Selective oxidation of 5-methylcytosine by TET-family proteins

Inventors

Rao, AnjanaTahiliani, MamtaKoh, Kian PengAgarwal, SuneetIyer, Aravind

Assignees

United States, Reprsented By Secretary Department Of Health & Human Services ASBoston Childrens HospitalUS Department of Health and Human Services

Publication Number

US-12331346-B2

Publication Date

2025-06-17

Expiration Date

2029-09-28

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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.

Core Innovation

The present invention provides novel methods for regulating and detecting the cytosine methylation status of DNA. It is based on the identification of a new catalytic activity of the TET family of proteins—TET1, TET2, TET3, and CXXC4—capable of converting 5-methylcytosine into 5-hydroxymethylcytosine via hydroxylation.

The invention addresses the problem that DNA methylation and demethylation are vital processes in mammalian development, differentiation, aging, and tumorigenesis, but the molecular basis of active DNA demethylation was unknown. Existing methods lacked tools to detect or regulate this process effectively, limiting the ability to study or manipulate stem cell pluripotency, reprogramming, or cancer-related aberrations in DNA methylation.

The invention further provides methods and reagents for enhancing reprogramming of somatic cells into pluripotent stem cells by increasing the rate and/or efficiency of induced pluripotent stem (iPS) cell generation, using TET family enzymes or their derivatives. It also provides novel ways to generate stable human regulatory Foxp3+ T cells and diagnostic and therapeutic methods for myeloid cancers connected with TET family mutations, alongside novel detection methods for 5-hydroxymethylcytosine.

Claims Coverage

The patent contains multiple independent claims covering methods, kits, and compositions involving TET family enzymes and their derivatives for detection, regulation, and therapeutic applications related to DNA methylation and hydroxymethylation.

Detection of methylated cytosine using TET family polypeptides

Contacting mammalian nucleic acids comprising methylated cytosine residues with polypeptides having at least 90% sequence identity to TET1, TET2, TET3, or CXXC4, or their catalytically active fragments or variants, to generate modified methylated cytosine residues for detection.

Conversion of 5-methylcytosine to 5-hydroxymethylcytosine for mapping methylation

Use of catalytically active TET family enzymes or derivatives to convert 5-methylcytosine into 5-hydroxymethylcytosine in nucleic acids, enabling robust detection methods.

Derivatization of modified methylated cytosine with glucose or derivatives

Derivatizing the modified methylated cytosine residues in nucleic acids produced by TET activity with glucose molecules or glucose-derivative donor substrates, such as using alpha- and beta-glucosyltransferases from bacteriophages, facilitating downstream detection or purification.

Improvement of somatic cell reprogramming to pluripotency

Contacting somatic cells with catalytically active TET family enzymes, derivatives, or catalytic fragments, optionally combined with pluripotency factor delivery, to improve efficiency or rate of induced pluripotent stem cell production.

Improving efficiency of mammalian cloning by nuclear transfer

Contacting nuclei extracted for nuclear transfer protocols with an effective hydroxylating amount of catalytically active TET family enzymes or derivatives to enhance cloning efficiency.

Generation of stable human Foxp3+ regulatory T cells using TET enzymes

Methods involving delivering catalytically active TET family proteins or derivatives to human T cells to increase conversion of 5-methylcytosine to 5-hydroxymethylcytosine and improve generation of stable human regulatory Foxp3+ T cells.

Diagnostic and therapeutic methods for cancers linked to TET activity

Use of modulators of TET family enzyme hydroxylase activity to diagnose, treat, or modulate progression of cancers, especially myeloid malignancies, including molecular screening for TET modulators and assessing 5-hydroxymethylcytosine levels.

Kits comprising TET enzymes and related reagents for methylation analysis and therapy

Kits including catalytically active TET family proteins or encoding nucleic acids, enzymes from T-even bacteriophages, glucose donor substrates, detection proteins, and reagents for cell reprogramming or cloning applications.

The claims comprehensively cover methods of detecting and modifying methylation status in nucleic acids using TET family proteins, compositions and kits including TET proteins and related enzymes for biochemical and therapeutic applications, methods enhancing cellular reprogramming and cloning, generation of regulatory T cells, and diagnosis and treatment of cancers related to TET function. The inventive features focus on the novel catalytic activity of TET enzymes converting 5-methylcytosine to 5-hydroxymethylcytosine and leveraging this for detection, regulation, and therapy.

Stated Advantages

The invention provides novel methods to regulate DNA methylation status by exploiting TET family enzymatic activity.

It enables increased efficiency and rate of induced pluripotent stem cell generation from somatic cells.

Stable human regulatory Foxp3+ T cells can be generated more effectively by modulating 5-methylcytosine conversion.

Novel diagnostic and therapeutic approaches for myeloid cancers utilizing detection of hydroxymethylcytosine and TET family activity are provided.

The invention offers improved detection methods for 5-hydroxymethylcytosine, which is not recognized by existing methylcytosine probes.

Compositions and kits facilitate manipulation and characterization of methylation and hydroxymethylation states, advancing research and clinical applications.

Documented Applications

Regulating cytosine methylation and hydroxymethylation status of DNA.

Enhancing reprogramming of somatic cells into pluripotent stem cells to improve induced pluripotent stem cell production.

Generation of stable human regulatory Foxp3+ T cells.

Improving efficiency of mammalian cloning via nuclear transfer or nuclear transplantation.

Detecting and mapping 5-hydroxymethylcytosine in genomic DNA using enzymatic glucosylation and chemical derivatization.

Diagnosing and treating myeloid cancers including myeloproliferative disorders, myelodysplastic syndromes, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia.

Screening for modulators of TET enzymatic activity for use in cancer therapies.

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