cells can be induced only by genes of three families excluding genes of KLF family (Park IH et al., Nature, 451 (7175): 141-6, 2008).
- KLF family Park IH et al., Nature, 451 (7175): 141-6, 2008.
- Klf gene Klf gene
- Sox gene Sox gene
- fetal-derived NPC a G9a histone methyltransferase inhibitor
- pluripotent stem cells can be induced by only three genes of Myc gene (Shi Y et al., Cell Stem Cell, 2 (6): 525-8, 2008).
- any of the Sox gene, KLF gene, and Oct gene, or any of the Sox gene, Myc gene, and Oct gene, or a combination of the Sox gene, Myc gene, and Klf gene are particularly useful for use in the induction of cell reprogramming in the present invention, and can be suitably used for the induction of pluripotent stem cells.
- Viral vectors encoding each gene may be prepared separately as a single unit. They can be used in combination at the time of use. Arbitrary combinations or all may be combined into a kit, or may be mixed to form a composition.
- the invention also relates to one or more non-chromosomal viral vectors containing any combination (or all) of these genes, and kits or compositions for reprogramming containing the vectors. Furthermore, some of the recombinant vectors included in this kit can be replaced with proteins, synthetic compounds, and the like having considerable functions.
- neural progenitor cells express endogenous Sox family genes, so that pluripotent stem cells could be induced only by introducing Oct3 / 4 and Klf4 (Shi Y et al., Cell Stem Cell, 2 (6): 525-8, 2008).
- pluripotent stem cells from mouse embryonic fibroblasts (MEF) using 3 genes of Oct4, Sox2, Esrrb (estrogen-related receptor beta, NM_004452.2, NP_004443.2, NM_011934.3, NP_036064.2) It has been reported that Esrrb ⁇ can complement the function of Klf (Feng, B. et al., Nat Cell Biol. 11 (2): 197-203, 2009).
- pluripotent stem cells can be induced from embryonic fibroblasts only by introducing Oct3 / 4 and Klf4 (Shi Y et al ., Cell Stem Cell, 3 (5): 568-574, 2008).
- Oct3 / 4 and Klf4 Sort al ., Cell Stem Cell, 3 (5): 568-574, 2008.
- NSCs neural stem cells
- pluripotent stem cells can be induced (Kim, JB et al., Nature, doi: 10.1038 / nature07061; Published online 29 June 2008; Nature. 2008, 454 (7204): 646-50). Also, by adjusting the culture period, pluripotent stem cells can be induced with only Oct4 (Jeong Beom Kim et al., Cell, 136 (3): 411-419, 2009). As needed, only non-chromosomal viral vectors encoding reprogramming factors may be used. In addition, if endogenous expression of the endogenous reprogramming factor is induced by expression of another gene or compound treatment, etc., it can be combined with the introduction of a vector that expresses the other gene or compound treatment.
- a non-chromosomal viral vector encoding a reprogramming factor that cannot be induced may be introduced.
- Combining vectors so that they are expressed endogenously or exogenously means, for example, that a reprogramming factor is not only endogenously expressed in the natural state but also introduces a vector or compound that expresses other genes,
- a reprogramming factor is not only endogenously expressed in the natural state but also introduces a vector or compound that expresses other genes
- combinations containing 4 genes, Oct gene, Sox gene, NANOG gene (NM_024865, AB093574), and LIN28 gene (NM_024674), also induce pluripotent stem cells.
- NM_024865, AB093574 NANOG gene
- LIN28 gene NM_024674
- a combination of the Myc gene and the KLF gene with these genes is also suitable (Liao J et al., Cell Res. 18 (5): 600-3, 2008).
- a non-chromosomal viral vector carrying any of these genes is particularly useful for use in inducing cell dedifferentiation in the present invention, and can be suitably used for inducing pluripotent stem cells.
- One or more non-chromosomal viral vectors containing any combination (or all) of these genes, and kits or compositions containing the vectors, can also be used for cell reprogramming, particularly for the production of pluripotent stem cells. Can be suitably used.
- a vector for expressing the genes may or may not be introduced.
- some of the recombinant vectors included in this kit can be replaced with proteins, synthetic compounds, and the like having considerable functions.
- genes include TERT (NM_198253, NM_009354) and / or SV40 large T antigen (NC_001669.1, Fiers, W. (05-11-1978) Nature 273: (5658) 113-120) ( Park IH. Et al., Nature, 451 (7175): 141-6, 2008).
- TERT NM_198253, NM_009354
- One or more genes selected from the group consisting of HPV16 E6, HPV16 E7, and Bmil NM_005180, NM_007552
- Fbx15 Mol Cell Biol.
- ECAT1 (AB211062, AB211060), DPPA5 (NM_001025290, NM_025274, XM_236761), DNMT3L (NM_013369, NM_019448), ECAT8 (AB211063, AB211061), GDF3 (NM_020634, NM_008108) _0 NM_028610), FTHL17 (NM_031894, NM_031261), SALL4 (NM_020436, NM_175303), Rex-1 (NM_174900, NM_009556), Utf1 (NM_003577, NM_009482), DPPA3 (NM_199286, NM_139218, STAT3, One or more genes selected from the group consisting of GRB2 (NM_002086, NM_008163) may be combined.
- pluripotent stem cells By additionally expressing these genes, induction of pluripotent stem cells can be promoted (WO2007 / 69666).
- the myeloid transcription factor C / EBP ⁇ CCAAT / enhancer-binding-protein ⁇
- the B cell transcription factor Pax5 paired box 5
- NM_016734 B cell transcription factor
- these factors can also be expressed using the non-chromosomal viral vector of the present invention.
- some of the recombinant vectors included in this kit can be replaced with proteins, synthetic compounds, and the like having considerable functions.
- the efficiency of reprogramming can be improved by combining, for example, addition of a compound.
- bFGF basic fibroblast growth factor
- SCF stem cell factor
- a compound for example, bFGF (basic fibroblast growth factor) and / or SCF (stem cell factor) can promote the induction of pluripotent stem cells, and can further replace the function of c-Myc in the induction of pluripotent stem cells (WO2007) / 69666).
- a MAP kinase inhibitor (PD98056) is also useful for establishing pluripotent stem cells closer to ES cells (WO2007 / 69666).
- DNA methylase (Dnmt) inhibitors and / or histone deacetylase (HDAC) inhibitors improve the induction efficiency of pluripotent stem cells (Huangfu D et al., Nat Biotechnol. (Published online: 22 June 2008, doi: 10.1038 / nbt1418); Nat. Biotechnol. 26, 795-797 (2008)).
- HDAC histone deacetylase
- pluripotent stem cells can be induced by introduction of only two genes, Oct4 and Sox2 (Huangfu, D. et al., Nat Biotechnol. 2008 26 (11): 1269 -75).
- the vector of the present invention is useful as an agent for expressing these genes or partial genes thereof.
- Dnmt inhibitor for example, 5-azacytidine and the like
- HDAC inhibitor for example, suberolanilide hydrozamic acid (SAHA), trichostatin A (TSA), valproic acid (VPA) and the like are useful.
- SAHA suberolanilide hydrozamic acid
- TSA trichostatin A
- VPA valproic acid
- the efficiency can be increased by using glucocorticoid (dexamethasone) in combination.
- the above-described vector or the like is introduced into the cell.
- the introduction is preferably performed simultaneously. Specifically, within 48 hours, preferably within 36 hours, more preferably after the first vector or compound is added. All vectors and / or encoding reprogramming factors within 24 hours, 18 hours, 12 hours, 10 hours, 8 hours, 6 hours, 3 hours, 2 hours, or 1 hour It is preferred to complete the addition of the compound.
- the dose of the vector can be appropriately adjusted, but preferably MOI 0.3-100, more preferably MOI 0.5-50, more preferably MOI 1-30, more preferably MOI 1-10, more preferably MOI 1-5 More preferably, the MOI is infected at about 3.
- the induced pluripotent stem cells form a flat colony similar to ES cells and express alkaline phosphatase.
- the induced pluripotent stem cells may express undifferentiated cell markers such as Nanog, Oct4, and / or Sox2.
- the induced pluripotent stem cells preferably express TERT and / or exhibit telomerase activity.
- the present invention relates to a method for producing a cell that expresses alkaline phosphatase, preferably further expressing the undifferentiated cell markers Nanog and / or TERT, and non-chromosomal integration in the production of the cell and in the manufacture of a drug that induces the cell It also relates to the use of type virus vectors.
- a colony of pluripotent stem cells from desired cells including adult skin cells and neonatal foreskin cells for example 0.3 ⁇ 10 ⁇ 5 or more, 0.5 ⁇ 10 ⁇ 5 or more, 0.8 ⁇ 10 ⁇ 5 or more, or Appearance rate of 1 ⁇ 10 -5 or higher (eg 1.7 ⁇ 10 -5 to 2.4 ⁇ 10 -3 ), preferably 1.5 ⁇ 10 -5 or higher, 1.7 ⁇ 10 -5 or higher, 2.0 ⁇ 10 -5 or higher, 2.5 ⁇ 10 -5 or more, 3 x 10 -5 or more, 4 x 10 -5 or more, 5 x 10 -5 or more, 8 x 10 -5 or more, 1 x 10 -4 or more, 2 x 10 -4 or more, 3 x 10 -4 or more, 5 x 10 -4 or more, 8 x 10 -4 or more, 1 x 10 -3 or more, 1.5 x 10 -3 or more, 2 x 10 -3 or more, or 2.3 x
- somatic cells there are no particular limitations on the differentiated cells that are the targets for inducing reprogramming, and desired somatic cells can be used. It has been shown that the generation of pluripotent stem cells from somatic cells is possible not only from cells derived from mouse embryos but also from differentiated cells collected from the tail of adult mice, hepatocytes and gastric mucosa cells This suggests that it does not depend on cell type or differentiation state (WO2007 / 069666; Aoi T. et al., Science [Published Online February 14, 2008]; Science. 2008; 321 (5889): 699-702) .
- pluripotent stem cells are not dependent on the underlying cells.
- the method of the present invention can be applied.
- differentiated cells to be reprogrammed include fibroblasts, synovial cells, mucosal cells such as the oral cavity or stomach, hepatocytes, bone marrow cells, tooth germ cells, and other desired cells. It is.
- the cells may also be derived, for example, from embryonic, fetal, neonatal, child, adult or elderly cells.
- the origin of the animal is not particularly limited, and includes humans and non-human primates (such as monkeys), rodents such as mice and rats, and mammals including non-rodents such as cows, pigs and goats, etc. It is.
- the cells produced by the method of the present invention are useful for differentiating into various tissues and cells, and can be used in desired tests, research, diagnosis, examinations, treatments, and the like.
- induced stem cells are expected to be used in stem cell therapy.
- reprogramming is induced using somatic cells collected from a patient, and then somatic stem cells and other somatic cells obtained by inducing differentiation can be transplanted into the patient.
- the method of inducing cell differentiation is not particularly limited, and differentiation can be induced by, for example, retinoic acid treatment, various growth factors / cytokine treatments, or hormone treatments.
- the obtained cells can be used for detecting the effect of a desired drug or compound, and through this, screening of the drug or compound can be performed.
- SeV18 + / TS ⁇ F refers to mutations of G69E, T116A, and A183S in the M protein, mutations of A262T, G264, and K461G in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein.
- This is a F gene-deficient Sendai virus vector having the expression (WO2003 / 025570).
- This vector has a transgene insertion site (NotI site) upstream of the NP gene (3 ′ side of genome; also referred to as “18 + position”).
- cell lysis buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgCl 2 , 0.65% NP-40) was added, pipetted, and suspended by vortex. Centrifugation was performed at 6000 rpm for 3 minutes, the supernatant was transferred to another 1.5 ml Eppendorf tube, and 200 ⁇ l of extraction buffer was added. After fully suspending with vortex, 400 ⁇ l of phenol / chloroform / isoamyl alcohol (25: 24: 1) was added, and further well suspended with vortex. Centrifugation was performed at 15000 rpm and 4 ° C.
- the supernatant was transferred to another 1.5 ml Eppendorf tube, 400 ⁇ l of isopropanol was added, and the suspension was sufficiently suspended by vortexing and then cooled at ⁇ 20 ° C. for 30 minutes. Centrifugation was performed at 15000 rpm, 4 ° C. for 15 minutes, the supernatant was removed, 1 ml of 70% ethanol was added to the precipitate, suspended by vortexing, and then centrifuged at 15000 rpm, 4 ° C. for 5 minutes. After removing the supernatant and drying at room temperature, it was dissolved in 100 ⁇ l of nuclease-free water to obtain a total RNA solution of Jurkat cells.
- Oct3 / 4 gene from human embryonic cancer cell NCCIT cells (ATCC number CRL-2073; Damjanov I, et al., Lab. Invest. 1993, 68 (2): 220-32)
- Total RNA was recovered by the same method as described above.
- CDNA was synthesized from the recovered total RNA using SuperScript III Reverse Transcriptase (Invitrogen catalog number 18080-044). 1 ⁇ g of total RNA was made up to 100 ⁇ g of random hexamer, 1 ⁇ l of 10 mM dNTP mixed solution and 13 ⁇ l with nuclease-free water. The mixture was heat-treated at 65 ° C. for 5 minutes and placed on ice for 1 minute to cool. Next, add 4 ⁇ l of 5xFirst-Strand Buffer, 1 ⁇ l of 0.1M DTT, 1 ⁇ l of RNaseOUT and 1 ⁇ l of Superscript III RT, mix by pipetting, spin down, 25 ° C for 5 minutes, 50 ° C for 60 minutes, 70 ° C. was reacted for 15 minutes. 180 ⁇ l of TE (pH 8.0) was added to obtain a cDNA library.
- TE pH 8.0
- This PCR product was diluted 100-fold with TE, and 1 ⁇ l was diluted with c-Myc-F (5′-GATGCCCCTCAACGTTAGCTTCACC-3 ′ (SEQ ID NO: 3)) and c-Myc-R (5′-GTTACGCACAAGAGTTCCGTAGCTG-3 ′ (PCR was performed using the primer of SEQ ID NO: 4)).
- PCR products were separated by 1% agarose gel electrophoresis, and a band of about 1.3 kbp was excised and purified with Qiaquick Gel Extraction Kit (QIAGEN, Cat. No. 28706).
- the clone was cloned into the SwaI site of pCAGGS-BSX (WO2005 / 071092), sequenced to select a clone with the correct sequence, and pCAGGS-BSX-c-Myc was obtained.
- NotI-c-Myc F (5'-ATTGCGGCCGCATGCCCCTCAACGTTAGCTTCAC-3 '(SEQ ID NO: 5)
- NotI-c-Myc R 5'-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACTCGTGTTAGTGTGTGTTTC PCR was carried out with the primer '(SEQ ID NO: 6)).
- the PCR product was purified using Qiaquick PCR Purification kit (Qiagen Cat. No. 28106), followed by Not I digestion (3 hours at 37 ° C.). Purify using Qiaquick PCR Purification kit (Qiagen, Catalog No. 28106), clone to Not I site of bluescript plasmid vector, confirm gene sequence by sequencing, select clone with correct sequence and select pBS-KS-c- Got Myc. pBS-KS-c-Myc was digested with Not I (3 hours at 37 ° C.), separated by 1% agarose gel electrophoresis, excised band of approximately 1.5 kbp, Qiaquick Gel Extraction Kit (Qiagen, Catalog No. 28706) ).
- the Not I fragment containing this c-Myc gene was cloned into the Not I site of the pSeV18 + / TS ⁇ F vector encoding the antigenome of the Sendai virus vector (SeV18 + / TS ⁇ F), and the correct sequence clone was selected by sequencing, and pSeV18 + c-Myc / TS ⁇ F was obtained.
- This PCR product was diluted 100-fold with TE, and 1 ⁇ l was diluted with Sox2-F (5′-GATGTACAACATGATGGAGACGGAGC-3 ′ (SEQ ID NO: 9)) and Sox2-R (5′-GTCACATGTGTGAGAGGGGCAGTG-3 ′ (SEQ ID NO: PCR was performed using the primers of 10)).
- Sox2-F 5′-GATGTACAACATGATGGAGACGGAGC-3 ′
- Sox2-R 5′-GTCACATGTGTGAGAGGGGCAGTG-3 ′
- the clone was cloned into the SwaI site of pCAGGS-BSX and sequenced to select a clone with the correct sequence to obtain pCAGGS-BSX-SOX2.
- Not I Sox-2F (5'-ATTGCGGCCGCATGTACAACATGATGGAGACG-3 '(SEQ ID NO: 11)
- Not I Sox-2R 5'-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCACATGTCGCGTGTTGTCTGCATGTCGTG : PCR was performed with the primers of 12)).
- the PCR product was purified using Qiaquick PCR Purification kit (Qiagen Cat. No. 28106), followed by Not I digestion (3 hours at 37 ° C.). Purify using the Qiaquick PCR Purification kit (Qiagen, Cat.No. 28106), clone it into the Not I site of the bluescript plasmid vector, confirm the gene sequence by sequencing, select the correct clone and select pBS-KS-Sox2. Obtained. pBS-KS-Sox2 digested with Not I (3 hours at 37 ° C), separated by 1% agarose gel electrophoresis, excised band of approximately 1k bp, purified with Qiaquick Gel Extraction Kit (Qiagen, catalog number 28706) did.
- the Not I fragment containing the Sox2 gene was cloned into the Not I site of the pSeV18 + / TS ⁇ F vector, and a clone with the correct sequence was selected by sequencing to obtain pSeV18 + Sox2 / TS ⁇ F.
- This PCR product was diluted 100-fold with TE, and 1 ⁇ l was diluted with KIF4-F (5′-GATGGCTGTCAGCGACGCGCTGCTCCC-3 ′ (SEQ ID NO: 15)) and KIF4-R (5′-GTTAAAAATGCCTCTTCATGTGTAAGGCGAG-3 ′ (SEQ ID NO: 16 )) Primers were used for PCR. PCR products were separated by 1% agarose gel electrophoresis, and a band of about 1.4 kbp was excised and purified with Qiaquick Gel Extraction Kit (QIAGEN, Cat. No. 28706).
- the PCR product was purified using a Qiaquick PCR Purification kit (Qiagen Cat. No. 28106) and cloned into the SwaI site of pCAGGS-BSX. After confirming the sequence, a clone with the correct sequence was selected to obtain pCAGGS-BSX-KLF4.
- NotI-KIF4-F (5'-ATTGCGGCCGCGACATGGCTGTCAGCGACGCGCTG-3 '(SEQ ID NO: 17)
- NotI-KIF4-R 5'-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTTAGGAAAATGCCTCTTCATGT : PCR was performed with the primers of 18).
- the PCR product was purified using Qiaquick PCR Purification kit (Qiagen Cat. No. 28106), followed by Not I digestion (3 hours at 37 ° C.).
- pBS-KS-KLF4 was digested with Not I (3 hours at 37 ° C), separated by 1% agarose gel electrophoresis, the approximately 1.5 kbp band was excised, and the Qiaquick Gel Extraction Kit (Qiagen, catalog number 28706) was used. Purified.
- the Not I fragment containing the KLF4 gene was cloned into the Not I site of the pSeV18 + / TS ⁇ F vector, and a clone with the correct sequence was selected by sequencing to obtain pSeV18 + KLF4 / TS ⁇ F.
- Not I-Oct-3 / 4F 5′-GCCGCGGCCGCACCATGGCGGGACACCTGGCTTC-3 ′ (SEQ ID NO: 23)
- Not I-Oct-3 / 4R 5 ′ -PCR with GCCGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCAGTTTGAATGCATGGGAGAGCCCAGAGTGGTGAC-3 '(SEQ ID NO: 24)) PCR (94 °C -3min ⁇ 98 °C -10sec, 55 °C -15sec, 72 °C -2min 25 cycles ⁇ 72 °C -7min) Went.
- the PCR product was purified using a Qiaquick PCR Purification kit (Qiagen Cat. No. 28106), followed by Not I digestion (2 hours at 37 ° C.). Purify using the Qiaquick PCR Purification kit (Qiagen, Cat.No. 28106), clone the Not I fragment containing the Oct3 / 4 gene into the Not I site of the pSeV18 + / TS ⁇ F vector, select the correct sequence clone by sequencing, and pSeV18 + Oct3 / 4 / TS ⁇ F was obtained.
- Qiaquick PCR Purification kit Qiagen Cat.No. 28106
- Sendai virus vector loaded with human transcription factor
- Sendai virus vector carrying pCAGGS-NP, pCAGGS-P4C (-), pCAGGS-L (TDK), pCAGGS-T7, pCAGGS-F5R (WO2005 / 071085) and the above-mentioned human transcription factors in 293T / 17 cells
- Sendai virus vector carrying pCAGGS-NP, pCAGGS-P4C (-), pCAGGS-L (TDK), pCAGGS-T7, pCAGGS-F5R (WO2005 / 071085) and the above-mentioned human transcription factors in 293T / 17 cells
- Mix plasmids (pSeV18 + c-Myc / TS ⁇ F or pSeV18 + Sox2 / TS ⁇ F or pSeV18 + KLF4 / TS ⁇ F or pSeV18 + Oct3 / 4 /
- the cells were cultured for 2 days in a 37 ° C. CO 2 incubator. Subsequently, cells expressing the Sendai virus fusion protein (F protein) LLC-MK2 / F / A (Li, H.-O. et al., J. Virology 74. 6564-6569 (2000), WO00 / 70070) was layered on 293T / 17 cells transfected at a rate of 10 6 cells per well and cultured in a CO 2 incubator at 37 ° C. for 1 day.
- F protein Sendai virus fusion protein
- the cell culture medium is removed, the cells are washed once with 1 ml of MEM medium (hereinafter referred to as PS / MEM) supplemented with penicillin streptomycin, and PS / MEM medium containing 2.5 ⁇ g / ml trypsin (hereinafter referred to as Try / PS / MEM).
- PS / MEM MEM medium
- Try / PS / MEM PS / MEM medium containing 2.5 ⁇ g / ml trypsin
- A F gene-deficient Sendai virus vector carrying the Oct3 / 4 gene (hereinafter referred to as “SeV18 + Oct3 / 4 / TS ⁇ F vector”)
- B F gene-deficient Sendai virus vector carrying the Sox2 gene (hereinafter referred to as “SeV18 + Sox2 / TS ⁇ F vector”)
- C F gene-deficient Sendai virus vector carrying the Klf4 gene (hereinafter referred to as “SeV18 + Klf4 / TS ⁇ F vector”)
- D F gene deletion type Sendai virus vector carrying the c-Myc gene (hereinafter referred to as “SeV18 + c-Myc / TS ⁇ F vector”)
- Example 1 Production of ES-like cells by Sendai virus vector carrying a foreign gene First, human neonatal foreskin-derived fibroblasts (BJ) (ATCC (http://www.atcc.org), CRL-2522) ( Human adult skin-derived fibroblasts HDF (Applications, Inc.
- mitomycin-treated feeder cells for example, MEF
- 5.0 ⁇ 10 5 (cells) prepared in a gelatin-coated 10 cm culture dish and 5.0 ⁇ 10 4 (cells) to 1.0 ⁇ 10 5 of the introduced cells detached with 0.25% trypsin. 6 pieces were cultured on it.
- the medium was changed from DMEM, 10% FBS to a medium for primate ES (ReproCell, RCHEMD001) (bFGF was added to 4 ng / ml), and cultured in a 3% CO 2 incubator.
- the medium was changed every day or once every two days.
- the medium may be a feeder cell conditioned medium.
- Example 2 Alkaline phosphatase staining of cells obtained by the above-described initialization experiment
- the activity of alkaline phosphatase, an undifferentiated marker of ES cells, is expressed in NBCT / BCIP (PIERCE, NBT / BCIP, 1-Step, # 34042). When stained, a blue-stained colony positive for alkaline phosphatase was observed (FIG. 2).
- Example 3 Verification of expression level of specific gene in cells of cells obtained by the above-mentioned culture Extracting RNA by collecting a plurality of alkaline phosphatase positive colonies (ALP (+)) shown in Example 2 above (ALP (+) in FIG. 3 (a)). Reverse transcription reaction was performed with random primers, and PCR was performed with each primer (FIG. 3 (a)).
- Primer sequences are Fw: 5'-GATCCTCGGACCTGGCTAAGC-3 '(SEQ ID NO: 25) and Rv: 5'-GCTCCAGCTTCTCCTTCTCCAGC-3' (SEQ ID NO: 26) for Oct3 / 4, Fw: 5'-AGCGCTGCACATGAAGGAGCACC for Sox2 -3 '(SEQ ID NO: 27) and Rv: 5'-ATGCGCTGGTTCACGCCCGCGCCCAGG-3' (SEQ ID NO: 28), for KLF4 Fw: 5'-GCTGCACACGACTTCCCCCTG-3 '(SEQ ID NO: 29) and Rv: 5'- GGGGATGGAAGCCGGGAGGAAGCGG-3 '(SEQ ID NO: 30), c-myc for Fw: 5'-TCTCAACGACAGCAGCTCGC-3' (SEQ ID NO: 31) and Rv: 5'-CAGGAGCCTGCCTCTTTTCCACAGA-3 '(SEQ ID NO:
- Nanog is a marker for ES cells, was induced to be induced like fetal carcinoma cells (NCCIT), which is a positive control (ALP (+) in FIG. 3 (a)).
- NCCIT fetal carcinoma cells
- ALP (+) in FIG. 3 (a) Nanog is a newly identified homeodomain protein (Cell, Vol.
- Example 4 Production of inducible pluripotent stem cells using mutant c-Myc Production of silent mutation-introduced human transcription factor c-Myc (hereinafter referred to as c-rMyc) Using pBS-KS-c-Myc as a template 6 types of primers (c-rMyc1-F (5'-CGGACGACGAGACCTTCATCAAGAACATCATCATCCAGGACTG-3 '(SEQ ID NO: 39)), c-rMyc1-F using PrimeStar HS DNA polymerase (Takara Bio Inc.
- the PCR product was treated with DpnI at 37 ° C. for 2 hours. Transformation was performed using 5 ⁇ l of this reaction solution E. coli DH5 ⁇ (ToYoBo Code No. DNA-903). Sixteen colonies of E. coli were picked up, minipreped, and clones with the correct sequence were selected by sequencing to obtain pBS-KS-c-rMyc. pBS-KS-c-rMyc was digested with Not I (3 hours at 37 ° C.), separated by 1% agarose gel electrophoresis, excised band of approximately 1.5 kbp, and Qiaquick Gel Extraction Kit (Qiagen, catalog number 28706). ).
- c-rMyc has the mutations a378g, t1122c, t1125c, a1191g, and a1194g.
- the PCR product is treated with Pac I and Dpn I continuously, and the product is ligated (self-ligated), sequenced to select a plasmid from which the correct GFP gene has been removed, and Litmus SalINheIfrg PmutMtsHNts ⁇ F-GFP DelGFP was obtained.
- HNLNOTI-F 5'-GGGTGAATGGGAAGCGGCCGCTAGGTCATGGATGG-3 '(SEQ ID NO: 49) and HNLNOTI-R: 5'-CCATCCATGACCTAGCGGCCGCTTCCCATTCACCC-3' PCR (94 ° C.-3 minutes ⁇ 98 ° C.-10 seconds, 55 ° C.-15 seconds, 72 ° C.-12 minutes, 25 cycles ⁇ 72 ° C.-7 minutes) was performed.
- Sendai virus vector (SeV (HNL) -c-rMyc / TS ⁇ F vector) loaded with c-rMyc Seed 10 6 293T / 17 cells per well in a 6-well plate the day before transfection at 37 ° C The cells were cultured in a CO 2 incubator (under 5% CO 2 condition).
- Sendai virus vector plasmid pSeV carrying pCAGGS-NP, pCAGGS-P4C (-), pCAGGS-L (TDK), pCAGGS-T7, pCAGGS-F5R and the human transcription factor c-rMyc shown above in the 293T / 17 cells (HNL) -c-rMyc / TS ⁇ F was mixed with 0.5 ⁇ g, 0.5 ⁇ g, 2 ⁇ g, 0.5 ⁇ g, 0.5 ⁇ g and 5.0 ⁇ g, respectively, and transfection was performed using 15 ⁇ l of TransIT-LT1 (Mirus). The cells were cultured for 2 days in a 37 ° C. CO 2 incubator.
- Example 5 iPS induction efficiency by Sendai virus vector loaded with reprogramming factor
- the iPS induction efficiency by Sendai virus vector loaded with reprogramming factor is shown in the table.
- the number of appearance of ES-like colonies is shown with respect to the number of Sendai virus-infected cells placed on feeder cells.
- the experiment was performed in the same manner as in Example 1 except that the above-mentioned c-rMyc-loaded vector was used as a vector.
- the reprogramming factors Oct3 / 4, Sox2, Klf4, and c-Myc
- the maximum number of colonies was obtained when the modified c-Myc (c-rMyc) mounted on the HNL site of the vector was used.
- Example 6 Expression of ES marker in iPS cell Expression of ES marker in iPS cell induced by Sendai virus vector loaded with reprogramming factor was confirmed. Induction of iPS cells was performed in the same manner as in Example 1 except that the above-mentioned c-rMyc-loaded vector was used as a vector. Each ES cell-like colony was isolated and passaged under a microscope using a stem cell knife (Nippon Medical Instruments). RNA was extracted from each strain, and RT reaction and PCR were performed as in FIG.
- TERT F2847 TGCCCGGACCTCCATCAGAGCCAG (SEQ ID NO: 37) and TERT R3399 (TCAGTCCAGGATGGTCTTGAAGTCTG (SEQ ID NO: 38)) for TERT
- GDF3 F GGCGTCCGCGGGAATGTACTTC (SEQ ID NO: 51)
- GDF3 R TGGCTTAGGGGT 52
- TDGF1-F1 ATGGACTGCAGGAAGATGGCCCGC (SEQ ID NO: 53)
- TDGF1-R567 TTAATAGTAGCTTTGTATAGAAAGGC (SEQ ID NO: 54)
- Zfp42-F1 ATGAGCCAGCAACTGAAGAAACGGGCAAAG (SEQ ID NO: 55)
- Zfp42 R933 CACTTTCCCTCTTGTTCATTCTTGTTCG (SEQ ID NO: 56)
- Sall4 F AAACCCCAGCACATCAACTC (SEQ ID NO: 57)
- Sall4 F AAACC
- telomerase activity of iPS cells Telomerase activity was measured in order to confirm the infinite proliferation ability of iPS cells induced by Sendai virus vector loaded with reprogramming factor. Induction of iPS cells was performed in the same manner as in Example 1 except that the above-described c-rMyc-loaded vector (indicated as HNL) was used as a vector.
- TRAPEZE TM Telomerase Detection Kit CHEMICON Cat. No. S7700
- the cells were collected, 200 ⁇ l of 1X CAPS Lysis buffer attached to the kit was added, and suspended by pipetting.
- the cells were cultured for 30 minutes on ice, and centrifuged at 12000 rpm for 20 minutes at 4 ° C. in a cooled microcentrifuge. 160 ⁇ l of the supernatant was transferred to another Eppendorf tube, and the protein concentration of this cell lysate was measured. Before performing the assay, an amount corresponding to 1 ⁇ g protein of the cell lysate was taken in an Eppendorf tube and heat-treated at 85 ° C. for 10 minutes. Samples with and without heat treatment were used in the TRAP assay.
- Example 8 Multipotency of iPS cells
- iPS cell induction was performed in the same manner as in Example 1.
- Three iPS clones 4BJ1, B1 (BJ-derived), and 7H5 (HDF-derived) colonies were detached from the petri dish with collagenase IV (Invitrogen, 17104-019), and the cell mass was transferred to MPC-coated wells (Nunc, 145383).
- RPMI1640 Suspension culture was performed for several days in 10% FBS, and the formation of embryoid bodies was confirmed under a microscope.
- Sendai virus vector-derived iPS has differentiation ability, and each iPS formed an embryoid body, and on the 7th day, a number of cyst-like embryoid bodies that were further differentiated were observed (FIG. 6). .
- SeV-iPS clones from which SeV vector was removed were detached from feeder cells with 1 mg / ml collagenase IV, and in the case of cardiomyocyte induction, DMEM, 20% FBS in the presence of 0.1 mM vitamin C on NPC coated plate The suspension was cultured for 6 days, and after embryoid body formation, it was transferred onto a 0.1% gelatin-coated plate and cultured for 1 week, and pulsating myocardium was obtained (Takahashi, T. et.al., Circulation 107, 1912). -1916, 2003).
- iPS cells are similarly isolated and seeded on confluent PA6 feeder cells (RIKEN BRC) on a 0.1% gelatin-coated plate, followed by 2 mM L-glutamine, non-essential amino acids, and 2-mercaptoethanol. Incubate for 16 days in 10% KSR, GMEM medium (Invitrogen) with a final concentration of 1 x 10 -4 M, fix the cells with 10% formalin solution, stain with anti- ⁇ III tubulin antibody (SantaCruz; 2G10) and anti-Tyrosine Hydroxilase antibody (Chemicon; P07101) staining confirmed that it was a dopaminergic neuron (Kawasaki, H.
- SeV-iPS cells were cultured on MMC-treated MEF feeder cells in the presence of RPMI1640, 2% FBS medium, 100 ng / ml activin A (R & D Systems) for 4 days, and N2 and B27 supplements, 2 Culturing was carried out for 8 days in a DMEM / F12 medium supplemented with mM L-glutamine, a non-essential amino acid, 2-mercaptoethanol 1 ⁇ 10 ⁇ 4 M and 0.5 mg / ml BSA (Invitrogen).
- SeV-iPS cells are inoculated subcutaneously in SCID mice. After about 1 month, the formation of mass is confirmed. After about 2 months, specimens are collected, fixed with 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. Performed and confirmed the three germ layer differentiation. ( Figure 8)
- Example 9 Promoter analysis of iPS cells Methylation analysis was performed by the following bisulfite sequencing method on activation states of gene promoters Oct3 / 4 and Nanog expressed in ES cells in iPS cells. As a result, the control parent strains BJ and HDF showed a lot of methylation of Oct3 / 4 promoter (-2330 to -21010 region) and Nanog promoter (-685 to -120 region), but each clone of SeV-iPS cells. In, many demethylations were observed, and it became clear that SeV-iPS cells were activated by Oct3 / 4 and Nanog promoters as in ES cells (FIG. 9).
- Genomic DNA was extracted from iPS cells using QIAamp DNA Mini Kit (50) (Qiagen, catalog number: 51304) according to the protocol of the kit.
- bisulfite modification was performed using BisulFast DNA Modification Kit for Methylated DNA Detection (Toyobo, catalog number: MDD-101) based on 1 ⁇ g of the extracted genomic DNA according to the protocol of the kit.
- PCR was carried out using specific primers with bisulfite modified genomic DNA as a template and targeting the promoter region of Oct3 / 4 gene and Nanog gene.
- PCR products were separated by agarose gel electrophoresis, and the target band was purified with QIAquick Gel Extraction Kit (Qiagen, catalog number: 28704).
- the purified PCR product was subjected to TA-cloning using pGEM-T Easy Vector System I (Promega, catalog number: A1360) according to the kit protocol.
- colony PCR was performed using specific primers targeting the promoter region of Oct3 / 4 gene and Nanog gene. Agarose gel electrophoresis was performed, and about 10 clones with the correct band size were selected.
- plasmid DNA was extracted by miniprep and sequenced using T7 primer and SP6 primer. Comparison with the target sequence after bisulfite modification was performed to evaluate the methylation state of the promoter region.
- Oct3 / 4 gene promoter region amplification and colony PCR primers J. Biol. Chem., 2005, Vol. 280, 6257-6260
- mOct4-5F 5'-AATAGATTTTGAAGGGGAGTTTAGG-3 '(SEQ ID NO: 73)
- mOct4-5R 5'-TTCCTCCTTCCTCTAAAAAACTCA-3 '(SEQ ID NO: 74)
- Primer for Nanog gene promoter region amplification and colony PCR (Stem cell Research, Vol. 1, 105-115; Cell, 2007, Vol.
- Nanog-z1-L 5'-GGAATTTAAGGTGTATGTATTTTTTATTTT-3 '(SEQ ID NO: 75) mehNANOG-F1-AS: 5'-AACCCACCCTTATAAATTCTCAATTA-3 '(SEQ ID NO: 76) Sequence primer T7: 5'-TAATACGACTCACTATAGGG-3 '(SEQ ID NO: 77) SP6: 5'-CATACGATTTAGGTGACACTATAG-3 '(SEQ ID NO: 78) Bisulfast DNA Modification Kit for Methylated DNA Detection BisalFast DNA Modification Kit for Methylated DNA Detection (Toyobo, catalog number: MDD-101)
- iPS cells The gene expression profiles of iPS cells (SeV-iPS cells) induced by Sendai virus vectors were analyzed using parental BJ cells, human ES cells, and already established human iPS cells (GSM241846; Takahashi , K. et al., Cell, 131, 1-12, 2007).
- Control gene expression information was obtained from GEO DetaSets and compared to that of SeV-iPS cells: human ES cells hES-H9 (GSM194390; Teser PJ, et al, Nature 448, 196-199, 2007), and human iPS The cells were hiPS derived from HDF (GSM241846; Takahashi, K. et al., Cell, 131, 1-12, 2007).
- a temperature-dependent inactivating mutagenesis vector (Vector production method) Construction of a plasmid for preparing a temperature-dependent inactivating mutation-introduced Sendai virus vector L Y942H-F (5'-CAAATGTTGGAGGATTCAACCACATGTCTACATCTAGATG-3 '(SEQ ID NO: 79)), L Y942H based on Litmus SalINheIfrg PmutMtsHNts ⁇ F-GFP (WO2003 / 025570) -R (5'- CATCTAGATGTAGACATGTGGTTGAATCCTCCAACATTTG-3 '(SEQ ID NO: 80)), and L Y942H-F, L Y942H-R, P2-F (5'- CATCACAGCTGCAGGTGGCGCGACTGACAAC -3' (SEQ ID NO: 81)), PCR (94 ° C-3 minutes ⁇ 98 ° C-10 seconds, 55
- the PCR product was digested with Dpn I at 37 ° C. for 1 hour, and 20 ⁇ l of this reaction solution was transformed with E. coli DH5 ⁇ (ToYoBo Code No. DNA-903). The colonies that emerged were picked up, minipreped, sequenced to select clones with the correct sequence, and Litmus38TS ⁇ F-GFP-LY942H and Litmus38TS ⁇ F-GFP-P2LY942H were obtained, respectively.
- Litmus38TS ⁇ F-GFP-P2LY942H was digested with StuI and separated by agarose gel electrophoresis, and a 1.9 kbp band was cut out and purified.
- Litmus SalINheIfrg PmutMtsHNts ⁇ F-GFP was digested with StuI and separated by agarose gel electrophoresis, and a 9.8 kbp band was cut out and purified. These two purified fragments were ligated to obtain Litmus38TS ⁇ F-GFP-P2.
- Litmus38TS ⁇ F-GFP-P2LY942H was digested with NcoI and separated by agarose gel electrophoresis, and a 7.1 kbp band was cut out and purified.
- Litmus SalINheIfrg PmutMtsHNts ⁇ F-GFP delGFP was digested with NcoI, separated by agarose gel electrophoresis, and a 3.7 kbp band was cut out and purified. Ligation was performed using these purified bands, and the structure was confirmed by colony PCR and NcoI-PacI double digestion to obtain Litmus38TS ⁇ F-P2LY942H ⁇ GFP.
- PSeV (HNL) / TS ⁇ F was digested with NcoI, separated by agarose gel electrophoresis, and a 3.7 kbp band was cut out and purified. This band was ligated with the 7.1 kbp NcoI fragment of Litmus38TS ⁇ F-GFP-P2LY942H described above to obtain Litmus38TS ⁇ F-P2LY942H (HNL) ⁇ GFP.
- Litmus38TS ⁇ F-GFP-P2 was digested with NcoI, separated by agarose gel electrophoresis, and a 7.1 kbp band was cut out and purified. This band was ligated with the above-mentioned Litmus SalINheIfrg PmutMtsHNts ⁇ F-GFP delGFP NcoI digested purified fragment (3.7kbp) or pSeV (HNL) / TS ⁇ F NcoI digested purified fragment (3.7kbp), respectively, and Litmus38TS ⁇ F-P2 ⁇ GFP and Litmus38TS ⁇ F-P (HNL) ⁇ GFP was obtained.
- L L1361C-F (5'- GGTTCCTTAGGGAAGCCATGTATATTGCACTTACATCTTA -3 '(SEQ ID NO: 83)) and L L1361C-R (5'- TAAGATGTAAGTGCAATATAACCGGGATCTCTAAGCATACCGACT84' )
- L L1558I-F (5'- CCTGTGTATGGGCCTAACATCTCAAATCAGGATAAGATAC -3 '(SEQ ID NO: 85)
- L L1558I-R (5'- GTATCTTATCCTGATTTGAGATGTTAGGCCCATACACAGG -3' (SEQ ID NO: 86)
- L L1361C-F, L L1361C-R, L L1558I-F and L L1558I-R PCR (94 ° C-3 min ⁇ 98 ° C-10 sec, 55 °
- the PCR product was digested with Dpn I at 37 ° C for 1 hour, and 20 ⁇ l of this reaction solution was transformed with E. coli DH5 ⁇ (ToYoBooCode No. DNA-903). The colonies that emerged were picked up, minipreped, sequenced to select clones with the correct sequence, and pSeV / TS ⁇ F-Linker L1361C, pSeV / TS ⁇ F-Linker L1558I, and pSeV / TS ⁇ F-Linker L1361CL1558I were obtained, respectively.
- Litmus38TS ⁇ F-P2LY942H (HNL) ⁇ GFP and pSeV / TS ⁇ F-Linker L1361CL1558I were each digested with SalI-NheI and separated by agarose gel electrophoresis, and the bands of 8.0 kbp and 8.3 kbp were cut out and purified. These purified fragments were ligated to obtain pSeV (HNL) / TS8 ⁇ F.
- PSeV (HNL) / TS8 ⁇ F and pSeV (HNL) / TS ⁇ F were digested with NotI-XhoI and separated by agarose gel electrophoresis, and the bands of 4.9 kbp and 11.4 kbp were cut out and purified. These purified fragments were ligated to obtain pSeV (HNL) / TS7 ⁇ F.
- pSeV (HNL) / TS7 ⁇ F vector Not I site was introduced by digestion with pNot-I digested from pBS-KS-c-rMyc by digestion with NotI. / TS7 ⁇ F was obtained.
- Litmus38TS ⁇ F-P2LY942H ⁇ GFP and pSeV / TS ⁇ F-Linker L1361CL1558I were each digested with SalI-NheI and then separated by agarose gel electrophoresis, and the bands of 8.0 kbp and 8.3 kbp were cut out and purified. These purified fragments were ligated to obtain pSeV18 + BSSHII / TS8 ⁇ F.
- the pSeV18 + BSSHII / TS8 ⁇ F and pSeV18 + Oct3 / 4 / TS ⁇ F were digested with AatII-SphI, respectively, and 15.2 kbp and 2.3 kbp bands were cut out and purified. These purified fragments were ligated to obtain pSeV18 + Oct3 / 4 / TS8 ⁇ F.
- pSeV18 + Oct3 / 4 / TS8 ⁇ F and pSeV18 + / TS ⁇ F were each digested with PacI-SphI and separated by agarose gel electrophoresis, and the bands of 13.3 kbp and 4.2 kbp were cut out and purified.
- Litmus38TS ⁇ F-P2 (HNL) ⁇ GFP and pSeV / TS ⁇ F-Linker L1361C were each digested with SalI-NheI and then separated by agarose gel electrophoresis, and the bands of 8.0 kbp and 8.3 kbp were cut out and purified. These purified fragments were ligated to obtain pSeV (HNL) / TS14 ⁇ F.
- Litmus38TS ⁇ F-P2 (HNL) ⁇ GFP and pSeV / TS ⁇ F-Linker L1558I were each digested with SalI-NheI and then separated by agarose gel electrophoresis, and the 8.0 kbp and 8.3 kbp bands were cut out and purified. These purified fragments were ligated to obtain pSeV (HNL) / TS13 ⁇ F.
- This pSeV (HNL) / TS13 ⁇ F Not I site was digested by digestion with NotI from pBS-KS-c-rMyc, and the Not I fragment containing the purified c-rMyc gene was introduced, and pSeV (HNL) -c-rMyc / TS13 ⁇ F was obtained.
- Litmus38TS ⁇ F-P2 (HNL) ⁇ GFP and pSeV / TS ⁇ F-Linker L1361CL1558I were each digested with SalI-NheI and separated by agarose gel electrophoresis, and the bands of 8.0 kbp and 8.3 kbp were cut out and purified. These purified fragments were ligated to obtain pSeV (HNL) / TS15 ⁇ F.
- This pSeV (HNL) / TS15 ⁇ F Not I site was introduced from pBS-KS-c-rMyc by digestion with NotI, and a Not I fragment containing the purified c-rMyc gene was introduced, and pSeV (HNL) -c-rMyc / TS15 ⁇ F was obtained.
- Litmus38TS ⁇ F-P2 ⁇ GFP and pSeV / TS ⁇ F-Linker L1361C were each digested with SalI-NheI and then separated by agarose gel electrophoresis, and the 8.0 kbp and 8.3 kbp bands were cut out and purified. These purified fragments were ligated to obtain pSeV18 + BSSHII / TS14 ⁇ F. This pSeV18 + BSSHII / TS14 ⁇ F AatII-SphI was digested to excise and purify a 15.2 kbp band.
- This purified fragment and the AatII-SphI fragment (2.3 kbp) of pSeV18 + Oct3 / 4 / TS ⁇ F described above were ligated to obtain pSeV18 + Oct3 / 4 / TS14 ⁇ F.
- pSeV18 + Oct3 / 4 / TS14 ⁇ F was digested with NotI, separated by agarose gel electrophoresis, and then a 16.4 kbp band was cut out and purified.
- Litmus38TS ⁇ F-P2 ⁇ GFP and pSeV / TS ⁇ F-Linker® L1558I were each digested with SalI-NheI and then separated by agarose gel electrophoresis, and the 8.0 kbp and 8.3 kbp bands were cut out and purified. These purified fragments were ligated to obtain pSeV18 + BSSHII / TS13 ⁇ F. This pSeV18 + BSSHII / TS13 ⁇ F AatII-SphI was digested, and a 15.2 kbp band was cut out and purified.
- This purified fragment was ligated with the AatII-SphI fragment (2.3 kbp) of pSeV18 + Oct3 / 4 / TS ⁇ F described above to obtain pSeV18 + Oct3 / 4 / TS13 ⁇ F.
- pSeV18 + Oct3 / 4 / TS13 ⁇ F was digested with NotI, separated by agarose gel electrophoresis, and then a 16.4 kbp band was cut out and purified.
- Litmus38TS ⁇ F-P2 ⁇ GFP and pSeV / TS ⁇ F-Linker L1361CL1558I were each digested with SalI-NheI and then separated by agarose gel electrophoresis, and the 8.0 kbp and 8.3 kbp bands were cut out and purified. These purified fragments were ligated to obtain pSeV18 + BSSHII / TS15 ⁇ F. This pSeV18 + BSSHII / TS15 ⁇ F AatII-SphI was digested, and a 15.2 kbp band was cut out and purified.
- This purified fragment was ligated with the above-mentioned ASeII-SphI fragment (2.3 kbp) of pSeV18 + Oct3 / 4 / TS ⁇ F to obtain pSeV18 + Oct3 / 4 / TS15 ⁇ F.
- pSeV18 + Oct3 / 4 / TS15 ⁇ F was digested with NotI, separated by agarose gel electrophoresis, and then a 16.4 kbp band was cut out and purified.
- Litmus38TS ⁇ F-P2 ⁇ GFP and pSeV / ⁇ SalINheIfrg Lmut were each digested with SalI-NheI and separated by agarose gel electrophoresis, and the 8.0 kbp and 8.3 kbp bands were cut out and purified. These purified fragments were ligated to obtain pSeV18 + BSSHII / TS12 ⁇ F. This pSeV18 + BSSHII / TS12 ⁇ F AatII-SphI was digested, and a 15.2 kbp band was cut out and purified.
- This purified fragment was ligated with the above-mentioned ASeII-SphI fragment (2.3 kbp) of pSeV18 + Oct3 / 4 / TS ⁇ F to obtain pSeV18 + Oct3 / 4 / TS12 ⁇ F.
- pSeV18 + Oct3 / 4 / TS12 ⁇ F was digested with NotI, separated by agarose gel electrophoresis, and then a 16.4 kbp band was cut out and purified.
- This purified product and the NotI fragment containing Sox2, KLF4, and c-rMyc gene were ligated to obtain pSeV18 + Sox2 / TS12 ⁇ F, pSeV18 + KLF4 / TS12 ⁇ F, and pSeV18 + c-rMyc / TS12 ⁇ F.
- the activated mutation-introduced F gene-deficient Sendai virus vector plasmid was mixed with 0.5 ⁇ g, 0.5 ⁇ g, 2 ⁇ g, 0.5 ⁇ g and 5.0 ⁇ g, respectively, and transfection was performed using 15 ⁇ l of TransIT-LT1 (Mirus).
- the cells were cultured for 2 to 3 days in a 37 ° C. CO 2 incubator. After that, cells transfected with Sendai virus fusion protein (F protein) LLC-MK2 / F / A were layered on 293T / 17 cells transfected at a rate of 10 6 cells per well, and CO 2 at 37 ° C. The cells were cultured for 1 day in an incubator.
- the cell culture medium is removed, the cells are washed once with 1 ml of MEM medium (hereinafter PS / MEM) supplemented with penicillin streptomycin, and then PS / MEM medium containing 2.5 ⁇ g / ml trypsin (hereinafter Try / PS / MEM). 1 ml per well was added and cultured in a CO 2 incubator at 32 ° C. While changing the medium every 3 to 4 days, in some cases, the culture was continued while being subcultured with LLC-MK2 / F / A cells.
- PS / MEM MEM medium
- Try / PS / MEM PS / MEM medium containing 2.5 ⁇ g / ml trypsin
- Example 12 Removal of vector Colonies from which SeV vectors were naturally removed from SeV-iPS cells were obtained. Moreover, Sendai virus negative clones were obtained when iPS was induced at 37 ° C using a temperature sensitive vector and then shifted to 39 ° C. Furthermore, SeV negative clones were obtained by using HN antigen expressed on the cell surface by infection with SeV as an index and negative selection with anti-HN antibody.
- SeV vector negative cells can be actively collected.
- SeV vector-removed clones can be obtained with anti-HN antibodies using HN antigen expressed on the cell surface as a result of infection with SeV.
- Anti-HN monoclonal antibody (IL4.1) was reacted on ice for 30 minutes with collagenase IV and trypsin treatment and suspension operation, and after washing with medium, secondary antibody bound to eg magnetic beads Anti-mouse IgG1 antibody (Anti-Mouse IgG1 Particles, BD) was similarly reacted on ice for 30 minutes, and unbound fraction was collected in a magnet (IMagnet Cell Separation Magnet, BD) (negative selection).
- IMagnet Cell Separation Magnet, BD IMagnet Cell Separation Magnet
- the mounted gene is most highly expressed at 32 ° C., is expressed at 35-36 ° C., is slightly weak at 37 ° C., and is not expressed at 38.5 ° C. or 39 ° C.
- These vectors were loaded with reprogramming factors in the same manner (previous description), iPS was induced at 37 ° C., and the temperature was shifted after iPS cells were prepared, allowing easy removal of SeV.
- HNL-Myc replication superiority As shown in 1 above, when iPS induction was performed with a combination of SeV-18 + Oct3 / 4, Klf4, Sox2 and SeV-HNL-c-rMyc, the c-rMyc gene was HN and L SeV-HNL-c-rMyc inserted in between is advantageous for replication compared to SeV vector with other factors inserted at 18+ position (upstream of NP gene), and c-Myc is the cell Of the four factors loaded on SeV, only SeV-HNL-c-rMyc remained at the end because of its advantageous growth characteristics.
- SeV-HNL-c-rMyc-induced SeV-iPS cells were easy to establish as clones due to their excellent proliferation ability, and only one vector remained at the end, which was also easy to remove spontaneously. . Therefore, to remove by temperature shift using a temperature sensitive strain, only HNL-c-rMyc needs to be temperature sensitive, and in fact the last remaining HNL-c-rMyc vector is removed by temperature shift. (Figs. 12 and 13).
- iPS cells could be induced by placing an initialization factor (Oct3 / 4, Sox2, Klf4, c-Myc) on TS7 ⁇ F, TS13 ⁇ F, and TS15 ⁇ F in Example 12-5. It was confirmed as follows that iPS cells can be similarly induced using another ⁇ F vector backbone L mutant Y1214F (WO2008 / 096811). (Construction of Lm ⁇ F / SeV) Plasmid construction pSeV18 + LacZ / ⁇ F-1214 (WO2008 / 096811) was digested with NotI and purified.
- an initialization factor Oct3 / 4, Sox2, Klf4, c-Myc
- pSeV18 + / ⁇ F-1214 also referred to as “Lm (Y1214F) ⁇ F / SeV” or simply “Lm ⁇ F / SeV”.
- pSeV18 + / ⁇ F-1214 was digested with NotI and purified, and loaded with NotI fragments of the above-mentioned initialization 4 factors Oct3 / 4, Klf4, Sox2 and c-rMyc, respectively, and plasmid pSeV18 + Oct3 for constructing a viral vector.
- the Sendai virus vector plasmid was mixed with 0.5 ⁇ g, 0.5 ⁇ g, 2 ⁇ g, 0.5 ⁇ g and 5.0 ⁇ g, respectively, and transfection was performed using 15 ⁇ l of TransIT-LT1 (Mirus). The cells were cultured for 2 to 3 days in a 37 ° C. CO 2 incubator.
- the cell culture medium is removed, the cells are washed once with 1 ml of MEM medium (hereinafter referred to as PS / MEM) to which penicillin streptomycin has been added, and PS / MEM medium containing 2.5 ⁇ g / ml trypsin (hereinafter referred to as Try / PS / 1 ml of MEM) was added per well, and cultured in a CO 2 incubator at 32 ° C. While changing the medium every 3 to 4 days, in some cases, the culture was continued while being subcultured with LLC-MK2 / F / A cells.
- PS / MEM MEM medium
- Example 14 Feeder-free iPS induction method Since the expression of reprogramming factor by Sendai virus vector is high, it can be induced not only by the conventional method of induction on feeder cells but also by feeder-free. Induction factor-incorporated SeV was induced on a plastic petri dish until 15 days after infection, and the culture medium was changed from DMEM / 10% FBS to the medium for ES cells from the time when iPS-like colonies appeared. After reaching the size, the cells were detached from the petri dish with collagenase IV, reapplied on new feeder cells, and iPS cells were established.
- Example 15 iPS induction by 4 factors of Thomson (Oct3 / 4, Sox2, Lin28, Nanog) 4 factors of Yamanaka (Oct3 / 4, Sox2, Klf4, c-Myc) (Takahashi, K. and Yamanaka S., In addition to Cell 126, 663-676, 2006), Thomson's four factors (Oct3 / 4, Sox2, Lin28, Nanog) (Yu J et al., Science. 2007, 318 (5858): 1917-20) Even when mounted on SeV, it was possible to induce iPS cells from human fibroblasts (FIG. 15B). Examples of the construction of Nanog and Lin28 vectors are shown below.
- NotI-Nanog-F (5'-GCGCGGCCGCACCACCATGAGTGTGGATCCAGCTTGTCC-3 '(SEQ ID NO: 89)
- NotI-Nanog-R 5'-GCGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCTAGACTTTTTACCATGGTGCAT : PCR was performed with the primers of 90)).
- the PCR product was purified using Qiaquick® PCR® Purification® kit® (Qiagen Cat. No. 28106), followed by NotI digestion. Purification was performed using Qiaquick PCR-Purification Kit (Qiagen, Cat. No.
- KS-Lin28 was obtained.
- NotI-Lin28-F (5'-GCGCGGCCGCACCACCATGGGCTCCGTGTCCAACCAGC-3 '(SEQ ID NO: 93)
- NotI-Lin28-R 5'-GCGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCGGTGTTCTTACGCGGTAGGG PCR was performed with the primer of No. 94)).
- the PCR product was purified using a Qiaquick PCR Purification kit (Qiagen Cat. No. 28106) followed by Not I digestion. It was purified using Qiaquick PCR Purification kit (Qiagen, catalog number 28106), cloned into the Not I site of the pSeV18 + / TS ⁇ F vector, and a clone with the correct sequence was selected by sequencing to obtain pSeV18 + Lin 28 / TS ⁇ F. Using this plasmid, the F gene-deficient Sendai virus vector (referred to as “SeV18 + Lin 28 / TS ⁇ F vector”) retaining the Lin 28 gene by the method described above.
- ES-like cells pluripotent stem cells
- the obtained ES-like cells do not have foreign genes integrated into their chromosomes, they are not only convenient for testing and research using these cells, but also for immune rejection and ethical issues in disease treatment. It is possible to avoid the risk of canceration based on genotoxicity.
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Abstract
Description
(1)Oct3/4遺伝子を含むガンマレトロウイルスベクター又はレンチウイルスベクター(以下、これらを合わせて「レトロウイルスベクター」と総称する)
(2)Klf4遺伝子を含むレトロウイルスベクター
(3)c-Myc遺伝子を含むレトロウイルスベクター
(4)Sox2遺伝子を含むレトロウイルスベクター
すなわち本発明は、染色体非組み込み型ベクターを用いた多能性幹細胞の製造方法、および本発明の方法により作製されたES様細胞等に関し、より具体的には請求項の各項に記載の発明に関する。なお同一の請求項を引用する請求項に記載の発明の2つまたはそれ以上の任意の組み合わせからなる発明も、本明細書において意図された発明である。すなわち本発明は、
〔1〕細胞のリプログラミングにおいて遺伝子を導入するための方法であって、染色体非組み込み型ウイルスベクターを用いて細胞に該遺伝子を導入することを特徴とする方法、
〔2〕リプログラミングが多能性幹細胞の誘導である、〔1〕に記載の方法、
〔3〕染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、〔1〕または〔2〕に記載の方法、
〔4〕RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、〔3〕に記載の方法、
〔5〕マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、〔4〕に記載の方法、
〔6〕パラミクソウイルスベクターがセンダイウイルスベクターである、〔5〕に記載の方法、
〔7〕該遺伝子が下記(1)~(8)からなる群より選択される、〔1〕から〔6〕のいずれかに記載の方法、
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子
〔8〕染色体非組み込み型ウイルスベクターを含む、細胞のリプログラミングにおける遺伝子導入に用いるための組成物、
〔9〕リプログラミングが多能性幹細胞の誘導である、〔8〕に記載の組成物、
〔10〕染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、〔8〕または〔9〕に記載の組成物、
〔11〕RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、〔10〕に記載の組成物、
〔12〕マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、〔11〕に記載の組成物、
〔13〕パラミクソウイルスベクターがセンダイウイルスベクターである、〔12〕に記載の組成物、
〔14〕該遺伝子が下記(1)~(8)からなる群より選択される、〔8〕から〔13〕のいずれかに記載の組成物、
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子
に関する。また本発明は、
〔1〕リプログラムされた細胞の製造方法であって、分化した細胞に少なくとも一つの染色体非組み込み型ウイルスベクターを接触させる工程を含むことを特徴とする方法、
〔2〕リプログラムされた細胞が人工多能性幹細胞である、〔1〕に記載の方法、
〔3〕該ベクターが、核初期化因子をコードする遺伝子を少なくとも一つ搭載する、少なくとも一つの染色体非組み込み型ウイルスベクターである、〔1〕または〔2〕に記載の方法、
〔4〕該遺伝子が、下記(1)~(8)からなる群より選択される、〔3〕に記載の方法、
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子
〔5〕細胞内で、少なくともOct遺伝子、Klf遺伝子およびSox遺伝子の3種、あるいは少なくともOct遺伝子、Sox遺伝子、Nanog遺伝子、Lin28遺伝子の4種が内在性または外来性に発現するようにベクターが組み合わされる、〔1〕から〔4〕のいずれかに記載の方法、
〔6〕細胞内で、少なくともOct遺伝子、Klf遺伝子、Sox遺伝子およびMyc遺伝子の4種が内在性または外来性に発現するようにベクターが組み合わされる、〔5〕に記載の方法、
〔7〕染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、〔1〕から〔6〕のいずれかに記載の方法、
〔8〕RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、〔7〕に記載の方法、
〔9〕マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、〔8〕に記載の方法、
〔10〕パラミクソウイルスベクターがセンダイウイルスベクターである、〔9〕に記載の方法、
〔11〕〔1〕から〔10〕のいずれかに記載の方法により製造された細胞を分化させる工程をさらに含む、分化した細胞の製造方法、
〔12〕〔1〕から〔11〕のいずれかに記載の方法により製造された細胞、
〔13〕リプログラミングの工程によりベクターが染色体に組み込まれていない、〔12〕に記載の細胞、
〔14〕染色体非組み込み型ウイルスベクターを発現ベクターとして含む、細胞のリプログラミングに用いるための組成物、
〔15〕リプログラミングが、分化した細胞からの多能性幹細胞の誘導である、〔14〕に記載の組成物、
〔16〕染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、〔14〕または〔15〕に記載の組成物、
〔17〕RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、〔16〕に記載の組成物、
〔18〕マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、〔17〕に記載の組成物、
〔19〕パラミクソウイルスベクターがセンダイウイルスベクターである、〔18〕に記載の組成物、
〔20〕ベクターが、下記(1)~(8)からなる群より選択される初期化因子をコードする遺伝子を少なくとも搭載する、〔14〕から〔19〕のいずれかに記載の組成物、
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子
〔21〕染色体非組み込み型ウイルスベクターの、分化した細胞のリプログラミングのための薬剤の製造における使用、
〔22〕リプログラミングが、分化した細胞からの多能性幹細胞の誘導である、〔21〕に記載の使用、
〔23〕染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、〔21〕または〔22〕に記載の使用、
〔24〕RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、〔23〕に記載の使用、
〔25〕マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、〔24〕に記載の使用、
〔26〕パラミクソウイルスベクターがセンダイウイルスベクターである、〔25〕に記載の使用、
〔27〕ベクターが、下記(1)~(8)からなる群より選択される初期化因子をコードする遺伝子を少なくとも搭載する、〔21〕から〔26〕のいずれかに記載の使用、
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子、
に関する。また本発明は、
〔1〕下記(1)~(8)からなる群より選択される遺伝子を搭載する、染色体非組み込み型ウイルスベクター、
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子
〔2〕染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、〔1〕に記載のベクター、
〔3〕RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、〔2〕に記載のベクター、
〔4〕マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、〔3〕に記載のベクター、
〔5〕パラミクソウイルスベクターがセンダイウイルスベクターである、〔4〕に記載のベクター、に関する。
ラッサウィルスなどのアレナウイルス科(Arenaviridae)
インフルエンザウイルスなどのオルソミクソウイルス科(Orthomyxoviridae)
SARSウイルスなどのコロナウイルス科(Coronaviridae)
風疹ウイルスなどのトガウイルス科(Togaviridae)
ムンプスウイルス、麻疹ウイルス、センダイウイルス、RSウイルスなどのパラミクソウイルス科(Paramyxoviridae)
ポリオウイルス、コクサッキーウイルス、エコーウイルスなどのピコルナウイルス科(Picornaviridae)
マールブルグウイルス、エボラウイルスなどのフィロウイルス科(Filoviridae)
黄熱病ウイルス、デング熱ウイルス、C型肝炎ウイルス、G型肝炎ウイルスなどのフラビウイルス科(Flaviviridae)
ブンヤウイルス科(Bunyaviridae; Bunyavirus, Hantavirus, Nairovirus, および Phlebovirus属等を含む)
狂犬病ウイルスなどのラブドウイルス科(Rhabdoviridae)
レオウイルス科(Reoviridae)
またP蛋白質の変異としては、SeV P蛋白質の433位(D433)、434位(R434)、および437位(K437)から任意に選択される部位のアミノ酸の他のアミノ酸への置換、または他のマイナス鎖RNAウイルスP蛋白質の相同部位の置換が挙げられる。上記と同様に、アミノ酸の置換は、側鎖の化学的性質の異なる他のアミノ酸への置換が好ましい。具体的には、433番目のアミノ酸のAla (A) への置換、434番目のアミノ酸のAla (A) への置換、437番目のアミノ酸のAla (A) への置換などが例示できる。特にこれら3つの部位全てが置換されたP蛋白質を好適に用いることができる。これらの変異により、P蛋白質の温度感受性を上昇させることができる。
1)コロナウイルス
Enjuanes L, Sola I, Alonso S, Escors D, Zuniga S.
Coronavirus reverse genetics and development of vectors for gene expression.
Curr Top Microbiol Immunol. 2005;287:161-97. Review.
2)トガウイルス
Yamanaka R, Zullo SA, Ramsey J, Onodera M, Tanaka R, Blaese M, Xanthopoulos KG.
Induction of therapeutic antitumor antiangiogenesis by intratumoral injection of genetically engineered endostatin-producing Semliki Forest virus.
Cancer Gene Ther. 2001 Oct;8(10):796-802.
Datwyler DA, Eppenberger HM, Koller D, Bailey JE, Magyar JP.
Efficient gene delivery into adult cardiomyocytes by recombinant Sindbis virus.
J Mol Med. 1999 Dec;77(12):859-64.
3)ピコルナウイルス
Lee SG, Kim DY, Hyun BH, Bae YS.
Novel design architecture for genetic stability of recombinant poliovirus: the manipulation of G/C contents and their distribution patterns increases the genetic stability of inserts in a poliovirus-based RPS-Vax vector system.
J Virol. 2002 Feb;76(4):1649-62.
Mueller S, Wimmer E.
Expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardioviruslike open reading frames.
J Virol. 1998 Jan;72(1):20-31.
4)フラビウイルス
Yun SI, Kim SY, Rice CM, Lee YM.
Development and application of a reverse genetics system for Japanese encephalitis virus.
J Virol. 2003 Jun;77(11):6450-65.
Arroyo J, Guirakhoo F, Fenner S, Zhang ZX, Monath TP, Chambers TJ.
Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE).
J Virol. 2001 Jan;75(2):934-42.
5)レオウイルス
Roner MR, Joklik WK.
Reovirus reverse genetics: Incorporation of the CAT gene into the reovirus genome.
Proc Natl Acad Sci U S A. 2001 Jul 3;98(14):8036-41. Epub 2001 Jun 26.
その他のRNAウイルスの増殖方法および組み換えウイルスの製造方法については、ウイルス学実験学 各論、改訂二版(国立予防衛生研究所学友会編、丸善、1982)を参照のこと。
本発明で用いた外来遺伝子を保持するセンダイウイルスベクターの作製方法を以下に示す。特に断らない限り、外来遺伝子の導入にはこのベクターを用いた。尚、下記SeV18+/TSΔFとは、M蛋白質にG69E,T116A,及びA183Sの変異を、HN蛋白質にA262T,G264,及びK461Gの変異を、P蛋白質にL511F変異を、そしてL蛋白質にN1197S及びK1795E変異を持つF遺伝子欠失型センダイウイルスベクターである(WO2003/025570)。このベクターは、NP遺伝子の上流(ゲノムの3'側;「18+位」とも言う)に導入遺伝子挿入部位(NotI部位)を有する。
c-Myc遺伝子、Sox2遺伝子、KLF4遺伝子、及びOct3/4遺伝子を単離するために、Jurkat細胞からトータルRNAを抽出した。1.0×106(個)細胞のJurkat細胞(Schneider U et al (1977) Int J Cancer 19 (5): 621-6)を8000rpm室温で1分間遠心して集めた。細胞溶解緩衝液(10 mM Tris-HCl(pH7.5), 150 mM NaCl, 1.5 mM MgCl2, 0.65 % NP-40)を200μl 添加し、ピペッティング後、ボルテクスにより懸濁した。6000rpm、で3分間遠心分離し、上清を別の1.5mlエッペンドルフチューブに移し、200μlの抽出緩衝液を添加した。ボルテクスで十分に懸濁した後、400μlのフェノール/クロロフォルム/イソアミルアルコール(25:24:1)を添加し、さらにボルテクスにより十分懸濁した。15000rpm、4℃、5分間遠心分離を行い、上清を別の1.5mlエッペンドルフチューブに移し、400μlのイソプロパノールを添加し、ボルテクスにより十分に懸濁後、-20℃にて30分間冷やした。15000rpm、4℃、15分間遠心分離を行い、上清を除き、沈殿物に70%エタノールを1ml添加し、ボルテクスにより懸濁後、15000rpm、4℃、5分間遠心分離を行った。上清を除き室温で乾燥後、100μlのヌクレアーゼフリー水に溶解し、Jurkat細胞のトータルRNA溶液を得た。
KLF4遺伝子を単離するために、ヒト胎児腎細胞由来の293T/17細胞(Human embryonic kidney subclone 17, ATCC CRL-11286, Pear, W. S. et al., 1993, Proc. Natl. Acad. Sci. USA 90:8392-8396)より上記と同じ方法でトータルRNAを回収した。
Oct3/4遺伝子を単離するために、ヒト胚性癌細胞のNCCIT細胞(ATCC number CRL-2073; Damjanov I, et al., Lab. Invest. 1993, 68(2):220-32)より上記と同じ方法でトータルRNAを回収した。
JurkatのcDNAライブラリーからPrimeStar(商標) HS DNA polymerase (タカラバイオ株式会社 カタログ番号R010A)を用いてc-Myc -21F(5’-AACCAGCAGCCTCCCGCGACG-3’(配列番号:1))およびc-Myc 1930R(5’-AGGACATTTCTGTTAGAAGGAATCG-3’(配列番号:2))のプライマーによりPCR(94℃-3分→98℃-10秒、55℃-15秒、72℃-2分を40サイクル→72℃-7分)を行った。このPCR産物をTEを用いて100倍希釈後、1μlをc-Myc-F(5’-GATGCCCCTCAACGTTAGCTTCACC-3’(配列番号:3))およびc-Myc-R(5’-GTTACGCACAAGAGTTCCGTAGCTG-3’(配列番号:4))のプライマーを使用して、PCRを行った。PCR産物を1%アガロースゲル電気泳動により分離し、約1.3 kbpのバンドを切り出し、Qiaquick Gel Extraction Kit (QIAGEN, Cat. No. 28706) で精製した。pCAGGS-BSX (WO2005/071092) のSwaIサイトにクローニングし、シークエンスを行い配列の正しいクローンを選択し、pCAGGS-BSX-c-Mycを得た。次に、pCAGGS-BSX-c-Mycを鋳型にして、NotI-c-Myc F(5’-ATTGCGGCCGCATGCCCCTCAACGTTAGCTTCAC-3’(配列番号:5))およびNotI-c-Myc R(5’-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTTACGCACAAGAGTTCCGTAGCTGTTCAAGTTTGTGTTTC-3’(配列番号:6))のプライマーでPCRを行った。PCR産物はQiaquick PCR Purification kit (キアゲンCat. No. 28106)を用いて精製し、その後Not I消化(37℃で3時間)を行った。Qiaquick PCR Purification kit (キアゲン、カタログ番号28106)を用いて精製し、ブルースクリプトプラスミドベクターのNot Iサイトにクローニングし、シークエンスにより遺伝子配列を確認し、配列の正しいクローンを選択しpBS-KS-c-Mycを得た。pBS-KS-c-MycをNot Iで消化(37℃で3時間)し、1%アガロースゲル電気泳動により分離し、約1.5k bpのバンドを切り出し、Qiaquick Gel Extraction Kit (キアゲン、 カタログ番号28706) で精製した。このc-Myc遺伝子を含むNot I断片を、センダイウイルスベクター(SeV18+/TSΔF)のアンチゲノムをコードするpSeV18+/TSΔFベクターのNot Iサイトにクローニングし、シークエンスにより配列の正しいクローンを選択し、pSeV18+c-Myc/TSΔFを得た。
JurkatのcDNAライブラリーからPrimeStar HS DNA polymerase (タカラバイオ株式会社 カタログ番号R010A)を用いてSOX2-64F(5’- CAAAGTCCCGGCCGGGCCGAGGGTCGG-3’(配列番号:7))およびSOX2-1404R(5’- CCCTCCAGTTCGCTGTCCGGCCC-3’(配列番号:8))のプライマーによりPCR(94℃-3分→98℃-10秒、55℃-15秒、72℃-2分を40サイクル→72℃-7分)を行った。このPCR産物をTEを用いて100倍希釈後、1μlをSox2-F(5’-GATGTACAACATGATGGAGACGGAGC-3’(配列番号:9))および、Sox2-R(5’-GTCACATGTGTGAGAGGGGCAGTG-3’(配列番号:10))のプライマーを使用して、PCRを行った。PCR産物を1%アガロースゲル電気泳動により分離し、約0.95 k bpのバンドを切り出し、Qiaquick Gel Extraction Kit (QIAGEN, Cat. No. 28706) で精製した。pCAGGS-BSXのSwaIサイトにクローニングし、シークエンスを行い配列の正しいクローンを選択し、pCAGGS-BSX-SOX2を得た。次に、pCAGGS-BSX-SOX2を鋳型にして、Not I Sox-2F(5’-ATTGCGGCCGCATGTACAACATGATGGAGACG-3’(配列番号:11))およびNot I Sox-2R(5’-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCACATGTGTGAGAGGGGCAGTGTGCCGTTAATGGCCGTG-3’(配列番号:12))のプライマーでPCRを行った。PCR産物はQiaquick PCR Purification kit (キアゲンCat. No. 28106)を用いて精製し、その後Not I消化(37℃で3時間)を行った。Qiaquick PCR Purification kit (キアゲン、カタログ番号28106)を用いて精製し、ブルースクリプトプラスミドベクターのNot Iサイトにクローニングし、シークエンスにより遺伝子配列を確認し、配列の正しいクローンを選択しpBS-KS-Sox2を得た。pBS-KS-Sox2をNot Iで消化(37℃で3時間)し、1%アガロースゲル電気泳動により分離し、約1k bpのバンドを切り出し、Qiaquick Gel Extraction Kit (キアゲン、カタログ番号28706) で精製した。このSox2遺伝子を含むNot I断片をpSeV18+/TSΔFベクターのNot Iサイトにクローニングし、シークエンスにより配列の正しいクローンを選択し、pSeV18+Sox2/TSΔFを得た。
JurkatのcDNAライブラリーからPrimeStar HS DNA polymerase (タカラバイオ株式会社 カタログ番号R010A)を用いてKIF-4 -35F(5’-CCACATTAATGAGGCAGCCACCTGGC-3’(配列番号:13))およびKIF-4 1772R(5’-GCAGTGTGGGTCATATCCACTGTCTG-3’(配列番号:14))のプライマーによりPCR(94℃-3分→98℃-10秒、55℃-15秒、72℃-2分を40サイクル→72℃-7分)を行った。このPCR産物をTEを用いて100倍希釈後、1μlをKIF4-F(5’-GATGGCTGTCAGCGACGCGCTGCTCCC-3’(配列番号:15))およびKIF4-R(5’-GTTAAAAATGCCTCTTCATGTGTAAGGCGAG-3’(配列番号:16))のプライマーを使用して、PCRを行った。PCR産物を1%アガロースゲル電気泳動により分離し、約1.4 k bpのバンドを切り出し、Qiaquick Gel Extraction Kit (QIAGEN, Cat. No. 28706) で精製した。pCAGGS-BSXのSwaIサイトにクローニングし、pCAGGS-BSX-KLF4#19を得た。シークエンスの結果、pCAGGS-BSX-KLF4#19は1箇所にサイレント変異(c19t)が認められた。そこで、pCAGGS-BSX-KLF4#19を鋳型にして、NotI-KIF4-F(5’-ATTGCGGCCGCGACATGGCTGTCAGCGACGCGCTG-3’(配列番号:17))およびNotI-KIF4-R(5’-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTTAAAAATGCCTCTTCATGTGTAAGGCGAGGTGGTC-3’(配列番号:18))のプライマーでPCRを行った。PCR産物はQiaquick PCR Purification kit (キアゲンCat. No. 28106)を用いて精製し、pCAGGS-BSXのSwaIサイトにクローニングした。シークエンスの確認を行い配列の正しいクローンを選択し、pCAGGS-BSX-KLF4を得た。次に、pCAGGS-BSX-KLF4を鋳型にして、NotI-KIF4-F(5’-ATTGCGGCCGCGACATGGCTGTCAGCGACGCGCTG-3’(配列番号:17))およびNotI-KIF4-R(5’-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTTAAAAATGCCTCTTCATGTGTAAGGCGAGGTGGTC-3’(配列番号:18))のプライマーでPCRを行った。PCR産物はQiaquick PCR Purification kit (キアゲンCat. No. 28106)を用いて精製し、その後Not I消化(37℃で3時間)を行った。Qiaquick PCR Purification kit (キアゲン、カタログ番号28106)を用いて精製し、ブルースクリプトプラスミドベクターのNot Iサイトにクローニングし、シークエンスにより遺伝子配列を確認し、配列の正しいクローンを選択しpBS-KS-KLF4を得た。pBS-KS-KLF4をNot Iで消化(37℃で3時間)し、1%アガロースゲル電気泳動により分離し、約1.5k bpのバンドを切り出し、Qiaquick Gel Extraction Kit (キアゲン、カタログ番号28706) で精製した。このKLF4遺伝子を含むNot I断片をpSeV18+/TSΔFベクターのNot Iサイトにクローニングし、シークエンスにより配列の正しいクローンを選択し、pSeV18+KLF4/TSΔFを得た。
NCCITのcDNAライブラリーからPrimeStar HS DNA polymerase (タカラバイオ株式会社 カタログ番号R010A)を用いてOct-3 -28F(5’-CACCATGCTTGGGGCGCCTTCCTTCC-3’(配列番号:19))およびOCT3/4 R301(5’-CATCGGAGTTGCTCTCCACCCCGAC-3’(配列番号:20))のプライマー、OCT3/4 F192(5’-CCCGCCGTATGAGTTCTGTGG-3’(配列番号:21))およびNotI-Oct-3/4R-DPN(5’-GCCGCGGCCGCGTTATCAGTTTGAATGCATGGGAGAGCCCAG-3’(配列番号:22))のプライマーによりPCR(94℃-3分→98℃-10秒、55℃-15秒、72℃-1分を35サイクル→72℃-7分)を行いOct3/4を2つの領域に分けて増幅した。これら2つのPCR産物はQiaquick PCR Purification kit (キアゲンCat. No. 28106)を用いて精製し、キットに付属の溶出液100μlに溶出した。溶出液をTEを用いて50倍に希釈した。これらのPCR産物1μlを混合し、 Not I-Oct-3/4F(5’-GCCGCGGCCGCACCATGGCGGGACACCTGGCTTC-3’(配列番号:23))およびNot I-Oct-3/4R-DPN(5’-GCCGCGGCCGCGTTATCAGTTTGAATGCATGGGAGAGCCCAG-3’(配列番号:22))のプライマーを使用して、PCRを行った(94℃-3分→98℃-10秒、55℃-15秒、72℃-1.5分を35サイクル→72℃-7分)。PCR産物はQiaquick PCR Purification kit (キアゲンCat. No. 28106)を用いて精製し、pCAGGS-BSXのSwaIサイトにクローニングし、シークエンスを行い配列の正しいクローンを選択しpCAGGS-BSX-Oct3/4を得た。次に、pCAGGS-BSX-Oct3/4を鋳型にして、Not I-Oct-3/4F(5’-GCCGCGGCCGCACCATGGCGGGACACCTGGCTTC-3’(配列番号:23))およびNot I-Oct-3/4R(5’- GCCGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCAGTTTGAATGCATGGGAGAGCCCAGAGTGGTGAC-3’(配列番号:24))のプライマーでPCR (94℃-3分→98℃-10秒、55℃-15秒、72℃-2分を25サイクル→72℃-7分)を行った。PCR産物はQiaquick PCR Purification kit (キアゲンCat. No. 28106)を用いて精製し、その後Not I消化(37℃で2時間)を行った。Qiaquick PCR Purification kit (キアゲン、カタログ番号28106)を用いて精製し、Oct3/4遺伝子を含むNot I断片をpSeV18+/TSΔFベクターのNot Iサイトにクローニングし、シークエンスにより配列の正しいクローンを選択し、pSeV18+Oct3/4/TSΔFを得た。
トランスフェクションの前日に6ウェルプレートに1ウェル当たり106細胞の293T/17細胞を播種し、37℃のCO2インキュベーター(5%CO2条件下)で培養した。その293T/17細胞にpCAGGS-NP, pCAGGS-P4C(-), pCAGGS-L(TDK), pCAGGS-T7, pCAGGS-F5R (WO2005/071085) および上記で示したヒト転写因子を搭載したセンダイウイルスベクタープラスミド(pSeV18+c-Myc/TSΔFまたはpSeV18+Sox2/TSΔFまたはpSeV18+KLF4/TSΔFまたはpSeV18+Oct3/4/TSΔF)をそれぞれ0.5μg, 0.5μg, 2μg, 0.5μg, 0.5μg および5.0μgを混合し、TransIT-LT1 (Mirus)を15μl使用してトランスフェクションを行った。37℃のCO2インキュベーターで2日間培養した。その後、センダイウイルスの融合タンパク質(Fタンパク質)を発現する細胞LLC-MK2/F/A (Li, H.-O. et al., J. Virology 74. 6564-6569 (2000), WO00/70070) を1ウェル当たり106細胞の割合でトランスフェクションを行った293T/17細胞に重層し、37℃のCO2インキュベーターで1日間培養した。翌日、細胞の培養液を除き、ペニシリンストレプトマイシンを添加したMEM培地(以下PS/MEM)1mlで細胞を1度洗浄し、2.5 μg/mlのトリプシンを含むPS/MEM培地(以下Try/PS/MEMとする)を1ウェル当たり1ml添加し、32℃のCO2インキュベーターで2日間培養した。3~4日毎に培地交換を行いながら、場合によっては、LLC-MK2/F/A細胞で継代を行いながら培養を継続した。培養上清の一部を赤血球凝集分析によりベクター回収の有無を確認し、十分な赤血球凝集反応が得られた後に培養上清を回収した。回収した培養上清よりQIAamp Viral RNA Mini Kit (キアゲン カタログ番号52906)を用いてRNAを回収し、搭載した転写因子の領域を標的にRT-PCRを行った。得られたRT-PCR産物はシークエンスにより正しい塩基配列であることを確認し、下記(a)から(d)のベクターを作製できた。
(a)Oct3/4遺伝子を保持するF遺伝子欠失型センダイウイルスベクター(以下「SeV18+Oct3/4/TSΔFベクター」という。)
(b)Sox2遺伝子を保持するF遺伝子欠失型センダイウイルスベクター(以下「SeV18+ Sox2/TSΔFベクター」という。)
(c)Klf4遺伝子を保持するF遺伝子欠失型センダイウイルスベクター(以下「SeV18+ Klf4/TSΔFベクター」という。)
(d)c-Myc遺伝子を保持するF遺伝子欠失型センダイウイルスベクター(以下「SeV18+ c-Myc/TSΔFベクター」という。)
先ず、ヒト新生児包皮由来線維芽細胞(BJ)( ATCC(http://www.atcc.org), CRL-2522) (ヒト成人皮膚由来線維芽細胞HDF(Applications, Inc. 106-05A-1388; 36歳成人白人女性頬由来), ヒト胎児肺細胞由来線維芽細胞(MRC5;ATCC, CCL-171)8.0x 105(個)を DMEM (GIBCO-BRL, 11995), 10 % FBS (GIBCO-BRL) 中で一日37℃、0.5 % CO2インキュベータにて培養した (DMEM (GIBCO-BRL, 11995), 10 % FBS (GIBCO-BRL))。
培養後、MOI=3の濃度の下記(a)~(d)のベクターを上記培養した細胞に投与した。
(a)SeV18+ Oct3/4/TSΔFベクター
(b)SeV18+ Sox2/TSΔFベクター
(c)SeV18+ Klf4/TSΔFベクター
(d)SeV18+ c-Myc/TSΔFベクター
上記ベクターを投与後、翌日に培地交換を行った(DMEM(GIBCO-BRL, 11995), 10 % FBS (GIBCO-BRL))。
その後、7日~8日間、37℃、0.5 % CO2インキュベータにて培養した。その後、ゼラチンコート10cm培養皿に用意したマイトマイシン処理済みフィーダー細胞(例えばMEF) 5.0×105(個)に対し、0.25 %トリプシンで剥がした上記導入細胞の5.0×104(個)から1.0×106(個)をその上で培養した。翌日、DMEM, 10 % FBSから霊長類ES用培地(ReproCell社, RCHEMD001)(4 ng/ml になるようbFGFを添加した)に培地交換し、3 % CO2インキュベータで培養した。培地交換は毎日もしくは二日に一度行った。培地はフィーダー細胞のコンディションドメディウムでも構わない。
コロニーは数日後から現れ、ほぼ20日程度培養することで、ヒトES細胞様のコロニーが出現した(図1;BJ由来)。
図1の写真を見ると、誘導前の線維芽細胞(BJ)と明らかに異なる、ヒトES細胞に見られるのと同様の扁平なコロニーが見られた(実験医学 Vol.26, No.5 (増刊): pp. 35-40, 2008)。このコロニーは、ピックアップし、新しいフィーダー細胞上で培養し、ES細胞様剥離液で剥がし(トリプシン、コラゲナーゼ混合:ReproCell社、RCHETP002)継代し、増殖することが可能であった。
上記初期化実験により得られた細胞が、ES細胞の特徴である未分化マーカーを発現しているか否かを明らかにするために、更に下記実験を行った。
ES細胞の未分化マーカーであるアルカリホスファターゼの活性をNBCT/BCIP(PIERCE, NBT/BCIP, 1-Step, # 34042)にて染色すると、アルカリホスファターゼ陽性の青色に染まったコロニーが観察された(図2)。
上記実施例2で示したアルカリホスファターゼ陽性の複数のコロニー(ALP(+))をまとめてRNAを抽出した(図3(a)のALP(+))。ランダムプライマーにて逆転写反応を行い、それぞれのプライマーでPCRを行った(図3(a))。プライマー配列は、Oct3/4については Fw:5'-GATCCTCGGACCTGGCTAAGC-3'(配列番号:25)および Rv:5'-GCTCCAGCTTCTCCTTCTCCAGC-3'(配列番号:26)、Sox2については Fw:5'-AGCGCTGCACATGAAGGAGCACC-3'(配列番号:27)および Rv:5'-ATGCGCTGGTTCACGCCCGCGCCCAGG-3'(配列番号:28)、KLF4については Fw:5'-GCTGCACACGACTTCCCCCTG-3'(配列番号:29)および Rv:5'-GGGGATGGAAGCCGGGAGGAAGCGG-3'(配列番号:30)、c-mycについては Fw:5'-TCTCAACGACAGCAGCTCGC-3'(配列番号:31)および Rv:5'-CAGGAGCCTGCCTCTTTTCCACAGA-3'(配列番号:32)、Nanogについては Fw:5'-TACCTCAGCCTCCAGCAGAT-3'(配列番号:33)および Rv:5'-TGCGTCACACCATTGCTATT-3'(配列番号:34)、β-アクチンについては Fw:5'-CAACCGCGAGAAGATGAC-3'(配列番号:35)および Rv:5'-AGGAAGGCTGGAAGAGTG-3'(配列番号:36)を用いた。
また単一のES様細胞コロニーを単離し、上記と同様の方法によりRT-PCRを行った(図3(b)の4BJ-1iPS)。このとき、プライマー Fw: 5'-tgcccggacctccatcagagccag-3'(配列番号:37)および Rv: 5'-tcagtccaggatggtcttgaagtctg-3’(配列番号:38)を用いてhTERTの発現も検出した。
サイレント変異導入ヒト転写因子c-Myc(以下c-rMycとする)の作製
pBS-KS-c-Mycを鋳型にして、PrimeStar HS DNA polymerase (タカラバイオ株式会社 カタログ番号R010A) を用いて変異導入用の6種類のプライマー(c-rMyc1-F(5'-CGGACGACGAGACCTTCATCAAGAACATCATCATCCAGGACTG-3' (配列番号:39))、c-rMyc1-R(5'-CAGTCCTGGATGATGATGTTCTTGATGAAGGTCTCGTCGTCCG-3' (配列番号:40))、c-rMyc2-F(5'-GAACGAGCTAAAACGGAGCTTCTTCGCCCTGCGTGACCAGATCC-3' (配列番号:41))、c-rMyc2-R(5'-GGATCTGGTCACGCAGGGCGAAGAAGCTCCGTTTTAGCTCGTTC-3' (配列番号:42))、c-rMyc3-F(5'-CCCAAGGTAGTTATCCTTAAGAAGGCCACAGCATACATCCTGTC-3' (配列番号:43))およびc-rMyc3-R(5'-GACAGGATGTATGCTGTGGCCTTCTTAAGGATAACTACCTTGGG-3' (配列番号:44)))を入れ、PCR(94℃-3分→98℃-30秒、55℃-30秒、72℃-6分を25サイクル→72℃-7分)を行った。PCR産物をDpn Iで37℃、2時間処理した。この反応液5μl大腸菌DH5α(ToYoBo Code No. DNA-903)を用いてトランスフォーメーションを行った。16個の大腸菌のコロニーをピックアップし、ミニプレップを行い、シークエンスにより配列の正しいクローンを選択し、pBS-KS-c-rMycを得た。pBS-KS-c-rMycをNot Iで消化(37℃で3時間)し、1%アガロースゲル電気泳動により分離し、約1.5k bpのバンドを切り出し、Qiaquick Gel Extraction Kit (キアゲン、カタログ番号28706) で精製した。このc-rMyc遺伝子を含むNot I断片をpSeV(HNL)/TSΔFベクターのNot Iサイトにクローニングし、シークエンスにより配列の正しいクローンを選択し、pSeV(HNL)-c-rMyc/TSΔFを得た。c-rMycの塩基配列およびアミノ酸配列を配列番号:45および46に示した。c-rMycは、a378g、t1122c、t1125c、a1191g、および a1194gの変異を有する。
トランスフェクションの前日に6ウェルプレートに1ウェル当たり106細胞の293T/17細胞を播種し、37℃のCO2インキュベーター(5%CO2条件下)で培養した。その293T/17細胞にpCAGGS-NP, pCAGGS-P4C(-), pCAGGS-L(TDK), pCAGGS-T7, pCAGGS-F5Rおよび上記で示したヒト転写因子c-rMycを搭載したセンダイウイルスベクタープラスミドpSeV(HNL)-c-rMyc/TSΔFをそれぞれ0.5μg, 0.5μg, 2μg, 0.5μg, 0.5μgおよび5.0μgを混合し、TransIT-LT1 (Mirus) を15μl使用してトランスフェクションを行った。37℃のCO2インキュベーターで2日間培養した。その後、センダイウイルスの融合タンパク質(Fタンパク質)を発現する細胞LLC-MK2/F/Aを1ウェル当たり106細胞の割合でトランスフェクションを行った293T/17細胞に重層し、37℃のCO2インキュベーターで1日間培養した。翌日、細胞の培養液を除き、ペニシリンストレプトマイシンを添加したMEM培地(以下PS/MEM)1 mlで細胞を一度洗浄し、2.5μg/mlのトリプシンを含むPS/MEM培地(以下Try/PS/MEMとする)を1ウェル当たり1 ml添加し、32℃のCO2インキュベーターで2日間培養した。3~4日毎に培地交換を行いながら、場合によっては、LLC-MK2/F/A細胞で継代を行いながら培養を継続した。培養上清の一部を赤血球凝集分析によりベクター回収の有無を確認し、十分な赤血球凝集反応が得られた後に培養上清を回収した。回収した培養上清よりQIAamp Viral RNA Mini Kit(キアゲン カタログ番号52906)を用いてRNAを回収し、搭載したc-rMycの領域を標的にRT-PCRを行った。得られたRT-PCR産物はシークエンスにより正しい塩基配列であることを確認し、SeV(HNL)-c-rMyc/TSΔFベクターを得た。
初期化因子搭載センダイウイルスベクターによるiPS誘導効率を表に示した。フィーダー細胞に載せるセンダイウイルス感染細胞数に対し、ES様コロニーの出現数を示している。実験は、ベクターとして上記のc-rMyc搭載ベクターも用いた以外は実施例1と同様に行った。初期化因子Oct3/4, Sox2, Klf4, c-Mycのうち、ベクターのHNL部位に搭載した改変型c-Myc (c-rMyc) を用いた場合に最大のコロニー出現数が得られた。これはレトロウイルスベクターを用いた誘導効率に比べ約10倍高い効率である。また、Myc無しの3因子でもセンダイウイルスベクターによるiPS誘導が可能であった。さらに、ヒト新生児包皮由来細胞(BJ)のみならず、ヒト成人皮膚由来細胞HDFからもセンダイウイルスベクターによりiPSの誘導がBJと類似の効率で可能であった。この結果は、本発明の方法が高効率でiPS細胞を誘導できる、従来に比較して簡易な方法であることを示している。
初期化因子搭載センダイウイルスベクターにより誘導されたiPS細胞のESマーカーの発現を確認した。iPS細胞の誘導は、ベクターとして上記のc-rMyc搭載ベクターも用いた以外は実施例1と同様に行った。それぞれES細胞様コロニーをステムセルナイフ(日本医化器械)を用いて顕微鏡下で単離し、継代した。それぞれの株からRNAを抽出し、RT反応およびPCRを図3同様に行った。ES細胞マーカーであるOct3/4, NanogおよびTert, ほか8遺伝子: GDF3, TDGF1, Zfp42, Sal4F, Dmmt3b, CABRB3, CYP26A1, FoxD3(Adewumi, O. et al, Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat. Biotechnol. 25, 803-816, 2007)の発現および初期化因子Sox2, Klf4, c-Mycの発現をRT-PCRで確認した。方法は実施例3の通りである。5つすべてのクローンに於いて、すべてのマーカーの発現が確認された(図4)。
なお使用した各プライマーは以下の通りである。TERTについてはTERT F2847(TGCCCGGACCTCCATCAGAGCCAG (配列番号:37))およびTERT R3399(TCAGTCCAGGATGGTCTTGAAGTCTG (配列番号:38))、GDF3についてはGDF3 F(GGCGTCCGCGGGAATGTACTTC (配列番号:51))およびGDF3 R(TGGCTTAGGGGTGGTCTGGCC (配列番号:52)、TDGF1についてはTDGF1-F1(ATGGACTGCAGGAAGATGGCCCGC (配列番号:53))およびTDGF1-R567(TTAATAGTAGCTTTGTATAGAAAGGC (配列番号:54))、Zfp42についてはZfp42-F1(ATGAGCCAGCAACTGAAGAAACGGGCAAAG (配列番号:55))およびZfp42-R933(CTACTTTCCCTCTTGTTCATTCTTGTTCG (配列番号:56))、Sall4についてはSall4 F(AAACCCCAGCACATCAACTC (配列番号:57))およびSall4 R(GTCATTCCCTGGGTGGTTC (配列番号:58))、Dmmt3bについてはDnmt3b F(GCAGCGACCAGTCCTCCGACT (配列番号:59))およびDnmt3b R(AACGTGGGGAAGGCCTGTGC (配列番号:60))、GABRB3についてはGABRB3 F(CTTGACAATCGAGTGGCTGA (配列番号:61))およびGABRB3 R(TCATCCGTGGTGTAGCCATA (配列番号:62))、CYP26A1についてはCYP26A1 F(AACCTGCACGACTCCTCGCACA (配列番号:63))およびCYP26A1 R(AGGATGCGCATGGCGATTCG (配列番号:64))、FOXD3についてはFoxD3-F418(GTGAAGCCGCCTTACTCGTAC (配列番号:65))およびFoxD3-R770(CCGAAGCTCTGCATCATGAG (配列番号:66))、SeV-Oct3/4についてはF6(ACAAGAGAAAAAACATGTATGG (配列番号:67))およびOCT3/4 R259(GAGAGGTCTCCAAGCCGCCTTGG (配列番号:68))、SeV-Sox2についてはSox2-F294(AGCGCTGCACATGAAGGAGCACC (配列番号:27))およびR150(AATGTATCGAAGGTGCTCAA (配列番号:69))、SeV-Klf4についてはF6(ACAAGAGAAAAAACATGTATGG (配列番号:67))およびKIF4-R405(CGCGCTGGCAGGGCCGCTGCTCGAC (配列番号:70))、SeV-c-MycについてはF6(ACAAGAGAAAAAACATGTATGG (配列番号:67))およびc-rMyc406(TCCACATACAGTCCTGGATGATGATG (配列番号:71))、c-Myc/HNLについてはF8424(TAACTGACTAGCAGGCTTGTCG (配列番号:72))およびc-rMyc406(TCCACATACAGTCCTGGATGATGATG (配列番号:71))。
初期化因子搭載センダイウイルスベクターにより誘導されたiPS細胞の無限増殖能を確認するため、テロメラーゼ活性の測定を行った。iPS細胞の誘導は、ベクターとして上記のc-rMyc搭載ベクター(HNLと表記)も用いた以外は実施例1と同様に行った。テロメラーゼ活性の検出には、TRAPEZE(商標) Telomerase Detection Kit (CHEMICON Cat. No. S7700) を使用した。細胞を回収し、キットに付属の1X CAPS Lysis bufferを200μl添加し、ピペッティングにより懸濁した。氷上で30分間培養し、冷却微量遠心機にて4℃で12000rpm 20分間遠心を行った。上清160μlを別のエッペンドルフチューブに移し、この細胞溶解液のタンパク質濃度を測定した。アッセイを行う前に細胞溶解液の1μgタンパク質に相当する量をエッペンドルフチューブに採り、85℃で10分間熱処理を行った。熱処理を行ったサンプル、熱処理を行っていないサンプル1μgをTRAPアッセイに使用した。各アッセイ当たり、10X TRAP Reaction Buffer 5.0μL, 50X dNTP Mix 1.0μL, TS Primer 1.0μL, TRAP Primer Mix 1.0μL, H2O+細胞溶解液(1μgタンパク質)=40.6μL, Taq Polymerase 0.4μLの割合で反応液を調製し、PCR(30℃-30分、94℃-30秒、59℃-30秒、72℃-60秒を30サイクル)を行った。PCR反応液に6Xローディングダイを添加し、10%または12.5%のポリアクリルアミドゲルに20μLアプライし、電気泳動を行いエチジウムブロマイドにより染色を行った。
すべてのiPSクローンに於いてテロメラーゼ活性が認められ、対照である親株のBJ, HDFおよび熱処理後のiPS細胞では活性が認められなかった(図5)。
初期化因子搭載センダイウイルスベクターにより誘導されたiPS細胞の多分化能を確認するため、in vitroの胚様体形成実験を行った。iPS細胞の誘導は実施例1と同様に行った。3つのiPSクローン4BJ1, B1(BJ由来)、および7H5(HDF由来)コロニーをコラゲナーゼIV (Invitrogen, 17104-019)でシャーレから剥がし、細胞塊をMPCコートのウェル(Nunc, 145383)に移しRPMI1640, 10 % FBSにて数日間浮遊培養を行い、胚様体の形成を顕微鏡下で確認した。センダイウイルスベクター誘導iPSは分化能を持っており、いずれのiPSも胚様体を形成し、7日目ではさらに分化の進んだ、多数の嚢胞状の胚様体が認められた(図6)。
ES細胞で発現する遺伝子プロモーターOct3/4とNanogのiPS細胞における活性化状況について、以下のバイサルファイトシークエンス法によりメチル化解析を行った。その結果、対照の親株BJおよびHDFではOct3/4プロモーター (-2330~-2110領域) およびNanogプロモーター (-685~-120領域) のメチル化が多く認められたが、SeV-iPS細胞の各クローンに於いては、多くの脱メチル化が認められ、SeV-iPS細胞はES細胞同様にOct3/4とNanogプロモーターの活性化が起きている事が明らかになった (図9)。
iPS細胞よりQIAamp DNA Mini Kit (50)(キアゲン、カタログ番号:51304)を用いて、キットのプロトコールに従いゲノムDNAを抽出した。次に、抽出したゲノムDNA 1μgを基にしてBisulFast DNA Modification Kit for Methylated DNA Detection(東洋紡、カタログ番号:MDD-101)を用いて、キットのプロトコールに従いバイサルファイト修飾を行った。バイサルファイト修飾ゲノムDNAを鋳型としてOct3/4遺伝子、Nanog遺伝子のプロモーター領域を標的として特異的プライマーを用いてPCRを行った。PCR産物をアガロースゲル電気泳動にて分離し、目的バンドをQIAquick Gel Extraction Kit(キアゲン、カタログ番号:28704)にて精製した。精製したPCR産物は、pGEM-T Easy Vector System I(プロメガ、カタログ番号:A1360)を用いて、キットのプロトコールに従い、TA-クローニングを行った。次に、Oct3/4遺伝子、Nanog遺伝子のプロモーター領域を標的とした特異的プライマーを用いてコロニーPCRを行った。アガロースゲル電気泳動を行いバンドサイズの正しいクローンを約10クローン選択した。そのクローンについて、ミニプレップによりプラスミドDNAを抽出し、T7プライマー、SP6プライマーを用いてシークエンスを行った。バイサルファイト修飾後の標的配列との比較を行いプロモーター領域のメチル化状態を評価した。
mOct4-5F: 5’-AATAGATTTTGAAGGGGAGTTTAGG-3’(配列番号:73)
mOct4-5R: 5’-TTCCTCCTTCCTCTAAAAAACTCA-3’(配列番号:74)
Nanog遺伝子プロモーター領域増幅用およびコロニーPCR用プライマー(Stem cell Research, Vol. 1, 105-115; Cell, 2007, Vol. 131, 861-72)
Nanog-z1-L: 5’-GGAATTTAAGGTGTATGTATTTTTTATTTT-3’(配列番号:75)
mehNANOG-F1-AS: 5’-AACCCACCCTTATAAATTCTCAATTA-3’(配列番号:76)
シークエンス用のプライマー
T7: 5’-TAATACGACTCACTATAGGG-3’(配列番号:77)
SP6: 5’-CATACGATTTAGGTGACACTATAG-3’(配列番号:78)
使用キット
バイサルファストディ-エヌエ-モディフィケーションキットフォーメチレイテットディーエヌエーディテクション
BisulFast DNA Modification Kit for Methylated DNA Detection
(東洋紡、カタログ番号:MDD-101)
センダイウイルスベクターにより誘導したiPS細胞(SeV-iPS細胞)の遺伝子発現プロファイルを親株BJ細胞、ヒトES細胞および、すでに樹立されたヒトiPS細胞 (GSM241846; Takahashi, K. et al., Cell, 131, 1-12, 2007) と比較した。SeV-iPSとBJ細胞のトータルRNAはRNeasy Mini Kit (Qiagen) を用いて抽出し、Agilent社Quick Amp Labeling Kitを用いてcDNAからCyanine色素標識cRNAを合成し、Agilent社Gene Expression Hybridization Kitを用いてWhole Human Genome Oligo Microarray (4 x 44K) 上で17時間ハイブリダイズし、洗浄後、Agilent Microarray Scanner でDNAマイクロアレイのイメージを読み取り、Feature Extraction Software (v.9.5.3.1) にて各スポットの蛍光シグナルを数値化し、解析を行った。チップの総probe数は重複を除き、41078 probeを解析対象として行った(バイオマトリックス研究所)。対照の遺伝子発現情報はGEO DetaSetsから取得しSeV-iPS細胞の遺伝子発現と比較した:ヒトES細胞hES-H9 (GSM194390; Teser P. J., et al, Nature 448, 196-199, 2007)、およびヒトiPS細胞はHDFより誘導されたhiPS (GSM241846; Takahashi, K. et al., Cell, 131, 1-12, 2007)。その結果、SeV-iPSとBJとはr=8732, ヒトES細胞とはr=0.9658、ヒトiPS細胞とはr=0.9580の相関があり、ES細胞に発現するNanog, Sox2, Oct3/4遺伝子はSeV-iPSに発現し、BJとは非相関していたが、SeV-iPSとそれぞれヒトES細胞もしくはヒトiPS細胞とは相関線上にあり、完全に一致していた(図9)。
(ベクターの作製方法)
温度依存不活化変異導入センダイウイルスベクター作製用プラスミドの構築
Litmus SalINheIfrg PmutMtsHNts ΔF-GFP(WO2003/025570)を基にL Y942H-F(5’- CAAATGTTGGAGGATTCAACCACATGTCTACATCTAGATG-3’(配列番号:79))、L Y942H-R (5’- CATCTAGATGTAGACATGTGGTTGAATCCTCCAACATTTG-3’(配列番号:80))の組み合わせ、およびL Y942H-F、L Y942H-R、P2-F(5'- CATCACAGCTGCAGGTGGCGCGACTGACAAC -3' (配列番号:81))、P2-R(5’- GTTGTCAGTCGCGCCACCTGCAGCTGTGATG -3’ (配列番号:82))の組み合わせでPCR(94℃-3分→98℃-10秒、55℃-15秒、72℃-11分を25サイクル→72℃-7分)を行った。PCR産物をDpn Iで37℃で1時間消化し、この反応液20μlを大腸菌DH5α(ToYoBo Code No. DNA-903)を用いてトランスフォーメーションを行った。出てきたコロニーを拾って、ミニプレップ後、シークエンスを行い配列の正しいクローンを選択し、それぞれLitmus38TSΔF-GFP-LY942HおよびLitmus38TSΔF-GFP-P2LY942Hを得た。
Litmus38TSΔF-P2(HNL)ΔGFPおよびpSeV/ΔSalINheIfrg LmutをそれぞれSalI-NheI消化後、アガロースゲル電気泳動により分離し、それぞれ8.0kbp、8.3kbpのバンドを切り出し精製した。これらの精製断片のライゲーションを行いpSeV(HNL)/TS12ΔFを得た。このpSeV(HNL)/TS12ΔFのNot IサイトにpBS-KS-c-rMycより NotI消化により、切り出し、精製したc-rMyc遺伝子を含むNot I断片を導入し、pSeV(HNL)-c-rMyc/TS12ΔFを得た。
トランスフェクションの前日に6ウェルプレートに1ウェル当たり106細胞の293T/17細胞を播種し、37℃のCO2インキュベーター(5%CO2条件下)で培養した。その293T/17細胞にpCAGGS-NP, pCAGGS-P4C(-), pCAGGS-L(TDK), pCAGGS-T7, pCAGGS-F5R (WO2005/071085) および上記で示したヒト転写因子を搭載した温度依存不活化変異導入F遺伝子欠失型センダイウイルスベクタープラスミドをそれぞれ0.5μg, 0.5μg, 2μg, 0.5μgおよび5.0μgを混合し、TransIT-LT1 (Mirus) を15μl使用してトランスフェクションを行った。37℃のCO2インキュベーターで2から3日間培養した。その後、センダイウイルスの融合タンパク質(Fタンパク質)を発現する細胞LLC-MK2/F/Aを1ウェル当たり106細胞の割合でトランスフェクションを行った293T/17細胞に重層し、37℃のCO2インキュベーターで1日間培養した。翌日、細胞の培養液を除き、ペニシリンストレプトマイシンを添加したMEM培地(以下PS/MEM)1mlで細胞を1度洗浄し、2.5μg/mlのトリプシンを含むPS/MEM培地(以下Try/PS/MEMとする)を1ウェル当たり1ml添加し、32℃のCO2インキュベーターで培養した。3~4日毎に培地交換を行いながら、場合によっては、LLC-MK2/F/A細胞で継代を行いながら培養を継続した。培養上清の一部を赤血球凝集分析によりベクター回収の有無を確認し、十分な赤血球凝集反応が得られた後に培養上清を回収した。回収した培養上清よりQIAamp Viral RNA Mini Kit (キアゲン カタログ番号52906)を用いてRNAを回収し、搭載した遺伝子の領域を標的にRT-PCRを行った。得られたRT-PCR産物はシークエンスにより正しい塩基配列であることを確認し、各種ヒト転写因子を搭載した温度依存不活化変異導入F遺伝子欠失型センダイウイルスベクターを得た。
SeV-iPS細胞からSeVベクターが自然除去するコロニーが得られた。また温度感受性ベクターを使ってiPSを37℃で誘導したのち39℃に温度シフトするとセンダイウイルス陰性クローンが得られた。さらに、SeVに感染する事で細胞表面に発現するHN抗原を指標にし、抗HN抗体でネガティブセレクションすることで、SeVネガティブなクローンが得られた。
SeV-iPS細胞を継代培養するとベクターが自然に除去された細胞が増えていった。SeV-iPS細胞コロニーからRNAを抽出し、RT-PCRを行い、SeV由来の外来遺伝子発現を確認すると、18+の位置(ゲノムの最も3'側であるNP遺伝子の3'側)にSeV-Oct3/4, Sox2, Klf4, c-Myc (c-rMycおよびc-Myc) を挿入した場合(それぞれSeV18+Oct3/4/TSΔF、SeV18+Sox2/TSΔF、SeV18+Klf4/TSΔF、SeV18+c-Myc/TSΔF (またはSeV18+c-rMyc/TSΔF))、外来遺伝子は細胞分裂に伴い希釈され、これらのうち1種類もしくは2種類の発現に減っていくことが多く、野生型c-Mycは複製の劣位から一番最初に除去された。またHNLの位置にc-rMycを挿入した場合(HNL-c-rMyc/TSΔF)は、18+の位置に目的因子を挿入したベクターに対する複製優位性から、Myc遺伝子を搭載したベクターのみ残存することが多く、さらに導入された外来性初期化因子がすべて完全に除去されたクローンも得られた。RT-PCRのみならず、蛋白レベルでも完全に除去されていることが抗SeV-NP抗体によるウェスタンブロットで示された(図10)。RT-PCRのプライマーは実施例6に示した通りである。
SeVベクターは細胞分裂による希釈と継代により、自然に減少していくが、積極的にSeVベクターネガティブ細胞を集めることも可能である。SeVに感染する事で細胞表面に発現するHN抗原を指標にし、抗HN抗体によりSeVベクターの除去クローンが取得可能である。コラゲナーゼIVとトリプシン処理、懸濁操作により小さな細胞集団にした細胞に、抗HNモノクローナル抗体(IL4.1)を氷上で30分間反応させ、培地で洗浄後、2次抗体として例えばマグネットビーズに結合した抗マウスIgG1抗体(Anti-Mouse IgG1 Particles, BD)を同様に氷上で30分間反応させ、磁石 (IMagnet Cell Separation Magnet, BD) に非結合画分を集めた(ネガティブセレクション)。その結果、SeVベクターの発現が減弱した細胞集団が得られ、同操作を繰り返すことによりベクターネガティブなiPS細胞が得られた(図11)。またFACSにより抗HN抗体陰性集団を集める事も可能である。
TS 7:L (Y942H/L1361C/L1558I)
TS 13:P(D433A/R434A/K437A), L(L1558I)
TS 14:P(D433A/R434A/K437A), L(L1361C)
TS 15:P(D433A/R434A/K437A), L(L1361C/L1558I)
これらの変異をSeV18+/TSΔFベクターに導入した。これらのベクターは温度感受性であり、温度シフトにより複製が阻害される。すなわち、搭載された遺伝子は32℃で最も高く発現し、35~36℃でも発現し、37℃で発現がやや弱く、38.5℃もしくは39℃で発現しない。
これらのベクターに初期化因子を同様(以前の記述)に搭載し、37℃でiPSを誘導し、iPS細胞が作製された後に温度シフトを行い、容易にSeVの除去を行うことができた。
1で示した通り、SeV-18+Oct3/4, Klf4, Sox2とSeV-HNL-c-rMycの組合せでiPS誘導を行った場合、c-rMyc遺伝子がHNとLの間に挿入されているSeV-HNL-c-rMycが、18+の位置(NP遺伝子の上流)に挿入した他の因子をもつSeVベクターに対して複製に有利であり、かつc-Mycが細胞増殖に有利であるという性質から、SeVに搭載した4因子のうちSeV-HNL-c-rMycだけが最後に残存した。またSeV-HNL-c-rMycで誘導したSeV-iPS細胞は、増殖能が優れているためクローンとして樹立しやすく、かつ最後に残ったベクターは1つだけであり、それも自然除去されやすかった。従って、温度感受性株を使った温度シフトによる除去を行うためには、HNL-c-rMycだけを温度感受性にすればよく、実際、最後に残ったHNL-c-rMycベクターは、温度シフトによって除去することができた(図12、13)。
上記TS7、TS13もしくはTS15のHN-Lの間にc-rMyc遺伝子を挿入したベクター(TS7ΔF、TS13ΔF、TS15ΔF)と、18+の位置にOct3/4, Klf4, Sox2をそれぞれ挿入したTSベクターを用いて線維芽細胞(BJ細胞)よりiPS誘導を行うと、上述のSeV-HNL-Myc複製優位により、温度感受性のSeV-HNL-Mycだけが残る。また温度感受性株は37℃での発現が弱いために、SeV-HNL-Mycだけになった場合、速やかに残存する最後のベクターも除去される。
このようにしてiPS誘導を行うと、TS/18+ Oct3/4, Sox2, Klf4/TSΔFと、TS13ΔF/HNL-c-rMycの組合せの場合、4/6クローンが、またTS15ΔF/HNL-c-rMycの組合せの場合、3/6のクローンが、TS7ΔF/HNL-c-rMycの場合、2/12のクローンが、誘導後1ヶ月以内にSeVベクター陰性となった(図13)。得られたSeV陰性クローンはすべてヒトES細胞特異的マーカーを発現していた(図14)。
この方法により、容易に染色体を傷つけず、かつSeV陰性な、無傷のiPS細胞を得る事が出来た。
上記TS ΔFベクター以外にも、実施例12の5にてTS7ΔF、TS13ΔF、TS15ΔFに初期化因子(Oct3/4, Sox2, Klf4, c-Myc)を載せてiPS細胞の誘導が可能であったが、別のΔFベクターバックボーンL変異体Y1214F(WO2008/096811)を用いても、同様にiPS細胞を誘導できることを以下のように確認した。
(LmΔF/SeVの構築)
プラスミド構築
pSeV18+LacZ/ΔF-1214(WO2008/096811)をNotIで消化後、精製した。そして、ライゲーションを行いLacZ遺伝子の入っていないプラスミドを選択し、pSeV18+/ΔF-1214を得た(「Lm(Y1214F) ΔF/SeV」または単に「LmΔF/SeV」とも表記する)。次にpSeV18+/ΔF-1214をNotIで消化後精製し、前出の初期化4因子Oct3/4, Klf4, Sox2およびc-rMycのNotI断片をそれぞれ搭載し、ウイルスベクター作成用のプラスミドpSeV18+Oct3/4/ΔF-1214、pSeV18+Sox2/ΔF-1214、pSeV18+KLF4/ΔF-1214、pSeV18+c-rMyc/ΔF-1214を構築した。
トランスフェクションの前日に6ウェルプレートに1ウェル当たり106細胞の293T/17細胞を播種し、37℃のCO2インキュベーター(5%CO2条件下)で培養した。その293T/17細胞にpCAGGS-NP, pCAGGS-P4C(-), pCAGGS-L(TDK), pCAGGS-T7, pCAGGS-F5R (WO2005/071085)および上記で示したヒト転写因子を搭載したLmΔF/SeVセンダイウイルスベクタープラスミドをそれぞれ0.5μg, 0.5μg, 2μg, 0.5μgおよび5.0μgを混合し、TransIT-LT1 (Mirus)を15μl使用してトランスフェクションを行った。37℃のCO2インキュベーターで2から3日間培養した。その後、センダイウイルスの融合タンパク質(Fタンパク質)を発現する細胞LLC-MK2/F/Aを1ウェル当たり106細胞の割合でトランスフェクションを行った293T/17細胞に重層し、37℃のCO2インキュベーターで1日間培養した。翌日、細胞の培養液を除き、ペニシリンストレプトマイシンを添加したMEM培地(以下PS/MEM)1 mlで細胞を1度洗浄し、2.5μg/mlのトリプシンを含むPS/MEM培地(以下Try/PS/MEMとする)を1ウェル当たり1ml添加し、32℃のCO2インキュベーターで培養した。3~4日毎に培地交換を行いながら、場合によっては、LLC-MK2/F/A細胞で継代を行いながら培養を継続した。培養上清の一部を赤血球凝集分析によりベクター回収の有無を確認し、十分な赤血球凝集反応が得られた後に培養上清を回収した。回収した培養上清よりQIAamp Viral RNA Mini Kit (キアゲン カタログ番号52906)を用いてRNAを回収し、搭載した遺伝子の領域を標的にRT-PCRを行った。得られたRT-PCR産物はシークエンスにより正しい塩基配列であることを確認し、各種ヒト転写因子を搭載したLmΔF/SeVセンダイウイルスベクターを得た。
ヒト線維芽細胞BJに4因子搭載LmΔF/SeVを感染させ、TSΔF SeVベクターと同様にiPS誘導を行った結果、TSΔF/SeVを用いた場合と同様、iPS様コロニーが出現し、ESマーカーであるALPを発現していた(図15A)。このことは、ひとつのベクターに限らずセンダイウイルスベクターの他の骨格でもiPS誘導可能であることを示す。
センダイウイルスベクターによる初期化因子発現が高いため、従来のフィーダー細胞上で誘導する方法だけでなく、フィーダーフリーでも誘導可能である。初期化因子搭載SeVを感染後15日まではそのままプラスチックシャーレ上で誘導し、iPS様コロニーが出現した頃から培養液をDMEM/10 % FBSからES細胞用の培地に変更し、コロニーが十分な大きさになった後、collagenase IVでシャーレから剥離し、新しいフィーダー細胞上にまき直し、iPS細胞を樹立できた。
Yamanakaの4因子(Oct3/4, Sox2, Klf4, c-Myc)(Takahashi, K. and Yamanaka S., Cell 126, 663-676, 2006)以外に、Thomsonの4因子(Oct3/4, Sox2, Lin28, Nanog)(Yu J et al., Science. 2007, 318(5858):1917-20)をTSΔF/SeVに搭載しても、ヒト線維芽細胞からiPS細胞誘導が可能であった(図15B)。以下に、NanogおよびLin28ベクターの構築例を示した。
NCCIT細胞のcDNAライブラリーからPrimeStar(商標) HS DNA polymerase (タカラバイオ株式会社 カタログ番号R010A) を用いてNANOF-F(5’-CCACCATGAGTGTGGATCCAGCTTGTCC-3’(配列番号:87))およびNANOF-R(5’-CTCACACGTCTTCAGGTTGCATGTTC-3’(配列番号:88))のプライマーによりPCRを行った。PCR産物はQiaquick PCR Purification kit (キアゲンCat. No. 28106) を用いて精製した。ブルースクリプトプラスミドベクターのEco RVサイトにクローニングし、シークエンスにより遺伝子配列を確認し、配列の正しいクローンを選択しpBS-KS-Nanogを得た。
NCCIT細胞のcDNAライブラリーからPrimeStar(商標) HS DNA polymerase (タカラバイオ株式会社 カタログ番号R010A)を用いてLIN28-F(5’-CCACCATGGGCTCCGTGTCCAACCAGC-3’(配列番号:91))およびLIN28-R(5’-GTCAATTCTGTGCCTCCGGGAGC-3’(配列番号:92))のプライマーによりPCRを行った。PCR産物はQiaquick PCR Purification kit (キアゲンCat. No. 28106)を用いて精製し、ブルースクリプトプラスミドベクターのEco RVサイトにクローニングし、シークエンスにより遺伝子配列を確認し、配列の正しいクローンを選択しpBS-KS-Lin28を得た。次に、pBS-KS-Lin 28を鋳型にして、NotI-Lin28-F(5’- GCGCGGCCGCACCACCATGGGCTCCGTGTCCAACCAGC-3’(配列番号:93))およびNotI-Lin28-R(5’- GCGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCAATTCTGTGCCTCCGGGAGCAGGGTAGGGCTGTG-3’(配列番号:94))のプライマーでPCRを行った。PCR産物はQiaquick PCR Purification kit (キアゲンCat. No. 28106)を用いて精製し、その後Not I消化を行った。Qiaquick PCR Purification kit (キアゲン、カタログ番号28106)を用いて精製し、pSeV18+/TSΔFベクターのNot Iサイトにクローニングし、シークエンスにより配列の正しいクローンを選択し、pSeV18+Lin 28/TSΔFを得た。このプラスミドを用いて前出の方法にてLin 28遺伝子を保持するF遺伝子欠失型センダイウイルスベクター(「SeV18+ Lin 28/TSΔFベクター」という。)
Claims (39)
- 細胞のリプログラミングにおいて遺伝子を導入するための方法であって、染色体非組み込み型ウイルスベクターを用いて細胞に該遺伝子を導入することを特徴とする方法。
- リプログラミングが多能性幹細胞の誘導である、請求項1に記載の方法。
- 染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、請求項1または2に記載の方法。
- RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、請求項3に記載の方法。
- マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、請求項4に記載の方法。
- パラミクソウイルスベクターがセンダイウイルスベクターである、請求項5に記載の方法。
- 該遺伝子が下記(1)~(8)からなる群より選択される、請求項1から6のいずれかに記載の方法。
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子 - 染色体非組み込み型ウイルスベクターを含む、細胞のリプログラミングにおける遺伝子導入に用いるための組成物。
- リプログラミングが多能性幹細胞の誘導である、請求項8に記載の組成物。
- 染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、請求項8または9に記載の組成物。
- RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、請求項10に記載の組成物。
- マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、請求項11に記載の組成物。
- パラミクソウイルスベクターがセンダイウイルスベクターである、請求項12に記載の組成物。
- 該遺伝子が下記(1)~(8)からなる群より選択される、請求項8から13のいずれかに記載の組成物。
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子 - 染色体非組み込み型ウイルスベクターの、分化した細胞のリプログラミングのための薬剤の製造における使用。
- リプログラミングが、分化した細胞からの多能性幹細胞の誘導である、請求項15に記載の使用。
- 染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、請求項15または16に記載の使用。
- RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、請求項17に記載の使用。
- マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、請求項18に記載の使用。
- パラミクソウイルスベクターがセンダイウイルスベクターである、請求項19に記載の使用。
- ベクターが、下記(1)~(8)からなる群より選択される初期化因子をコードする遺伝子を少なくとも搭載する、請求項15から20のいずれかに記載の使用。
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子 - 下記(1)~(8)からなる群より選択される遺伝子を搭載する、染色体非組み込み型ウイルスベクター。
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子 - 染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、請求項22に記載のベクター。
- RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、請求項23に記載のベクター。
- マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、請求項24に記載のベクター。
- パラミクソウイルスベクターがセンダイウイルスベクターである、請求項25に記載のベクター。
- リプログラムされた細胞の製造方法であって、分化した細胞に少なくとも一つの染色体非組み込み型ウイルスベクターを接触させる工程を含むことを特徴とする方法。
- リプログラムされた細胞が人工多能性幹細胞である、請求項27に記載の方法。
- 該ベクターが、核初期化因子をコードする遺伝子を少なくとも一つ搭載する、少なくとも一つの染色体非組み込み型ウイルスベクターである、請求項27または28に記載の方法。
- 該遺伝子が、下記(1)~(8)からなる群より選択される、請求項29に記載の方法。
(1)Oct遺伝子
(2)Klf遺伝子
(3)Myc遺伝子
(4)Sox遺伝子
(5)Nanog遺伝子
(6)Lin28遺伝子
(7)SV40 LargeT抗原遺伝子
(8)TERT遺伝子 - 細胞内で、少なくともOct遺伝子、Klf遺伝子およびSox遺伝子の3種、あるいは少なくともOct遺伝子、Sox遺伝子、Nanog遺伝子、Lin28遺伝子の4種が内在性または外来性に発現するようにベクターが組み合わされる、請求項27から30のいずれかに記載の方法。
- 細胞内で、少なくともOct遺伝子、Klf遺伝子、Sox遺伝子およびMyc遺伝子の4種が内在性または外来性に発現するようにベクターが組み合わされる、請求項31に記載の方法。
- 染色体非組み込み型ウイルスベクターがRNAウイルスベクターである、請求項27から32のいずれかに記載の方法。
- RNAウイルスベクターがマイナス鎖RNAウイルスベクターである、請求項33に記載の方法。
- マイナス鎖RNAウイルスベクターがパラミクソウイルスベクターである、請求項34に記載の方法。
- パラミクソウイルスベクターがセンダイウイルスベクターである、請求項35に記載の方法。
- 請求項27から36のいずれかに記載の方法により製造された細胞を分化させる工程をさらに含む、分化した細胞の製造方法。
- 請求項27から37のいずれかに記載の方法により製造された細胞。
- リプログラミングの工程によりベクターが染色体に組み込まれていない、請求項38に記載の細胞。
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EP09797978.5A EP2322611B1 (en) | 2008-07-16 | 2009-07-16 | Method for production of reprogrammed cell using chromosomally unintegrated virus vector |
EP16161002.7A EP3075850B1 (en) | 2008-07-16 | 2009-07-16 | Method for production of reprogrammed cell using chromosomally unintegrated virus vector |
CA2731007A CA2731007A1 (en) | 2008-07-16 | 2009-07-16 | Method for production of reprogrammed cell using chromosomally unintegrated virus vector |
JP2010520896A JP5763340B6 (ja) | 2008-07-16 | 2009-07-16 | 染色体非組み込み型ウイルスベクターを用いてリプログラムされた細胞を製造する方法 |
DK09797978.5T DK2322611T3 (en) | 2008-07-16 | 2009-07-16 | A process for producing reprogrammed cells using chromosomally non-integrated viral vector |
KR1020117003451A KR20110046472A (ko) | 2008-07-16 | 2009-07-16 | 염색체 비삽입형 바이러스 벡터를 사용해서 리프로그램된 세포를 제조하는 방법 |
CN200980136168.2A CN102159710B (zh) | 2008-07-16 | 2009-07-16 | 使用染色体非整合型病毒载体制造经初始化的细胞的方法 |
US13/054,022 US9127256B2 (en) | 2008-07-16 | 2009-07-16 | Method for production of reprogrammed cell using chromosomally unintegrated virus vector |
US14/812,108 US9695445B2 (en) | 2008-07-16 | 2015-07-29 | Method for production of reprogrammed cell using chromosomally unintegrated virus vector |
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US9695445B2 (en) | 2017-07-04 |
JP2016105729A (ja) | 2016-06-16 |
US11136594B2 (en) | 2021-10-05 |
EP2322611B1 (en) | 2016-06-01 |
JP5763340B2 (ja) | 2015-08-12 |
CN102159710A (zh) | 2011-08-17 |
CA2731007A1 (en) | 2010-01-21 |
CN102159710B (zh) | 2015-09-02 |
US20110287538A1 (en) | 2011-11-24 |
JP6402129B2 (ja) | 2018-10-10 |
KR20110046472A (ko) | 2011-05-04 |
CN104962583A (zh) | 2015-10-07 |
JP2015164434A (ja) | 2015-09-17 |
EP3075850B1 (en) | 2019-02-06 |
EP2322611A4 (en) | 2012-04-18 |
US9127256B2 (en) | 2015-09-08 |
DK2322611T3 (en) | 2016-09-05 |
EP2322611A1 (en) | 2011-05-18 |
EP3075850A1 (en) | 2016-10-05 |
CN104962583B (zh) | 2019-11-01 |
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US20160177337A1 (en) | 2016-06-23 |
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