Claims for Patent: 8,524,498

Summary for Patent: 8,524,498

Title:	Methods and compositions for homologous recombination in human cells
Abstract:	The methods and compositions described herein are based, in part, on the discovery of a stem cell state in human cells that resembles the morphology observed in murine-derived stem cells. Induction of such a state in human stem cells permits an increase in the efficiency of homologous recombination. Thus, the methods and compositions described herein relate to cells and methods for increasing the efficiency of homologous recombination in human stem cells.
Inventor(s):	Geijsen; Niels (West Newbury, MA), Buecker; Christa (Somerville, MA)
Assignee:	The General Hospital Corporation (Boston, MA)
Application Number:	12/791,696
Patent Claims:	1. A composition comprising: (a) a human pluripotent stem cell in admixture with a GSK3.beta. inhibitor and a protein kinase A (PKA) inhibitor, and (b) a homologous recombination cassette. 2. The composition of claim 1, wherein the human pluripotent stem cell in the admixture is an iPS cell or an embryonic stem cell. 3. The composition of claim 1, wherein the GSK3.beta. inhibitor is selected from the group consisting of: CHIR99021, BIO, Kenpaullone, and SB216763. 4. The composition of claim 1, wherein the human pluripotent stem cell in the admixture has an enhanced efficiency of homologous recombination compared to a corresponding human pluripotent stem cell not in an admixture with the GSK3.beta. inhibitor. 5. The composition of claim 4, wherein the efficiency of homologous recombination is enhanced by at least 50% compared to a corresponding human pluripotent stem cell not in the admixture with the GSK3.beta. inhibitor. 6. The composition of claim 1, wherein the human pluripotent stem cell in the admixture survives expansion from, or can be expanded from, a single cell and wherein a corresponding human pluripotent stem cell not in an admixture with the GSK3.beta. inhibitor does not survive expansion from, or cannot be expanded from, a single cell. 7. The composition of claim 1, wherein the human pluripotent stem cell in the admixture survives passaging from a single cell by a non-collagenase digest, and wherein the corresponding human pluripotent stem cell not in an admixture with the GSK3.beta. inhibitor does not survive passaging from a single cell by a non-collagenase digest. 8. The composition of claim 1, wherein the human pluripotent stem cell in the admixture survives passaging from a single cell by trypsin digest, and wherein the corresponding human pluripotent stem cell not in an admixture with the GSK3.beta. inhibitor does not does not survive passaging from a single cell by trypsin digest. 9. The composition of claim 1, wherein the human pluripotent stem cell has a faster growth rate compared to the growth rate of a corresponding human pluripotent stem cell not in an admixture with the GSK3.beta. inhibitor. 10. The composition of claim 9, wherein the growth rate is assessed by measuring doubling time. 11. The composition of claim 10, wherein the doubling time is at least 2 hours faster than the growth rate of a corresponding human pluripotent stem cell not in an admixture with the GSK3.beta. inhibitor. 12. The composition of claim 1, further comprising a MEK inhibitor. 13. The composition of claim 12, wherein the MEK inhibitor is PD98059. 14. A stem cell composition comprising: (a) a human pluripotent stem cell clone isolated from a population of pluripotent stem cells treated with a GSK3.beta. inhibitor and a protein kinase A (PKA) inhibitor having the following characteristics: (i) survives propagation from a single cell, (ii) an efficiency of homologous recombination at least 1-fold higher than the efficiency of homologous recombination in a corresponding human pluripotent stem cell not treated with the GSK3.beta. inhibitor, (iii) a doubling time at least 2 hours faster than the growth rate of a corresponding human pluripotent stem cell not treated with the GSK3.beta. inhibitor, (iv) survives passaging as a single cell using trypsin digest, and (b) a homologous recombination cassette. 15. The composition of claim 14, wherein the GSK3.beta. inhibitor is selected from the group consisting of: CHIR99021, BIO, Kenpaullone, and SB216763. 16. The composition of claim 14, wherein the population of pluripotent stem cells is further treated with a MEK inhibitor. 17. The composition of claim 16, wherein the MEK inhibitor is PD98059. 18. The composition of claim 14, wherein the human pluripotent stem cell is an iPS cell or an embryonic stem cell. 19. The composition of claim 14, wherein the doubling time is 15-30 hours. 20. The composition of claim 14, wherein the doubling time is 20-24 hours. 21. A kit for enhancing the efficiency of homologous recombination in a human stem cell, the kit comprising: (a) a GSK3.beta. inhibitor, (b) a protein kinase A (PKA) inhibitor, (c) a homologous recombination cassette, and (d) instructions and packaging thereof. 22. The kit of claim 21, wherein the GSK3.beta. inhibitor is selected from the group consisting of: CHIR99021, BIO, Kenpaullone, and SB216763. 23. The kit of claim 21, further comprising a MEK inhibitor. 24. The kit of claim 23, wherein the MEK inhibitor is PD98059.

Details for Patent 8,524,498

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2029-05-29