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

• US 9,102,921 B2
• Filed: 04/12/2013
• Issued: 08/11/2015
• 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:
  US13/862,087
• Publication Number:
  US 20130288374A1
• Time in Patent Office: 851 Days
• Field of Search: None
• US Class Current: 1/1
• 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 method for generating induced Pluripotent Stem (iPS) cells, comprising:

contacting somatic cells with a construct that comprises 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 method of claim 1, further comprising the step of assessing the pluripotency of the somatic cells after contact with the construct.

3. The method of claim 1, wherein the construct is transfected into the somatic cells during contact.

4. The method of claim 1, wherein the Clostridium toxin is a C. difficile toxin.

5. The method of claim 4, wherein the C. difficile toxin is TcdA.

6. The method of claim 4, wherein the C. difficile toxin is TcdB.

7. The method of claim 1, wherein said cargo bearing domain is an inactive exotoxin domain.

8. The method of claim 1, wherein the Clostridium toxin is a C. botulinum toxin.

9. The method of claim 8, wherein the C. botulinum toxin is selected from the group consisting of BoNTA, BoNTB, BoNTC, BoNTD, BoNTE, BoNTF and BoNTG.

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

11. The method of claim 1, wherein the Clostridium toxin is a clostridial C2 toxin.

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

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

1.

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

2.

15. The method 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 method of claim 1, wherein the inducer is Sox2.

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

5.

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

6.

19. The method 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 and SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7.

20. The method 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.