Selective oxidation of 5-methylcytosine by TET-family proteins
Inventors
Rao, Anjana • Tahiliani, Mamta • Koh, Kian Peng • Agarwal, Suneet • Iyer, Aravind
Assignees
Boston Childrens Hospital • US Department of Health and Human Services
Publication Number
US-10465234-B2
Publication Date
2019-11-05
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 invention identifies a novel and surprising enzymatic hydroxylase activity for the family of TET proteins including TET1, TET2, TET3, and CXXC4. Specifically, these enzymes catalyze the conversion of the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation. This discovery is used in novel methods for regulating and detecting the cytosine methylation status of DNA.
The problem being solved addresses the lack of molecular entities capable of active DNA demethylation. While passive replication-dependent demethylation is understood, enzymes that actively demethylate DNA were previously unknown. Additionally, no techniques or reagents could distinguish or specifically detect 5-hydroxymethylcytosine in DNA, as it is not recognized by existing 5-methylcytosine-binding proteins or antibodies. This has limited the understanding, detection, and modulation of DNA methylation processes involved in mammalian development, cell differentiation, tumorigenesis, and cancer.
Claims Coverage
The patent claims center on methods and compositions involving TET family proteins and their catalytic activity converting 5-methylcytosine to 5-hydroxymethylcytosine, and on detecting and modulating this DNA modification. Three independent claims focus on cancer treatment, as well as gene-derived siRNA modulation.
Method of treating cancer by modulating TET enzyme activity with siRNA
A method of administering an agent comprising an siRNA that modulates the activity of an enzyme converting methylated cytosine to hydroxymethylated cytosine in a subject with or at risk of cancer.
Use of agents targeting TET family enzymes for cancer treatment
Use of agents, including siRNA, to inhibit or activate TET family enzyme catalytic activity (TET1, TET2, TET3, or CXXC4) to treat cancer, including leukemia and myeloid leukemia.
Specific sequences and protein fragments of TET enzymes as therapeutic agents
Definition and use of functional TET family derivatives and catalytic fragments comprising specific sequences (SEQ ID NOs: 1 to 5) as agents modulating DNA hydroxymethylation involved in cancer and other applications.
The independent claims cover methods of modulating TET enzyme activity using agents such as siRNA for treating cancers, especially leukemia, involving TET family proteins and their catalytic functions. The inventive features define key molecular components and their use for diagnosis and therapy.
Stated Advantages
The TET family enzymes provide novel, non-transcription factor targets to modulate and regulate cellular differentiation status and pluripotency.
The discovery of TET enzymatic activity enables improved methods for reprogramming somatic cells into pluripotent stem cells and enhances stem cell therapies.
Methods and reagents based on TET proteins allow for improved detection and mapping of 5-hydroxymethylcytosine, enabling novel diagnostic and therapeutic strategies particularly in cancer.
Use of TET family activity modulators offers new avenues for cancer diagnosis and treatment, especially myeloid cancers characterized by aberrant methylation patterns.
Documented Applications
Regulating and detecting the cytosine methylation status of DNA; specifically modulating 5-methylcytosine conversion to 5-hydroxymethylcytosine.
Improving the efficiency or rate of generation of induced pluripotent stem (iPS) cells from somatic cells.
Enhancing the efficiency of cloning mammals by nuclear transfer or nuclear transplantation by treating isolated nuclei with TET enzymes during cloning protocols.
Producing stable human regulatory Foxp3+ T cells by delivering catalytically active TET proteins or derivatives to human T cells.
Methods for diagnosing and treating myeloid cancers including myeloproliferative disorders, myelodysplastic syndromes, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia.
Using antibodies and biochemical reagents to detect and isolate 5-hydroxymethylcytosine and its derivatives in genomic DNA for expanded methylation studies and diagnostics.
Screening methods to identify modulators of TET family activity for therapeutic use, including high-throughput screening for anti-cancer agents.
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