Stem cell differentiation

• US 20040053869A1
• Filed: 08/05/2003
• Published: 03/18/2004
• Est. Priority Date: 08/19/2000
• Status: Abandoned Application

• Current Assignee: Axordia Limited (Regenerative Medicine Assets Ltd.)
• Original Assignee: Axordia Limited (Regenerative Medicine Assets Ltd.)
• Inventors: Andrews, Peter, Walsh, James, Gokhale, Paul
• Application Number:
  US10/344,928
• Publication Number:
  US 20040053869A1
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• 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 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

The invention relates to a method to modulate stem cell differentiation comprising introducing inhibitory RNA (RNAi) into a stem cell to ablate mRNA'"'"'s which encode polypeptides which are involved in stem cell differentiation; RNAi molecules, DNA molecules encoding said RNAi molecules; and cells obtained by said method.

Claims

1. An in vitro method to modulate the differentiation state of a pluripotential stem cell selected from the group consisting of:

an embryonic stem cell, an embryonic germ cell or a teratocarcinoma stem cell comprising;

(i) contacting said stem cell with at least one inhibitory RNA molecule (RNAi) comprising a sequence of a gene, or the effective part thereof, which mediates at least one step in the differentiation of said cell;

(ii) providing conditions conducive to the growth and differentiation of the cell treated in (i) above; and

optionally

(iii) maintaining and/or storing the cell in a differentiated state.

2. A method according to claim 1 wherein said stem cell is an embryonic stem cell.

3. A method according to claim 1 wherein said stem cell is an embryonic germ cell.

4. A method according to claim 1 wherein said stem cell is a teratocarcinoma cell.

5. A method according to any of claims 1-4 wherein said cell surface receptor is selected from the group consisting of:

human Notch 1(hNotch

     1);

hNotch 2;

hNotch 3;

hNotch 4;

TLE-1;

TLE-2;

TLE-3;

TLE-4;

TCF7;

TCF7L1;

TCFFL2;

TCF3;

TCF19;

TCF1;

mFringe;

lFringe;

rFringe;

sel 1;

Numb;

Numblike;

LNX;

FZD1;

FZD2;

FZD3;

FZD4;

FZD5;

FZD6;

FZD7;

FZD8;

FZD9;

FZD10; and

FRZB.

6. A method according to any of claims 1-4 wherein said ligand is selected from the group consisting of:

D11-1;

D113;

D114;

Dlk-1;

Jagged 1;

Jagged 2;

Wnt 1;

Wnt 2;

Wnt 2b;

Wnt 3;

Wnt 3a;

Wnt5a;

Wnt6;

Wnt7a;

Wnt7b;

Wnt8a;

Wnt8b;

Wnt10b;

Wnt11;

Wnt14; and

Wnt15.

7. A method according to any of claims 1-4 wherein said gene is selected from the group consisting of:

SFRP1;

SFRP2;

SFRP4;

SFRP5;

SK;

DKK3;

CER1;

WIF-1;

DVL1;

DVL2;

DVL3;

DVL1L1;

mFringe;

lFringe;

rFringe;

sel11;

Numb;

LNX Oct4;

NeuroD1;

NeuroD2;

NeuroD3;

Brachyury;

MDFI;

CBF-1; and

CIR.

8. A method according to any of claims 1-7 wherein said gene comprises at least one of the genes identified by the DNA database accession numbers in Table 4.

9. A method according to claim 8 wherein said gene is selected from the group consisting of:

DLK1;

Oct 4;

hNotch 1;

hNotch 2;

RBPJk; and

CIR.

10. A method according to claim 9 wherein said gene is DLK1.

11. A method according to claim 10 wherein the RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2a.

12. A method according to claim 9 wherein said gene is Oct 4.

13. A method according to claim 12 wherein the RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2b.

14. A method according to claim 9 wherein said gene is hNotch 1.

15. A method according to claim 14 wherein said RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2c.

16. A method according to claim 9 wherein said gene is hNotch 2.

17. A method according to claim 16 wherein said RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2d.

18. A method according to claim 9 wherein said gene is RBBJk.

19. A method according to claim 18 wehrein said RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2e.

20. A method according to claim 9 wherein said gene is CIR.

21. A method according to claim 20 wherein said RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2f.

48. A cell obtainable by the method according to any of claims 1-21.

49. A cell obtainable by the method according to claim 48 wherein said cell is selected from the group consisting of:

a nerve cell;

a muscle cell;

liver cell;

a kidney cell;

a blood cell (eg erythrocyte, CD4+ cell, CD8+ cell;

panceatic β

cell;

epithelial cell (eg lung, gastric, intestinal).

50. A cell culture obtainable by the method according to any of claims 1-21.

51. An organ comprising at least one cell according to claim 48 or 49.

22. A RNAi molecule characterised in that said molecule comprises the coding sequence of at least one gene which mediates at least one step in stem cell differentiation.

23. A RNAi molecule according to claim 22 wherein said coding sequence is an exon.

24. A RNAi molecule according to claim 22 or 23 wherein said molecule is between 100 bp-1000 bp in length.

25. A RNAi molecule according to claim 24 wherein the length of said molecule is selected from the group consisting of:

100 bp;

200 bp;

300 bp;

400 bp;

500 bp;

600 bp;

700 bp;

800 bp;

900 bp;

or 1000 bp.

26. A RNAi molecule according to claim 22 or 23 wherein the length of said molecule is at least 1000 bp.

27. A RNAi molecule according to claim 22 or 23 wherein the length of said molecule is between 15 bp and 25 bp.

28. A RNAi molecule according to claim 27 wherein the length of said molecule is 21 bp.

29. A RNAi molecule according to any of claims 22-28 wherein said molecule comprises a sequence identified by the DNA database accession numbers in Table 4.

30. A RNAi molecule according to claim 29 wherein said RNAi is derived from a gene selected from the group consisting of:

DLK1;

Oct 4;

hNotch 1;

hNotch 2;

RBPJk; and

CIR.

31. A RNAi molecule according to claim 30 wherein said RNAi molecule comprises the nucleic acid sequence selected from the group consisting of the nucleic acid sequences presented in FIGS. 2a-2f.

32. A RNAi molecule according to any of claims 22-31 wherein said molecule comprises modified ribonucleotide bases.

42. A method to promote the differentiation of stem cells comprising administering to an animal an effective amount of RNAi according to any of claims 22-32 sufficient to effect differentiation of a target stem cell.

43. A therapeutic composition comprising at least one RNAi molecule according to any of claims 22-32.

44. The use of at least one RNAi molecule according to any of claims 22-32 for the manufacture of a medicament for use in promoting the differentiation of stem cells to provide differentiated cells/tissues to treat diseases where cell/tissues are destroyed by said disease.

45. The use according to claim 44 wherein said disease is selected from the group consisting of:

pernicious anemia;

stroke, neurodegenerative diseases such as Parkinson'"'"'s disease, Alzhiemer'"'"'s disease;

coronary heart disease;

cirrhosis; and

diabetes.

46. A therapeutic composition according to claim 43 or the use according to claim 44 or 45 which further comprises a diluent, carrier or excipient.

47. A therapeutic cell composition comprising a differentiated cell produced by introduction of a RNAi molecule according to any of claims 22-32.

33. Use of an isolated DNA molecule comprising a sequence of a gene which mediates at least one step in stem cell differentiation as represented by the DNA accession numbers identified in Table 4 wherein said DNA molecule is operably linked to at least one further DNA molecule capable of promoting transcription of said DNA linked thereto for the manufacture of a composition for use in mediating in vitro stem cell differentiation.

34. Use according to claim 33 wherein said gene is DLK1.

35. An isolated DNA molecule comprising a sequence of a gene which mediates at least one step in stem cell differentiation as represented by the accession numbers identified in Table 4, excluding NM_—

003836, wherein said DNA molecule is operably linked to at least one further DNA molecule capable of promoting transcription of said DNA linked thereto.

36. An isolated DNA molecule according to claim 35 wherein said DNA molecule is selected from the group consisting of:

Oct 4;

hNotch 1;

hNotch 2;

RBPJk; and

CIR.

37. An isolated DNA molecule according to claim 36 wherein said molecule comprises a sequence selected from the group consisting of the sequences as represented in FIGS. 2a-2f.

38. An isolated DNA molecule according to any of claims 35-37 wherein said gene is provided with at least two promoters characterised in that said promoters are oriented such that both DNA strands comprising said DNA molecule are transcribed into RNA.

39. A vector including a DNA molecule according to any of claims 35-38.

40. A method to manufacture RNAi molecules comprising:

(i) providing at least one isolated DNA molecule according to any of claims 35-38 or a vector according to claim 39;

(ii) providing reagents and conditions which allow the synthesis of each RNA strand comprising said RNAi molecule; and

(iii) providing conditions which allow each RNA strand to associate over at least part of their length, or at least that part corresponding to the nucleic acid sequence encoding said stem cell gene which mediates stem cell differentiation.

41. A method according to claim 40 wherein said gene is selected from those genes identified by the DNA database accession numbers in Table 4.