REPROGRAMMING OF SOMATIC CELLS

• US 20110076678A1
• Filed: 04/07/2008
• Published: 03/31/2011
• Est. Priority Date: 04/07/2007
• Status: Active Grant

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Welstead, G. Grant, Jaenisch, Rudolf, Hanna, Jaqub, Wernig, Marius, Brambrink, Oliver Tobias, Meissner, Alexander, Lengner, Christopher J., Foreman, Ruth
• Granted Patent: US 9,382,515 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 67/0273 Cloned vertebrates
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 5/02 of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
• CPC Class Codes: A61P 5/14 of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 5/18 of the parathyroid hormones
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/4702 : Regulators; Modulating activity
• CPC Class Codes: C12N 15/8509 : for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/8775 : Murine embryos
• CPC Class Codes: C12N 2501/602 : Sox-2
• CPC Class Codes: C12N 2501/603 : Oct-3/4
• CPC Class Codes: C12N 2501/604 : Klf-4
• CPC Class Codes: C12N 2501/605 : Nanog
• CPC Class Codes: C12N 2501/606 : c-Myc
• CPC Class Codes: C12N 2506/11 : from blood or immune system cells
• CPC Class Codes: C12N 2506/13 : from connective tissue cells, from mesenchymal cells
• CPC Class Codes: C12N 2800/30 : Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/003 : tet inducible
• CPC Class Codes: C12N 2830/006 : tet repressible
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: G01N 2333/91011 : Methyltransferases (general) (2.1.1.)
• CPC Class Codes: G01N 33/5023 : on expression patterns

Abstract

The disclosure relates to a method of reprogramming one or more somatic cells, e.g., partially differentiated or fully/terminally differentiated somatic cells, to a less differentiated state, e.g., a pluripotent or multipotent state. In further embodiments the invention also relates to reprogrammed somatic cells produced by methods of the invention, to uses of said cells, and to methods for identifying agents useful for reprogramming somatic cells.

Claims

1-175. -175. (canceled)

176. A purified preparation of isolated pluripotent mammalian cells, wherein the cells

(a) express endogenous Oct4 and Nanog;

(b) differentiate into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice;

(c) do not express an exogenously introduced pluripotency gene or selectable marker operably linked to an endogenous pluripotency gene;

(d) are reprogrammed mammalian somatic cells.

177. The purified preparation of cells of claim 176, wherein at least 50% of the cells are resistant to DNA demethylation.

178. The purified preparation of cells of claim 176, wherein the cells survive under conditions in which expression of DNA methyltransferase I is reduced by at least 50%.

179. The purified preparation of cells of claim 176, wherein said cells are not genetically modified.

180. The purified preparation of cells of claim 176, wherein said cells comprise at least one genetic modification.

181. The purified preparation of cells of claim 176, wherein the cells are genetically matched to a donor of said somatic cells or a donor of a precursor cell of said somatic cells, wherein said donor is an individual in need of cell therapy.

182. A method of generating a reprogrammed somatic cell comprising:

providing a somatic cell that contains one or more exogenously introduced genes that contribute to reprogramming said cell to a pluripotent state, wherein said cell has been reprogrammed to an pluripotent state in which the cells are (i) resistant to DNA demethylation;

(ii) express endogenous Oct4; and

(iii) differentiate into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice.

183. The method of claim 182, wherein two or more exogenously introduced genes are selected from the group consisting of:

Oct-4, Nanog, Sox-2, c-Myc, Lin28, and Klf4.

184. The method of claim 183, wherein the two or more exogenously introduced genes are Oct-4 and Nanog.

185. The method of claim 183, wherein the two or more exogenously introduced genes are Oct-4 and Sox-2.

186. The method of claim 183, wherein the two or more exogenously introduced genes are Oct-4 and Klf-4.

187. The method of claim 182, wherein the one or more exogenously introduced genes are selected from the group consisting of:

Oct-4, Sox-2, c-Myc, Klf4, and combinations thereof.

188. The method of claim 182, wherein the cell contains exogenously introduced genes encoding Oct-4, Sox-2, c-Myc, and Klf4 or the proteins themselves.

189. The method of claim 182, wherein the exogenously introduced genes are introduced by transfection.

190. The method of claim 182, wherein the exogenously introduced genes are introduced by viral infection.

191. The method of claim 182, further comprising a step of:

functionally inactivating at least one of the exogenously introduced genes.

192. The method of claim 191, comprising excising at least a portion of said exogenously introduced gene by introducing a site-specific recombinase in said cell or expressing a site-specific recombinase in the cell.

193. A method of reprogramming a differentiated somatic cell to a pluripotent state, comprising the steps of:

(a) contacting a differentiated somatic cell with at least one reprogramming agent that contributes to reprogramming of said cell to a pluripotent state; and

(b) maintaining said cell under conditions appropriate for proliferation of said cell and for activity of said at least one reprogramming agent for a period of time sufficient to begin reprogramming of said cell.

194. The method of claim 193, wherein said at least one reprogramming agent is a polynucleotide.

195. The method of claim 193, wherein said at least one reprogramming agent is a polypeptide.

196. The method of claim 194 or claim 195, wherein said at least one reprogramming agent is selected from the group consisting of Oct4, Sox2, Klf-4, Nanog, Lin28, c-Myc and combinations thereof.

197. The method of claim 193, wherein the reprogramming agent is selected from the group consisting of:

5-aza-cytidine, Trichostatin A (TSA) and valproic acid.

198. The method of claim 193, wherein said differentiated somatic cell is a partially differentiated cell.

199. The method of claim 193, wherein said differentiated somatic cell is fully differentiated.

200. A method of identifying an agent that reprograms somatic cells to a less differentiated state, the method comprising:

(a) contacting somatic cells with a candidate reprogramming agent, wherein the somatic cells are sensitive to reduced DNA methylation; and

(b) determining the amount of cells that are resistant to reduced DNA methylation, where an increased amount of cells that are resistant to reduced DNA methylation, as compared to a control, is indicative of the candidate agent being a reprogramming agent.