# US 10,370,646 B2 · Generation of human iPS cells by a synthetic self-replicative RNA > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,646 B2 - Document ID: US10370646B2 - Status: Granted - Kind: B2 - Application number: 15/860,356 - Filed: 2018-01-02 - Published: 2018-08-02 - Granted: 2019-08-06 - Earliest claimed: 2012-05-21 - Assignees: The Regents of the University of California - Applicants: The Regents of the University of California - Inventors: Steven F. Dowdy; Naohisa Yoshioka - Web page: https://patentgrep.com/patent/US10370646B2 ## CPC - C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS} - C07K 14/4702: {Regulators; Modulating activity} - C07K 14/4705: {stimulating, promoting or activating activity} - C12N 9/127: {RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase} - C12N 15/86: Viral vectors - G01N 33/56966: {Animal cells} - C12N 2501/60: Transcription factors - C12N 2501/602: Sox-2 - C12N 2501/603: Oct-3/4 - C12N 2501/604: Klf-4 - C12N 2501/605: Nanog - C12N 2501/606: c-Myc - C12N 2506/1307: from adult fibroblasts - C12N 2770/36122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2770/36143: viral genome or elements thereof as genetic vector - C12N 2840/206: having multiple IRES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The disclosure provides methods and compositions useful for obtaining induced stem cells, methods of making and use thereof. ## Claims ### Claim 1 A method comprising: contacting a somatic cell with an ectopic self-replicating RNA replicon comprising a plurality of non-structural replicase domains from an alphavirus and comprising polynucleotides encoding at least four reprogramming factors (RFs) selected from the group consisting of a (i) KLF4, (ii) OCT4, (iii) SOX2, (iv) c-MYC or n-MYC or L-MYC, (v) GLIS1 and (vi) NANOG, wherein the RNA replicon comprises from 5′ to 3′: (i) polynucleotide sequences encoding the plurality of non-structural replicase domains from an alphavirus; (ii) a promoter; (iii) RF1; (iv) a coding sequence for a first self-cleaving peptide; (v) RF2; (vi) a coding sequence for a second self-cleaving peptide; (vii) RF3; (viii) an IRES; (ix) RF4; (x) an optional IRES or an optional promoter; (xi) an optional sequence encoding an optional selectable marker; and (xii) an alphavirus 3′ UTR and polyA tail, culturing the somatic cell to express the at least four reprogramming factors (RFs); selecting cells that display a pluripotent stem cell morphology and/or pluripotent stem cell markers; and subculturing the cells to obtain a population of induced pluripotent stem cells. ### Claim 2 (depends on 1) The method of claim 1, wherein the cells are selected by detecting expression of a Tumor Rejection Antigen 1-60and/or 1-81. ### Claim 3 (depends on 1) The method of claim 1, further comprising transforming somatic cells with the ectopic self-replicating RNA replicon; culturing the transformed somatic cells, for at least 30 days under conditions to express the RFs; and isolating pluripotent stem cells. ### Claim 4 (depends on 1) The method of claim 1, wherein the culturing comprises culturing the cells in media conditioned with B18R. ### Claim 5 (depends on 4) The method of claim 4, wherein the B18R conditioned media is produced by transfection of B18R mRNA into primary human fibroblasts. ### Claim 6 (depends on 1) The method of claim 1, wherein the polynucleotide sequences encoding the plurality of non-structural replicase domain sequences are obtained from an alphavirus selected from the group consisting of Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Equine Encephalitis virus (WEE), Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus and Buggy Creek virus. ### Claim 7 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the KLF4 polypeptide encodes a KLF4 polypeptide selected from the group consisting of: (a) a polypeptide having a sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:8 and having KLF activity; and (b) a polypeptide having the sequence as set forth in SEQ ID NO:8. ### Claim 8 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the KLF polypeptide comprises the sequence as set forth in SEQ ID NO:7, wherein the thymidine residues are replaced with uracil resides. ### Claim 9 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the SOX-2 polypeptide encodes a SOX-2 polypeptide selected from the group consisting of: (a) a polypeptide having a sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:6 and having SOX-2 activity, and (b) a polypeptide having the sequence as set forth in SEQ ID NO:6. ### Claim 10 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the SOX-2 polypeptide comprises the sequence as set forth in SEQ ID NO:5, wherein the thymidine residues are replaced with uracil resides. ### Claim 11 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the OCT-4 polypeptide encodes an OCT-4 polypeptide selected from the group consisting of: (a) a polypeptide having a sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:4 and having OCT-4 activity; and (b) a polypeptide having the sequence as set forth in SEQ ID NO:4. ### Claim 12 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the OCT-4 polypeptide comprises the sequence as set forth in SEQ ID NO:3, wherein the thymidine residues are replaced with uracil resides. ### Claim 13 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the c-MYC polypeptide encodes a c-MYC polypeptide selected from the group consisting of: (a) a polypeptide having a sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:10 and having c-MYC activity; and (b) a polypeptide having the sequence as set forth in SEQ ID NO:10. ### Claim 14 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the c-MYC polypeptide comprises the sequence as set forth in SEQ ID NO:9, wherein the thymidine residues are replaced with uracil resides. ### Claim 15 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the GLIS1 polypeptide encodes a GLIS1 polypeptide selected from the group consisting of: (a) a polypeptide having a sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:34 and having GLIS1 activity; and (b) a polypeptide having the sequence as set forth in SEQ ID NO:34. ### Claim 16 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the GLIS1 polypeptide comprises the sequence as set forth in SEQ ID NO:33, wherein the thymidine residues are replaced with uracil resides. ### Claim 17 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the NANOG polypeptide encodes a NANOG polypeptide selected from the group consisting of: (a) a polypeptide having a sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:2 and having NANOG activity; and (b) a polypeptide having the sequence as set forth in SEQ ID NO:2. ### Claim 18 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the NANOG polypeptide comprises the sequence as set forth in SEQ ID NO:1, wherein the thymidine residues are replaced with uracil resides. ### Claim 19 (depends on 1) The method of claim 1, wherein the replicon comprises a sequence that has at least 90% sequence identity to SEQ ID NO:29, 30, 31, or 32 from about position 1 to about position 7561 wherein “T” of the sequence is substituted with “U”. ### Claim 20 (depends on 1) The method of claim 1, wherein the promoter located 3′ to the polynucleotide sequences encoding the plurality of non-structural replicase domains and 5′ to RF1, is a 26S internal promoter and wherein the first and second self-cleaving peptides are independently selected from T2A and E2A self-cleaving peptides.