# US 11,898,169 B2 · Synergistic genome-nonintegrating reprogramming by microRNAs and transcription factors > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,898,169 B2 - Document ID: US11898169B2 - Status: Granted - Kind: B2 - Application number: 17/239,059 - Filed: 2021-04-23 - Published: 2021-08-05 - Granted: 2024-02-13 - Earliest claimed: 2013-11-15 - Assignees: The McLean Hospital Corporation - Applicants: The McLean Hospital Corporation - Inventors: Kwang-Soo Kim; Young Cha - Web page: https://patentgrep.com/patent/US11898169B2 ## CPC - C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS} - A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00) - C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} - C12N 2310/141: MicroRNAs, miRNAs - C12N 2501/602: Sox-2 - C12N 2501/603: Oct-3/4 - C12N 2501/604: Klf-4 - C12N 2501/606: c-Myc - C12N 2501/65: MicroRNA - C12N 2506/1307: from adult fibroblasts - C12N 2510/00: Genetically modified cells - C12N 2799/022: where the vector is derived from an adenovirus - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract Disclosed herein are methods of generating induced pluripotent stem cells. The method involves providing a quantity of somatic or non-embryonic cells, contacting the contacting the somatic or non-embryonic cells with a quantity of one or more programming factors and one or more RNA molecules, and culturing the somatic or non-embryonic cells for a period of time sufficient to generate at least one induced pluripotent stem cell. Various reprogramming factors and RNA molecules for use in the methods are disclosed herein. Also disclosed are cell lines and pharmaceutical compositions generated by use of the methods. ## Claims ### Claim 1 A method of generating mammalian patient-specific induced pluripotent stem cells for treating a patient, comprising: providing a quantity of mammalian somatic or non-embryonic cells from the patient; contacting the mammalian somatic or non-embryonic cells with a quantity of one or more reprogramming factors and one or more microRNA molecules; and culturing the somatic or non-embryonic cells for a period of time sufficient to generate at least one induced pluripotent stem cell, wherein the one or more microRNA molecules consist of (i) at least one miR-302 cluster member and (ii) at least one miR-200 cluster member, and wherein the quantity of reprogramming factors comprises at least Oct-4, Sox-2, Klf-4, and at least one of c-Myc or 1-Myc. ### Claim 2 (depends on 1) The method of claim 1, wherein contacting the cells with a quantity of the one or more reprogramming factors and one or more microRNA molecules comprises transduction, nucleofection, electroporation, direct injection and/or transfection. ### Claim 3 (depends on 1) The method of claim 1, wherein the quantity of one or more reprogramming factors further comprise one or more factors selected from the group consisting of: Nanog, Sa114, Fbx-15, Utf-1, Tert, Lin-28, SV40 Large T Antigen (“SV40LT”), and short hairpin RNAs targeting p53 (“shRNA-p53”). ### Claim 4 (depends on 1) The method of claim 1, wherein the at least one miR-200 cluster member comprises microRNAs selected from the group consisting of miR-200a, miR-200b, miR-200c, miR-141, miR-429, and derivatives and orthologs thereof. ### Claim 5 (depends on 1) The method of claim 1, wherein the at least one miR-302 cluster member comprises at least one microRNA selected from the group consisting of miR-302a, miR-302b, miR-302c, miR-302d, miR-367, and derivatives and orthologs thereof. ### Claim 6 (depends on 1) The method of claim 1, wherein the at least one miR-302 cluster member comprises at least one microRNA selected from the group consisting of miR-302a, miR-302b, miR-302c, miR-302d, and miR-367, and the at least one miR-200 cluster member comprises at least one microRNA selected from the group consisting of miR-200a, miR-200b, miR-200c, miR-141, and miR-429. ### Claim 7 (depends on 1) The method of claim 1, wherein the one or more microRNA molecules further comprise one or more microRNAs selected from the group consisting of miR-106a, miR-106b, miR-106b25, miR-20b, miR-93, miR-17, miR-291a, miR-291b-5p, miR-294, miR-295, miR-25, miR-32, miR92a-1, miR92a-2, miR92b, miR-363, miR-19a, miR-19b, miR-290-5p, miR-292, miR-20a, miR-290-3p, miR-18b, miR-291b-3p, miR-293, miR-369-5p, and derivatives and orthologs thereof. ### Claim 8 (depends on 1) The method of claim 1, wherein the quantity of one or more reprogramming factors and one or more microRNA molecules are encoded in one or more viruses or in one or more non-integrative vectors. ### Claim 9 (depends on 8) The method of claim 8, wherein the one or more viruses are non-integrative viruses. ### Claim 10 (depends on 9) The method of claim 9, wherein the non-integrative virus is an Adenovirus or Sendai virus. ### Claim 11 (depends on 1) The method of claim 1, wherein the one or more microRNA molecules are mature microRNA molecules. ### Claim 12 (depends on 8) The method of claim 8, wherein the non-integrative vector is an episomal or minicircle vector. ### Claim 13 (depends on 1) The method of claim 1, wherein the culturing is in a reprogramming media and the reprogramming media comprises at least one chemical induction molecule. ### Claim 14 (depends on 1) The method of claim 1, wherein the culturing is in a reprogramming media for at least 7 days. ### Claim 15 (depends on 13) The method of claim 13, wherein culturing the somatic or non-embryonic cells in a reprogramming media is for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. ### Claim 16 (depends on 1) The method of claim 1, wherein generating induced pluripotent stem cells comprises further culturing the somatic or non-embryonic cells in an induction media is for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. ### Claim 17 (depends on 16) The method of claim 16, wherein the induction media is serum-free media. ### Claim 18 (depends on 1) The method of claim 1, further comprising isolating at least one induced pluripotent stem cell. ### Claim 19 (depends on 11) The method of claim 11, wherein the mature microRNAs are selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20, and SEQ ID NO:25. ### Claim 20 (depends on 1) The method of claim 1, wherein the quantity of one or more reprogramming factors are proteins or mRNAs encoding the quantity of reprogramming factors. ### Claim 21 (depends on 1) A population of mammalian patient-specific induced pluripotent stem cells produced by the method of claim 1.