METHODS FOR CREATING INTEGRATION-FREE, VIRUS-FREE, EXOGENOUS ONCOGENE-FREE IPS CELLS AND COMPOSITIONS FOR USE IN SUCH METHODS

• US 20190316095A1
• Filed: 04/16/2018
• Published: 10/17/2019
• Est. Priority Date: 04/16/2018
• Status: Active Grant

• Current Assignee: Cellular Engineering Technologies, Inc.
• Original Assignee: Cellular Engineering Technologies, Inc.
• Inventors: MOY, Alan B., KAMATH, Anant
• Granted Patent: US 11,268,070 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2330/51 Specially adapted vectors
• CPC Class Codes: C12N 2500/92 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/09 from epidermal cells, from skin cells, from oral mucosa cells
• CPC Class Codes: C12N 2506/11 from blood or immune system cells
• CPC Class Codes: C12N 2506/115 from monocytes, from macrophages
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2710/00041 Use of virus, viral particle or viral elements as a vector
• 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 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Methods are disclosed for reprogramming a somatic cell, including an adherent cell and a cell in suspension, into an induced pluripotent stem comprising expressing exogenous Sox-2, exogenous Klf-4, exogenous Oct3/4 from DNA that has not integrated into the genome of the somatic cell, suppressing p53 activity within the somatic cell, and exposing the somatic cell to reprogramming-assistance factors comprising an exogenous Alk-5 inhibitor, an exogenous histone deacetylase inhibitor, and an exogenous activator of glycolysis. Compositions and kits for use in such methods are also disclosed as are cells made by such a method.

Claims

1. A method for reprogramming a somatic cell into an induced pluripotent stem (iPS) cell in vitro comprising:

expressing exogenous sex determining region Y-box 2 (Sox-2), Kruppel-like factor 4 (Klf-4), and octamer-binding transcription factor 3/4 (Oct3/4) in the somatic cell from DNA that has not integrated into genomic DNA of the somatic cell;

inhibiting p53 activity in the somatic cell; and

culturing the somatic cell in a reprogramming medium comprising an exogenous activating receptor-like kinase 5 (Alk-5) inhibitor, an exogenous histone deacetylase inhibitor, and an exogenous activator of glycolysis to obtain an iPS cell.

2. The method according to claim 1, wherein the method further comprises expressing exogenous Epstein-Bar nuclear antigen-1 (EBNA-1) in the somatic cell from DNA that has not integrated into the genomic DNA of the somatic cell, and wherein the DNA that has not integrated into the genomic DNA of the somatic cell comprises at least one plasmid with an Epstein-Barr virus origin of replication (oriP).

3. The method according to claim 1, wherein one or more of lung myelocytomatosis oncogene (L-Myc), c-myelocytomatosis oncogene (c-Myc), Lin28, simian virus 40 large T antigen, and Nanog are not exogenously expressed.

4. The method according to claim 1, further comprising maintaining the cell in a dedifferentiation maintenance medium comprising basic fibroblast growth factor and transforming growth factor beta after being cultured in the reprogramming medium.

5. The method according to claim 1, wherein the culturing does not require feeder cells.

6. The method according to claim 3, wherein the reprogramming efficiency exceeds 0.0006%.

7. The method according to claim 1, wherein inhibiting p53 activity in the somatic cell inhibits p53-induced cell cycle arrest or p53-induced apoptosis.

8. The method according to claim 1, wherein inhibiting p53 activity in the somatic cell comprises suppressing p53 expression in the somatic cell.

9. The method according to claim 8, wherein suppressing p53 expression comprises expressing antisense p53 RNA in the somatic cell from DNA that has not integrated into the genomic DNA of the somatic cell.

10. The method according to claim 9, wherein the Alk-5 inhibitor comprises 3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), the histone deacetylase inhibitor comprises sodium butyrate or valproic acid, and the activator of glycolysis comprises a phosphoinositide-dependent protein kinase-1 inhibitor selected from 5-(4-Chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48);

α

,α

,-Dimethyl-4-[2-methyl-8-[2-(3-pyridinyl)ethynyl]-1H-imidazo[4,5-c]quinolin-1-yl]-benzeneacetonitrile (BAG956);

N-[3-[[5-Iodo-4-[[3-[(2-thienylcarbonyl)amino]propyl]amino]-2-pyrimidinyl]amino]phenyl]-1-pyrrolidinecarboxamide (BX795);

(3S,6R)-1-[6-(3-Amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide (GSK

     2334470);

2-Amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]acetamide (OSU03012); and

4-Dodecyl-N-1,3,4-thiadiazol-2-yl-benzenesulfonamide (PHT427).

11. The method according to claim 1, wherein the somatic cell is an isolated cord blood or peripheral blood mononuclear cell, and wherein the method further comprises pre-culturing the isolated cord blood or peripheral blood mononuclear cell in hematopoietic stem cell expansion media.

12. The method according to claim 2, which yields an integration-free, virus-free, exogenous oncogene-free iPS cell.

13. The method according to claim 12, wherein the iPS cell is a human iPS cell.

14. The method according to claim 12, wherein the iPS cell is differentiated into an endodermal, mesodermal, or ectodermal cell.

21. The method according to claim 1, the somatic cell being free of exogenous oncogenes and retroviruses.

22. The method according to claim 3, wherein the reprogramming efficiency exceeds 0.001%.

23. The method according to claim 1, wherein colonies of iPS cells are obtained and 100% of the colonies express stage-specific embryonic antigen-4 (SSEA-4).

24. The method according to claim 1, wherein colonies of iPS cells are obtained, the colonies expressing SSEA-4, Nanog, Oct3/4, and TRA160.

25. The method according to claim 1, the somatic cell being an adherent cell.

15. A method for reprogramming a somatic cell into an iPS cell in vitro comprising:

expressing exogenous Sox-2, Klf-4, and Oct-3/4 in the somatic cell from episomal DNA;

suppressing p53 expression in the somatic cell; and

culturing the somatic cell in a reprogramming medium comprising at least three different exogenous reprogramming-assistance factors, the exogenous reprogramming-assistance factors being at least an Alk-5 inhibitor, a histone deacetylase inhibitor, and an activator of glycolysis, to obtain an iPS cell free of exogenous oncogenes and exogenous viral elements.

17. The method according to claim 15, further comprising expressing EBNA-1 in the somatic cell and wherein Sox-2, Klf-4, Oct-3/4, and EBNA-1 are expressed from at least one plasmid with an Epstein-Barr virus origin of replication (oriP).

18. The method according to claim 15, wherein suppressing p53 expression in the somatic cell comprises expressing antisense p53 RNA in the somatic cell.

16. (canceled)

19. (canceled)

20. (canceled)