SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

• US 20190233885A1
• Filed: 04/10/2019
• Published: 08/01/2019
• Est. Priority Date: 09/26/2008
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation, United States Department Of Health And Human Services (Government of the United States of America)
• Original Assignee: Children's Medical Center Corporation
• Inventors: Rao, Anjana, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet, Iyer, Aravind
• Granted Patent: US 11,072,818 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/71 Oxidoreductases (EC 1.)
• CPC Class Codes: C12N 2501/724 Glycosyltransferases (EC 2.4.)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2522/10 Nucleic acid binding proteins
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.

Claims

1. A method for improving the generation of stable human Foxp3+ T cells, the method comprising contacting with, or delivering to, a human T cell an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET family catalytically active fragment thereof, or combination thereof.

2. The method of claim 1, wherein the human T cell is a purified human CD4+ T cell.

3. The method of claim 1, further comprising contacting with, or delivering to, the human T cell a composition comprising at least one cytokine, growth factor, activating reagent, or combination thereof.

4. The method of claim 3, wherein the composition comprises TGF-β

.

5. A method for improving efficiency or rate with which an induced pluripotent stem (iPS) cell is produced from an adult somatic cell, the method comprising contacting with, or delivering to a somatic cell, an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active thereof, or combination thereof.

6. The method of claim 5, wherein the catalytically active TET family enzyme is TET1 or TET2.

7. The method of claim 5, further comprising contacting with, or delivering to, the somatic cell, an effective amount of a TET family inhibitor.

8. The method of claim 7, wherein the TET family inhibitor is a TET3 inhibitor.

9. The method of claim 5, further comprising contacting the somatic cell with or delivering to the somatic cell a combination of nucleic acid sequences encoding Oct-4, Sox2, c-MYC, and Klf4.

10. The method of claim 5, wherein the combination of nucleic acid sequences encoding Oct-4, Sox2, c-MYC, and Klf4 are delivered in a viral vector.

11. The method of claim 5, wherein the somatic cell is a fibroblast.

12. A method for improving efficiency of cloning a mammal by nuclear transfer or nuclear transplantation, the method comprising contacting a nucleus extracted from a cell to be cloned with an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, during a nuclear transfer protocol.

13. The method of claim 12, wherein the catalytically active TET family enzyme is TET1 or TET2.

14. The method of claim 12, further comprising contacting the nucleus from the cell to be cloned with a TET family inhibitor.

15. The method of claim 14, wherein the TET family inhibitor is a TET3 inhibitor.

16. A kit for modulating gene transcription via hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine, the kit comprising the following separate components:

a. at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, or nucleic acid molecule that comprises a sequence encoding at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment, or combination thereof in an appropriate buffer or solution; and

b. packaging materials and instructions therein to use the kit to hydroxylate 5-methylcytosine to 5-hydroxymethylcytosine, for the purpose of modulating gene transcription.

17. The kit of claim 16, further comprising a composition comprising at least one cytokine, growth factor, activating reagent, or combination thereof for the purposes of generating stable human Foxp3+ regulatory T cells.

18. The kit of claim 17, wherein the composition comprises TGF-β

.

19. The kit of claim 16, further comprising at least one nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding Oct-4, Sox2, c-MYC, and Klf4, or any combination thereof.

20. The kit of claim 19, wherein the nucleic acid sequences encoding Oct-4, Sox2, c-MYC, and Klf4 are incorporated into a viral vector.

21. The kit of claim 16, further comprising at least one reagent suitable for the detection of 5-hydroxymethylcytosine.

22. The kit of claim 21, wherein the reagent suitable for the detection of 5-hydroxymethylcytosine is an antibody, an antigen-binding portion thereof, an intrabody, or a protein, that specifically binds to 5-hydroxymethylcytosine.

23. The kit of claim 21, wherein the reagent suitable for the detection of 5-hydroxymethylcytosine is an antibody, an antigen-binding portion thereof, an intrabody, or a protein that is specific for cytosine-5-methylsulfonate.