Compositions and Methods of Chromosomal Silencing

• US 20200131530A1
• Filed: 03/12/2018
• Published: 04/30/2020
• Est. Priority Date: 03/15/2017
• Status: Active Application

• Current Assignee: Carrygenes Bioengineering LLC
• Original Assignee: SynPloid Biotek, LLC
• Inventors: Perkins, Edward, Greene, Amy, Broccoli, Dominique
• Application Number:
  US16/494,252
• Publication Number:
  US 20200131530A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/208 of mammalian origin, e.g. minichromosome
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)

Abstract

The present invention encompasses compositions and methods to create a whole chromosome regulatable “kill” switch to inactivate expression of one or more therapeutic genes expressed from a synthetic chromosome.

Claims

1. A method for silencing genes expressed from a synthetic chromosome comprising:

providing mammalian target cells;

transfecting the mammalian target cells with a synthetic chromosome comprising at least one stably-integrated expression cassette expressing gene products from Xic; and

expressing the gene products from Xic, wherein the synthetic chromosome is inactivated and

expression of the gene products expressed from the synthetic chromosome are silenced.

2. The method of claim 1, wherein an entire Xic locus is stably integrated onto the synthetic chromosome.

3. The method of claim 1, wherein Xist has been stably integrated onto the synthetic chromosome.

4. The method of claim 3, wherein Tsix has been stably integrated onto the synthetic chromosome.

5. The method of claim 1, wherein the synthetic chromosome comprises mammalian centromere sequences.

6. The method of claim 5, wherein the synthetic chromosome comprises human centromere sequences.

7. The method of claim 1, wherein Xi st gene product from Xic is under control of an reguatable promoter.

8. The method of claim 7, wherein the regulatable promoter is regulated by a small molecule or regulatory RNA.

9. The method of claim 7, wherein the promoter is induced by the presence of tetracycline or a derivative thereof.

10. The method of claim 7, wherein the regulatable promoter is inactive in the presence of tetracycline or a derivative thereof.

11. The method of claim 1, wherein Tsix gene product from Xic is under control of a regulatable promoter.

12. The method of claim 11, wherein the regulatable promoter is regulated by a small molecule or regulatory RNA.

13. The method of claim 11, wherein the regulatable promotor is induced by the presence of tetracycline or a derivative thereof.

14. The method of claim 11, wherein the regulatable promoter is inactive in the presence of tetracycline or a derivative thereof.

15. The method of claim 1, wherein the mammalian target cells are human cells, and the synthetic chromosome comprises human centromere sequences.

16. The method of claim 15, wherein the mammalian target cells are human mesenchymal stem cells.

17. The method of claim 1, wherein Xic is delivered to the synthetic chromosome via λ

INTR integrase mediated targeting (catty x attB recombination).

18. A synthetic chromosome comprising a whole chromosome kill switch.

19. The synthetic chromosome of claim 18, comprising an entire Xic region from an X chromosome.

20. The synthetic chromosome of claim 18, comprising a Xist locus.

21. The synthetic chromosome of claim 20, further comprising a Tsix locus.

22. The synthetic chromosome of claim 18, further comprising at least one gene of interest.

23. The synthetic chromosome of claim 22, further comprising at least ten genes of interest.

24. The synthetic chromosome of claim 23, further comprising at least twenty genes of interest.

25. The synthetic chromosome of claim 24, further comprising at least 25 genes of interest.

26. The synthetic chromosome of claim 18, wherein the synthetic chromosome comprises mammalian centromere sequences.

27. The synthetic chromosome of claim 26, wherein the synthetic chromosome comprises human centromere sequences.

28. A human cell comprising the synthetic chromosome of claim 26.

29. A cell comprising the synthetic chromosome of claim 18.

30. A method for silencing genes expressed from a synthetic chromosome comprising:

providing mammalian target cells;

transfecting the mammalian target cells with a synthetic chromosome comprising at least one stably-integrated expression cassette expressing gene products from Xic;

expressing the gene products from Xic; and

inactivating the synthetic chromosome where expression of the gene products expressed from the synthetic chromosome are silenced.