Compositions comprising glucosylated hydroxymethylated bases
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-10774373-B2
Publication Date
2020-09-15
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 provides novel methods and compositions for regulating and detecting the cytosine methylation status of DNA, based on the identification of a novel catalytic activity for the family of TET proteins (TET1, TET2, TET3, and CXXC4). This activity converts the cytosine nucleotide 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) by hydroxylation.
This novel enzyme activity enables new approaches to modulate and regulate pluripotency and cellular differentiation status, including reprogramming somatic cells into induced pluripotent stem (iPS) cells, improving stem cell function and therapies, generating stable human regulatory Foxp3+ T cells, and enhancing cloning efficiency by nuclear transfer. The invention also provides new diagnostic and therapeutic methods for cancers, notably myeloid cancers, by detecting and modulating hydroxymethylation defects linked to TET family mutations.
Claims Coverage
The patent includes multiple inventive features focusing on compositions of nucleic acids with glucosylated hydroxymethylated cytosines, and methods and compositions related to TET enzymes' activity and detecting such modifications.
Isolated nucleic acid containing glucosylated hydroxymethylated cytosine
Isolation of nucleic acids from extracellular fluid samples comprising glucosylated hydroxymethylated cytosines, including genomic DNA with glucosylated 5-hydroxymethylcytosine, optionally labeled.
Compositions for modification and detection of glucosylated nucleic acid
Compositions comprising glucosylated nucleic acids and isolated dioxygenase enzymes or TET family enzymes or fragments, optionally with cofactors such as iron, to enable modulation of hydroxymethylcytosine levels.
Methods and kits for detection and purification of modified DNA
Methods and kits for detecting glucosylated 5-hydroxymethylcytosine in nucleic acids, including enzymatic labeling, affinity purification using biotinylated moieties or DNA-binding proteins, and sequencing techniques.
The claims cover isolated nucleic acids with glucosylated hydroxymethylated cytosines, compositions including TET enzymes for modulating or detecting these modifications, and various methods and kits facilitating detection, purification, and application of methylation status modulation.
Stated Advantages
The invention provides novel tools for regulating DNA methylation status to improve cellular reprogramming efficiency and pluripotency.
It enables generation of stable human regulatory Foxp3+ T cells.
It allows improved efficiency of cloning by nuclear transfer or nuclear transplantation.
Novel detection methods for 5-hydroxymethylcytosine enable accurate epigenetic profiling and disease diagnosis, including cancers.
Provides methods to identify and modulate TET enzyme activity for research and therapeutic purposes.
Documented Applications
Enhancement of reprogramming of somatic cells into induced pluripotent stem cells (iPS cells).
Generation of stable human regulatory Foxp3+ T cells for immunological tolerance and therapeutic uses.
Improved cloning efficiency of mammals by nuclear transfer or nuclear transplantation.
Stem cell therapy treatments involving increased pluripotency or differentiation to desired cell types.
Diagnosis and treatment methods for myeloid cancers including myelodysplastic syndrome, myeloproliferative disorders, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia.
Screening methods for identifying modulators of TET family enzyme activity for anticancer therapies.
Research tools and methods for detecting, purifying, and mapping 5-methylcytosine and 5-hydroxymethylcytosine in nucleic acids.
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