METHODS OF REPROGRAMMING CELLS

• US 20110280842A1
• Filed: 08/24/2009
• Published: 11/17/2011
• Est. Priority Date: 08/22/2008
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Zhou, Qiao, Melton, Douglas A.
• Granted Patent: US 9,683,215 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2799/022 where the vector is derived from an adenovirus
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides methods of reprogramming cells, for example, directly reprogramming a somatic cell of a first cell type into a somatic cell of a second cell type, are described herein. In particular, the present invention generally relates to methods for reprogramming a cell of an endoderm origin to a cell having pancreatic β-cell characteristics. The present invention also relates to an isolated population comprising reprogrammed cells, compositions and their use in the treatment of diabetes mellitus. In particular, the present invention relates to reprogramming a cell of an endoderm origin to a cell having pancreatic β-cell characteristics by increasing the protein expression of at least one transcription factor selected from Pdx1, Ngn3 or MafA in the cell of endoderm origin to reprogram the cell of an endoderm cell to a cell which exhibits at least one or at least two characteristics of an endogenous pancreatic β-cell.

Claims

1. A method for reprogramming a cell of endoderm origin, the method comprising increasing the protein expression of at least two transcription factors selected from Pdx1, Ngn3, or MafA in the cell of enodermal origin, wherein the cell of enodermal origin is reprogrammed to exhibit at least two characteristics of a pancreatic β

-cell.

3. The method of claim 1, wherein the cell of endoderm origin is selected from the group consisting of:

a pancreatic cell, a liver cell, and a gall bladder cell.

9. The method of claim 1, wherein a characteristic of a pancreatic β

-cell phenotype is secreting insulin in response to glucose.

10. The method of claim 1, wherein a characteristic of a pancreatic β

-cell phenotype is expression of at least one marker selected from the group consisting of;

Ngn3−

, Pdx1+ and MafA+.

11. The method of claim 1, wherein the protein expression of a transcription factor selected from Pdx1, Ngn3, or MafA is increased by contacting the cell of endoderm origin with an agent which increases the expression of the transcription factor.

12. The method of claim 11, wherein the agent is selected from the group consisting of:

a nucleotide sequence, a protein, an aptamer and small molecule, ribosome, RNAi agent and peptide-nucleic acid (PNA) and anologues or varients thereof.

20. The method of claim 1, wherein the cell of endoderm origin is in vitro.

21. The method of claim 1, wherein the cell of endoderm origin is ex vivo.

22. The method of claim 1, wherein the cell of endoderm origin is in vivo or present in a subject.

24. The method of claim 22, wherein the subject has, or is at risk of developing, diabetes or a metabolic disorder.

27. The method of claim 1, wherein the cell of endoderm origin is a mammalian cell.

29. The method of claim 1, wherein the method further comprises contacting the cell of endoderm origin with at least one agent which increases the protein expression of at least one of the transcription factors selected from the group consisting of:

NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6 or Isl1.

31. The method of claim 1, wherein the pancreatic β

-cell is a pancreatic β

-like cell.

32. The method of claim 31, wherein the pancreatic β

-like cell has an increased expression of a marker selected from the group consisting of;

c-peptide, glucose transporter 2 (Glut2), glucokinase (GCK), prohormone convertase 1/3 (PC1/3), β

-cell transcription factors NeuroD, Nkx2.2 and Nkx6.1 by a statistically significant amount relative to the cell of endoderm origin from which the pancreatic β

-like cell was derived.

33. The method of claim 31, wherein the pancreatic β

-like cell has a decreased expression of a marker selected from the group consisting of;

Amylase (Amy), glucagon, somatostatin/pancreatic polypeptide (SomPP), Ck19, Nestin, Vimentin and Tuji by a statistically significant amount relative to the cell of endoderm origin from which the pancreatic β

-like cell was derived.

2. (canceled)

4-8. -8. (canceled)

13-19. -19. (canceled)

23. (canceled)

25-26. -26. (canceled)

28. (canceled)

30. (canceled)

34. An isolated population of pancreatic β

-like cells obtained from a population of non-insulin producing cells of endoderm origin by a process comprising increasing the protein expression of at least two transcription factors selected from Ngn3, Pdx1 or MafA, or a functional fragment thereof, in the non-insulin producing cells of endoderm origin.

36. The isolated population of pancreatic β

-like cells of claim 34, wherein the pancreatic β

-like cells secrete insulin in response to an increase in glucose.

44. The isolated population of pancreatic β

-like cells of claim 34, wherein the population of non-insulin producing cells of endoderm origin is selected from the group consisting of;

a population of pancreatic cells, a population of liver cells, and a population of gall bladder cells.

48. The isolated population of pancreatic β

-like cells of claim 34, wherein the population of non-insulin producing cells of endoderm origin is a population of mammalian cells.

50. The isolated population of pancreatic β

-like cells of claim 34, wherein the population of non-insulin producing cells of endoderm origin is obtained from a subject that has diabetes, or has an increased risk of developing diabetes.

52. The isolated population of pancreatic β

-like cells of claim 34, wherein the population of non-insulin producing cells of endoderm origin is obtained from a subject that has a metabolic disorder, or has an increased risk of developing a metabolic disorder.

53. A method for the treatment of a subject with diabetes, the method comprising administering a composition comprising an isolated population of pancreatic β

-like cells according to claim 34.

35. (canceled)

37-43. -43. (canceled)

45-47. -47. (canceled)

49. (canceled)

51. (canceled)

54-64. -64. (canceled)

65. A composition comprising at least one non-insulin producing endodermal cell and at least one agent which increases the protein expression of at least two transcription factors selected from Ngn3, Pdx1 or MafA.

66-69. -69. (canceled)