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