EPIGENETIC MODIFICATION OF MAMMALIAN GENOMES USING TARGETED ENDONUCLEASES

• US 20170051354A1
• Filed: 04/24/2015
• Published: 02/23/2017
• Est. Priority Date: 04/28/2014
• Status: Abandoned Application

• Current Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation), Simga-Aldrich Co. LLC
• Original Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Inventors: DAVIS, Gregory D., KANG, Qiaohua
• Application Number:
  US15/306,720
• Publication Number:
  US 20170051354A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The present disclosure provides genetically engineered cell lines comprising chromosomally integrated synthetic sequences having predetermined epigenetic modifications, wherein a predetermined epigenetic modification is correlated with a known diagnosis, prognosis or level of sensitivity to a disease treatment. Also provided are kits comprising said epigenetically modified synthetic nucleic acids or cells comprising said epigenetically modified synthetic nucleic acids that can be used as reference standards for predicting responsiveness to therapeutic treatments, diagnosing diseases, or predicting disease prognosis.

Claims

1. A genetically modified cell line comprising at least one chromosomally integrated nucleic acid having a predetermined cytosine modification, wherein the cytosine modification is correlated with a known diagnosis, prognosis, or level of sensitivity to a disease treatment.

2. The genetically modified cell line of claim 1, wherein the cytosine modification is chosen from 5-methylcytosine (5mC), 3-methylcytosine (3mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxylcytosine (5caC).

3. The genetically modified cell line of claim 1, wherein the chromosomally integrated nucleic acid has a sequence with substantial sequence identity to that of a control element, a portion of a control element, a coding region, or a portion of a coding region of a gene associated with a disease.

4. The genetically modified cell line of claim 3, wherein the gene is a gene listed in Table 1.

5. The genetically modified cell line of claim 4, wherein the gene is chosen from MGMT, BRCA1, BRCA2, PITX2, GSTP1, APC, RASSF1, or HER2.

6. The genetically modified cell line of claim 1, wherein the chromosomally integrated nucleic acid is inserted into a chromosomal location in the cell using a targeting endonuclease.

7. The genetically modified cell line of claim 6, wherein the targeting endonuclease is chosen from a zinc finger nuclease, a CRISPR-based endonuclease, a meganuclease, a transcription activator-like effector nuclease (TALEN), a I-TevI nuclease or related monomeric hybrid, or an artificial targeted DNA double strand break inducing agent.

8. The genetically modified cell line of claim 1, wherein the chromosomally integrated nucleic acid replaces an endogenous chromosomal sequence from which the chromosomally integrated nucleic acid is derived.

9. The genetically modified cell line of claim 1, wherein the chromosomally integrated nucleic acid is inserted at a locus possessing adjacent insulating elements or other elements that assist in maintaining the original cytosine modification status of the chromosomally integrated nucleic acid.

10. The genetically modified cell line of claim 9, wherein the locus is chosen from AAVS1, CCR5, HPRT, or ROSA26.

11. The genetically modified cell line of claim 9, wherein endogenous chromosomal sequence corresponding to the chromosomally integrated nucleic acid is inactivated or deleted.

12. The genetically modified cell line of claim 1, further comprising at least one nucleic acid sequence encoding a recombinant protein.

13. The genetically modified cell line of claim 1, wherein the cell line is a human cell line.

14. The genetically modified cell line of claim 1, wherein the predetermined cytosine modification is stable.

15. The genetically modified cell line of claim 1, wherein the predetermined cytosine modification is metastable.

16. A kit for diagnosing a disease or predicting prognosis for a disease in a subject or for predicting responsiveness of a disease in a subject to a therapeutic treatment, the kit comprising at least one nucleic acid having a predetermined cytosine modification for use as a reference standard, wherein the cytosine modification is correlated with a known diagnosis, prognosis, or level of sensitivity to a disease treatment.

19. The kit of claim 16 comprising at least two nucleic acids, wherein each nucleic acid has a different predetermined cytosine modification.

20. The kit of claim 16, wherein the cytosine modification is chosen from 5-methylcytosine (5mC), 3-methylcytosine (3mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxylcytosine (5caC).

21. The kit of claim 16, wherein the nucleic acid has a sequence with substantial sequence identity to that of a control element, a portion of a control element, a coding region, or a portion of a coding region of a gene associated with a disease.

22. The kit of claim 21, wherein the gene is a gene listed in Table 1.

23. The kit of claim 22, wherein the gene is chosen from MGMT, BRCA1, BRCA2, PITX2, GSTP1, APC, RASSF1, or HER2.

24. The kit of claim 16, wherein the nucleic acid is located within a chromosome of a cell.

25. The kit of claim 24, wherein the cell is a fixed cell.

26. The kit of claim 24, wherein the nucleic acid is within genomic DNA isolated from the cell.

17-18. -18. (canceled)