MICRORNAS AND CELLULAR REPROGRAMMING

• US 20150247125A1
• Filed: 11/01/2013
• Published: 09/03/2015
• Est. Priority Date: 11/02/2012
• Status: Abandoned Application

• Current Assignee: Lonza Walkersville
• Original Assignee: Lonza Walkersville
• Inventors: Fellner, Thomas, Friedrich Ben Nun, Inbar
• Application Number:
  US14/439,245
• Publication Number:
  US 20150247125A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/60 Transcription factors
• 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 2501/608 Lin28
• CPC Class Codes: C12N 2501/65 MicroRNA
• CPC Class Codes: C12N 2506/11 from blood or immune system cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Methods and compositions for improved cellular reprogramming to generate induced pluripotent stem cells.

Claims

1. A method for generating induced pluripotent stem cells, the method comprising introducing at least one nucleic acid encoding an miRNA and/or at least one nucleic acid encoding an anti-miRNA into a differentiated cell, and treating the differentiated cell under conditions suitable for development of an iPSC.

2. The method of claim 1, wherein the at least one miRNA is capable of hybridizing to an mRNA selected from the group consisting of CDKN1A, DOT1L, SUV39H1.

3. The method of claim 1, wherein the at least one miRNA is selected from the group consisting of miR-302 (a, b, c &

d), miR-367(3p &

5p), miR372.

4. The method of claim 1, wherein the at least one anti-miRNA is capable of hybridizing to an miRNA selected from the group consisting of Let7, miR-29.

5. The method of claim 1, wherein the anti-miRNA targets an miRNA capable of hybridizing to an mRNA selected from the group consisting of MYC, LIN28, BCL2, DNM3B, DNM3A, BCL2, and CDK6.

6. The method of claim 1, wherein the at least one anti-miRNA is selected from the group consisting of Anti-Let7a and Anti-miR29a.

7. The method of claim 1, further comprising with at least one additional reprogramming factor.

8. The method of claim 7, wherein the additional reprogramming factor is selected from the group consisting of Oct4, Sox2, Klf4, Myc, Lin28, and SV40 Large T Antigen, and/or nucleic acids encoding the same.

9. The method of claim 8, wherein the culture medium comprises Oct4, Sox2, Klf4, Myc, Lin28, miR-302 (a, b, c &

d), miR-367(3p &

5p), miR372, Anti-Let7a, Anti-miR29a, and optionally SV40 Large T antigen, or nucleic acids encoding the same.

10. An iPSC culture obtained by the method of claim 1.

12. The culture medium of claim 9, wherein the at least one miRNA is capable of hybridizing to an mRNA selected from the group consisting of CDKN1A, DOT1L, SUV39H1.

13. The culture medium of claim 9, wherein the at least one miRNA is selected from the group consisting of miR-302 (a, b, c &

d), miR-367(3p &

5p), miR372.

14. The culture medium of claim 9, wherein the at least one anti-miRNA is capable of hybridizing to an miRNA selected from the group consisting of Let7, miR-29.

15. The culture medium of claim 9, wherein the anti-miRNA targets an miRNA capable of hybridizing to an mRNA selected from the group consisting of MYC, LIN28, BCL2, DNM3B, DNM3A, BCL2, and CDK6.

16. The culture medium of claim 9, wherein the at least one anti-miRNA is selected from the group consisting of Anti-Let7a and Anti-miR29a.

17. The culture medium of claim 9, further comprising with at least one additional reprogramming factor.

18. The culture medium of claim 17, wherein the additional reprogramming factor is selected from the group consisting of Oct4, Sox2, Klf4, Myc, Lin28, and SV40 Large T Antigen, and/or nucleic acids encoding the same.

19. The culture medium of claim 18, wherein the culture medium comprises Oct4, Sox2, Klf4, Myc, Lin28, miR-302 (a, b, c &

d), miR-367(3p &

5p), miR372, Anti-Let7a, and Anti-miR29a, and optionally SV-40 Large T antigen, or nucleic acids encoding the same.

11. A culture medium for generating induced pluripotent stem cells, the culture medium comprising at least one nucleic acid encoding an miRNA and/or at least one nucleic acid encoding an anti-miRNA into a differentiated cell, and optionally other factors suitable for development and growth of an iPSC.

20. A kit for reprogramming a cell to generate an iPSC, the kit comprising at least one nucleic acid encoding an miRNA and/or at least one nucleic acid encoding an anti-miRNA, and a suitable delivery system.

21. A method for identifying miRNA capable of inducing expression of factors involved in inducing development of a pluripotent phenotype is also provided, the method comprising monitoring expression of miRNA in human embryonic stem cells (“

hESC”

) and isolating miRNA that are over-expressed in the hESC relative to differentiated cells.

22. A method for identifying miRNA capable of inhibiting expression of factors involved in inducing development of a pluripotent phenotype, the method comprising the method comprising monitoring expression of miRNA in a differentiated cell and isolating miRNA that are over-expressed in the differentiated cell relative to hESC.