Methods of increasing insulin content in cells

Patent metadata

Field	Value
Publication number	US-2016017290-A1
Application number	US-201514803270-A
Country	US
Kind code	A1
Filing date	Jul 20, 2015
Priority date	Jul 20, 2014
Publication date	Jan 21, 2016
Grant date	—

Abstract

A method of ex-vivo increasing insulin content in progenitor cells which express Zinc Finger E-Box Binding Homeobox 1 (ZEB-1) is disclosed. The method comprises contacting the progenitor cells with an inhibitory agent directed against a polypeptide, wherein the RNA transcript encoding said polypeptide is targeted by miRNA-200c, said polypeptide being selected from the group consisting of ZEB-1, SOX-2 and SOX-6.

First claim

What is claimed is:

1 . A method of ex-vivo increasing insulin content in progenitor cells which express Zinc Finger E-Box Binding Homeobox 1 (ZEB-1), comprising contacting the progenitor cells with an inhibitory agent directed against a polypeptide, wherein the RNA transcript encoding said polypeptide is targeted by miRNA-200c, said polypeptide being selected from the group consisting of ZEB-1, SOX-2 and SOX-6, thereby increasing insulin content in the progenitor cells.

2 . The method of claim 1 , wherein said progenitor cells are selected from the group consisting of dedifferentiated adult islet beta cells, mesenchymal stem cells and induced pluripotent stem cells dedifferentiated from beta cells.

3 . The method of claim 1 , wherein said inhibitory agent is a polynucleotide agent.

4 . The method of claim 1 , further comprising culturing the progenitor cells in a medium comprising nicotinamide, exendin-4, activin A and glucose, said culture medium being devoid of serum.

5 . The method of claim 4 , which said culturing is effected following or concomitant with said contacting.

6 . The method of claim 4 , comprising:
(a) exposing said progenitor cells to a culture medium comprising said nicotinamide, said exendin-4, said activin A, wherein said glucose is present at a concentration of 10-100 mM; and subsequently (b) exposing said progenitor cells in an additional culture medium comprising said nicotinamide and said exendin-4, wherein said glucose is present at a concentration of 0.5-10 mM.

7 . The method of claim 6 , wherein said additional medium is devoid of activin A.

8 . The method of claim 1 , further comprising contacting the progenitor cells with an agent that down-regulates an activity and/or amount of HES-1.

9 . The method of claim 2 , wherein said dedifferentiated adult islet beta cells comprise induced pluripotent stem cells generated from beta cells.

10 . The method of claim 2 , wherein said dedifferentiated adult islet beta cells are generated by culturing said adult islet beta cells for at least 10 passages.

11 . The method of claim 10 , wherein said culturing is effected in CMRL medium.

12 . An isolated population of cells, comprising a heterologous polynucleotide which down-regulates a polypeptide, wherein the RNA transcript encoding said polypeptide is targeted by miRNA-200c, said polypeptide being selected from the group consisting of ZEB-1, SOX-2 and SOX-6, wherein said cells secrete insulin.

13 . An isolated population of cells generated according to the method of claim 1 .

14 . A method of treating diabetes in a subject, comprising transplanting a therapeutically effective amount of the population of adult islet beta cells of claim 12 into the subject, thereby treating diabetes.

15 . The isolated population of cells of claim 12 , being genetically modified to express a pharmaceutical agent.

16 . The isolated population of cells of claim 12 , being adult islet beta cells.

17 . A pharmaceutical composition comprising as an active ingredient the population of cells of claim 12 and a pharmaceutically acceptable carrier.

18 . A method of treating Diabetes in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitory agent directed against a polypeptide, wherein the RNA transcript encoding said polypeptide is targeted by miRNA-200c, said polypeptide being selected from the group consisting of ZEB-1, SOX-2 and SOX-6, thereby treating the Diabetes.

19 . The method of claim 18 , wherein said inhibitory agent is a polynucleotide agent.

20 . The method of claim 18 , wherein said inhibitory agent is a small molecule agent.

Assignees

• Univ Ramot

Inventors

• Efrat Shimon
• Sintov Elad

Classifications

• C12N15/113

  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)} · CPC title

• C12N5/0678 Primary

  Stem cells; Progenitor cells; Precursor cells · CPC title

• C12N2501/335

  Glucagon; Glucagon-like peptide [GLP]; Exendin · CPC title

• C12N2310/141

  MicroRNAs, miRNAs · CPC title

• C12N2501/16

  Activin; Inhibin; Mullerian inhibiting substance · CPC title