METHODS FOR THE PRODUCTION OF IPS CELLS USING NON-VIRAL APPROACH

• US 20100003757A1
• Filed: 06/04/2009
• Published: 01/07/2010
• Est. Priority Date: 06/04/2008
• Status: Active Grant

• Current Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Original Assignee: Cellular Dynamics International, Inc. (Fujifilm Holdings Corporation)
• Inventors: Thomson, James, Mack, Amanda
• Granted Patent: US 8,546,140 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/605 Nanog
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2501/608 Lin28
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2710/16222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2800/107 for mammalian
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2800/24 Vectors characterised by the absence of particular element, e.g. selectable marker, viral origin of replication
• CPC Class Codes: C12N 2820/60 from viruses
• CPC Class Codes: C12N 2840/105 enhancing translation
• CPC Class Codes: C12N 2840/206 having multiple IRES
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Methods and composition of induction of pluripotent stem cells and other desired cell types are disclosed. For example, in certain aspects methods for generating essentially vector-free induced pluripotent stem cells are described. Furthermore, the invention provides induced pluripotent stem cells and desired cell types essentially free of exogenous vector elements with the episomal expression vectors to express differentiation programming factors.

Claims

1. An iPS cell population that is essentially free of exogenous retroviral elements, the cell population comprising the genome of a selected human individual.

3. The iPS cell population of claim 1 or 2, wherein the iPS cell population is essentially free of exogenous viral vector elements.

17. The method of claim 1, wherein the trans-acting factor corresponds to EBNA-1 of EBV.

18. The method of claim 1, wherein the trans-acting factor is a derivative of a wild-type protein corresponding to EBNA-1 of EBV, which derivative has a reduced ability to activate transcription from an integrated template as compared to wild-type EBNA-1.

19. The method of claim 1, wherein the trans-acting factor is a derivative of a wild-type protein corresponding to EBNA-1 of EBV, which derivative activate transcription at levels at least 5% that of the corresponding wild-type protein from an extra-chromosomal template after the derivative binds the replication origin.

20. The method of claim 1, wherein the trans-acting factor is a derivative of wild-type protein corresponding to EBNA-1 of EBV, which derivative has a deletion of residues corresponding to residues about 65 to about 89 of EBNA-1, and/or a deletion of residues corresponding to residues about 90 to about 328 of EBNA-1.

21. The method of claim 18, wherein the derivative encodes a protein with at least 80% amino acid sequence identity to residues 1 to about 40 and residues about 328 to 641 of EBNA-1.

2. An iPS cell population comprising cells whose genome is derived from a terminally differentiated human cell and essentially free of exogenous retroviral elements.

4. A method of providing a cell population having an altered differentiation status relative to a starting cell population and having cells that are essentially free of programming vector genetic elements, the method comprising the steps of:

a) obtaining a starting population of cells having a first differentiation status;

b) obtaining one or more differentiation programming vectors, each vector comprising a replication origin and one or more expression cassettes encoding one or more differentiation programming factors that, in combination, can alter the differentiation status of the starting cell population to a second differentiation status, wherein one or more of said expression cassettes comprise a nucleotide sequence encoding a trans-acting factor that binds to the replication origin to replicate an extra-chromosomal template, and/or wherein the cells of the starting population express such a trans-acting factor;

c) introducing the differentiation programming vector(s) into cells of the starting population;

d) culturing the cells to effect expression of said one or more reprogramming factors such that traits consistent with the second differentiation status arise in at least a portion of cells in the cultured cells; and

e) further culturing cells having the traits for a sufficient number of generations to provide a target cell population that comprise cells having the second differentiation status but which cells are essentially free of programming vector genetic elements.

5. The method of claim 4, wherein said altering the differentiation status is reprogramming.

6. The method of claim 4, wherein said altering the differentiation status is differentiation.

7. The method of claim 4, wherein said altering the differentiation status is transdifferentiation.

8. The method of claim 5, wherein said starting cell is a somatic cell and said traits are defined as one or more characteristics of embryonic stem cells.

9. The method of claim 5, wherein the starting cell is a fibroblast, a keratinocyte, a hematopoietic cell, a mesenchymal cell, a liver cell, a stomach cell, or a β

cell.

10. The method of claim 5, wherein the differentiation programming factors are further defined as reprogramming factors comprising Sox-2 and Oct-4.

11. The method of claim 10, wherein the reprogramming factors further comprise Nanog, Lin28, Klf4, or c-Myc.

12. The method of claim 6, wherein the starting cell is an embryonic stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a neural stem cell, a mesenchymal stem cell, a hematopoietic progenitor, an endoderm progenitor, a pancreatic progenitor, or an endothelial progenitor.

13. The method of claim 7, wherein the first and the second differentiation status are terminally differentiated.

14. The method of claim 4, wherein the replication origin is a replication origin of a lymphotrophic herpes virus, an adenovirus, SV40, a bovine papilloma virus, or a yeast.

15. The method of claim 14, the replication origin is a replication origin of a lymphotrophic herpes virus and corresponds to oriP of EBV.

16. The method of claim 15, wherein the lymphotrophic herpes virus is Epstein Barr virus (EBV), Kaposi'"'"'s sarcoma herpes virus (KSHV), Herpes virus saimiri (HS), or Marek'"'"'s disease virus (MDV).

22. The method of claim 4, further comprising an additional step of selecting cells of said cultured cells in step d) or e), which cells are essentially free of differentiation programming vector genetic elements.

23. The method of claim 4, further comprising an additional step of selecting cells of said cultured cells in step d) or e), which cells are essentially free of a selection marker comprised in the differentiation programming vector.

24. The method of claim 23, wherein the selection marker is herpes simplex virus-thymidine kinase, a antibiotic resistance factor, or a fluorescent protein.

25. The method of claim 8, further comprising a step of differentiating the target cell population.

26. A differentiation programming vector comprising a replication origin, and one or more expression cassettes encoding a trans-acting factor which binds to the replication origin to replicate the vector extra-chromosomally;

and one or more differentiation programming factors.

27. The differentiation programming vector of claim 26, wherein the differentiation programming factors are selected from the group consisting of Sox-2, Sox-7, Sox-17, Oct-4, Nanog, Lin-28, c-Myc, Klf4, Esrrb, EBF1, C/EBPα

, C/EBPβ

, Ngn3, Pdx and Mafa.

28. The differentiation programming vector of claim 26, further defined as a reprogramming vector comprising a Sox family member and an Oct family member.

29. The differentiation programming vector of claim 28, wherein the differentiation programming factors further comprise one or more selected from the group consisting of Nanog, Lin-28, Klf4, and c-Myc.

30. The differentiation programming vector of claim 26, wherein the differentiation programming vector lacks the ability to be integrated into a host cell genome.

31. The differentiation programming vector of claim 26, wherein the replication origin is a replication origin of a lymphotrophic herpes virus, an adenovirus, SV40, a bovine papillomavirus, or a yeast.

32. The differentiation programming vector of claim 31, wherein the replication origin is a replication origin of a lymphotrophic herpes virus and corresponds to oriP of EBV.

33. The differentiation programming vector of claim 32, wherein the lymphotrophic herpes virus is Epstein Barr virus (EBV), Kaposi'"'"'s sarcoma herpes virus (KSHV), Herpes virus saimiri (HS), or Marek'"'"'s disease virus (MDV).

34. The differentiation programming vector of claim 26, wherein the trans-acting factor corresponds to EBNA-1 of EBV.

35. The differentiation programming vector of claim 26, wherein the trans-acting factor is a derivative of a wild-type protein corresponding to EBNA-1 of EBV, which derivative activates transcription at least 5% that of the corresponding wild-type protein from an extra-chromosomal template after binding to the replication origin and has a reduced ability to activate transcription from an integrated template as compared to wild-type EBNA-1.

36. The differentiation programming vector of claim 35, wherein the derivative lacks sequences present in the wild-type EBNA-1 protein that activate transcription from an integrated template.

37. The differentiation programming vector of claim 35, wherein the derivative has a deletion of residues corresponding to residues about 65 to about 89 of EBNA-1 and/or a deletion of residues corresponding to residues about 90 to about 328 of EBNA-1.

39. The differentiation programming vector of claim 35, wherein the derivative comprises a first nucleotide sequence encoding residues 1 to about 40 of the corresponding wild-type EBNA-1 and a second nucleotide sequence encoding residues about 328 to 641 of the corresponding wild-type EBNA-1.

38. The differentiation programming vector of 35, wherein the derivative encodes a derivative with at least 80% amino acid sequence identity to residues 1 to about 40 and residues about 328 to 641 of EBNA-1.