Protein delivery system to generate pluripotent stem (iPS) cells or tissue specific cells

• US 8,420,352 B2
• Filed: 08/27/2010
• Issued: 04/16/2013
• Est. Priority Date: 08/27/2009
• Status: Active Grant

• Current Assignee: Ipsen Pharma Sas (Ipsen Sa)
• Original Assignee: Synaptic Research, LLC
• Inventors: Oyler, George A., Chang, Yung-Nien
• Primary Examiner(s): Bunner, Bridget E
• Application Number:
  US12/870,782
• Publication Number:
  US 20110053244A1
• Time in Patent Office: 963 Days
• Field of Search: None
• US Class Current: 435/69.7
• CPC Class Codes: A61K 35/74 Bacteria therapeutic use of a bacterial protein A61K38/00
• CPC Class Codes: A61K 38/164 from bacteria
• CPC Class Codes: C07K 14/33 from Clostridium (G)
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/10 containing a tag for extracellular membrane crossing, e.g. TAT or VP22
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 5/00 Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; plant reproduction by tissue culture techniques A01H4/00
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A novel protein delivery system to generate induced pluripotent stem (iPS) cells is described. The delivery system comprises a construct with a receptor binding domain that recognizes a receptor in a somatic cell, a translocation domain that allows the transfer of an inducer into the cytosolic space, and a cargo bearing domain to which the inducer is attached and facilitates transfer of the inducer into the cell.

Claims

1. A construct for generating induced Pluripotent Stem (iPS) cells, comprising:

a receptor binding domain, a translocation domain, a cargo bearing domain, and an inducer, wherein the receptor binding domain, translocation domain, and cargo domain are atoxic Clostridium toxin domains.

2. The construct of claim 1, wherein the Clostridium toxin is a C. difficile toxin.

3. The construct of claim 2, wherein the C. difficile toxin is TcdA.

4. The construct of claim 2, wherein the C. difficile toxin is TcdB.

5. The construct of claim 2, wherein the inducer is selected from the group consisting of Sox2, Oct 3/4, Klf4, c-Myc, Nanog, lin28, hTERT (human telomerase), and SV40 large T-antigen.

6. The construct of claim 1, wherein the Clostridium toxin is a C. botulinum toxin.

7. The construct of claim 6, wherein the C. botulinum toxin is selected from the group consisting of BoNTA, BoNTB, BoNTC, BoNTD, BoNTE, BoNTF and BoNTG.

8. The construct of claim 6, wherein a receptor binding domain of the C. botulinum toxin is replaced with a non-specific receptor binding domain.

9. The construct of claim 6, wherein the inducer is selected from the group consisting of Sox2, Oct 3/4, Klf4, c-Myc, Nanog, lin28, hTERT (human telomerase), and SV40 large T-antigen.

10. The construct of claim 1, wherein the Clostridium toxin is a clostridial C2 toxin.

11. The construct of claim 10, wherein the inducer is selected from the group consisting of Sox2, Oct 3/4, Klf4, c-Myc, Nanog, lin28, hTERT (human telomerase), and SV40 large T-antigen.

12. The construct of claim 1, wherein the inducer is Oct3/4.

13. The construct of claim 12, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO:

1.

14. The construct of claim 12, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO:

2.

15. The construct of claim 12, wherein the construct has a sequence substantially identical to a sequence selected from the group consisting of SEQ ID NO:

1 and SEQ ID NO;

2, wherein no more than 20% of the amino acid residues vary between the construct sequence and SEQ ID NO;

1 or SEQ ID NO;

2.

16. The construct of claim 1, wherein the inducer is Sox2.

17. The construct of claim 16, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO:

5.

18. The construct of claim 16, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO:

6.

19. The construct of claim 16, wherein the construct has a sequence substantially identical to a sequence selected from the group consisting of SEQ ID NO:

5, SEQ ID NO;

6, and SEQ ID NO;

7, wherein no more than 20% of the amino acid residues vary between the construct sequence and SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7.

20. The construct of claim 1, wherein the inducer is selected from the group consisting of Klf4, c-Myc, Nanog, lin28, hTERT (human telomerase), and SV40 large T-antigen.

21. The construct of claim 1, wherein said cargo bearing domain is an inactive exotoxin domain.

22. A construct for generating induced Pluripotent Stem (iPS) cells, comprising:

(i) an atoxic Clostridium toxin comprising a receptor binding domain and a translocation domain; and

(ii) an inducer.

23. The construct of claim 22, wherein the Clostridium toxin is a C. difficile toxin.

24. The construct of claim 23, wherein the C. difficile toxin is TcdA.

25. The construct of claim 23, wherein the C. difficile toxin is TcdB.

26. The construct of claim 23, wherein the inducer is selected from the group consisting of Sox2, Oct 3/4, Klf4, c-Myc, Nanog, lin28, hTERT (human telomerase), and SV40 large T-antigen.

27. The construct of claim 22, wherein the Clostridium toxin is a C. botulinum toxin.

28. The construct of claim 27, wherein the C. botulinum toxin is selected from the group consisting of BoNTA, BoNTB, BoNTC, BoNTD, BoNTE, BoNTF and BoNTG.

29. The construct of claim 27, wherein a receptor binding domain of the C. botulinum toxin is replaced with a non-specific receptor binding domain.

30. The construct of claim 27, wherein the inducer is selected from the group consisting of Sox2, Oct 3/4, Klf4, c-Myc, Nanog, lin28, hTERT (human telomerase), and SV40 large T-antigen.

31. The construct of claim 22, wherein the Clostridium toxin is a clostridial C2 toxin.

32. The construct of claim 31, wherein the inducer is selected from the group consisting of Sox2, Oct 3/4, Klf4, c-Myc, Nanog, lin28, hTERT (human telomerase), and SV40 large T-antigen.

33. The construct of claim 22, wherein the inducer is Oct3/4.

34. The construct of claim 33, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO:

1.

35. The construct of claim 33, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO:

2.

36. The construct of claim 33, wherein the construct has a sequence substantially identical to a sequence selected from the group consisting of SEQ ID NO:

1 and SEQ ID NO;

2, wherein no more than 20% of the amino acid residues vary between the construct sequence and SEQ ID NO;

1 or SEQ ID NO;

2.

37. The construct of claim 22, wherein the inducer is Sox2.

38. The construct of claim 37, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO:

5.

39. The construct of claim 37, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO:

6.

40. The construct of claim 37, wherein the construct has a sequence substantially identical to a sequence selected from the group consisting of SEQ ID NO:

5, SEQ ID NO;

6, and SEQ ID NO;

7, wherein no more than 20% of the amino acid residues vary between the construct sequence and SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7.

41. The construct of claim 22, wherein the inducer is selected from the group consisting of Klf4, c-Myc, Nanog, lin28, hTERT (human telomerase), and SV40 large T-antigen.

42. The construct of claim 22, further comprising a cargo domain.