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Principal Investigator: Michael-Christopher Keogh
Organization: EPICYPHER, INC.
Fiscal Year: 2022
Award: $500,000
Funding agency: National Institute on Aging
PROJECT SUMMARY
EpiCypher is collaborating with Dr. Jessica Tyler (an expert in aging, DNA repair and epigenetics), to
develop CUT&RUssNTM (Cleavage Under Targets and Release Using single-stranded Nuclease), a first-in-class
single-stranded DNA (ssDNA) mapping technology for research into the early pathogenesis of and possible
interventions for Alzheimer’s Disease (AD). The double-stranded conformation of genomic DNA (dsDNA) is
essential to maintain genome stability. ssDNA forms during many cellular processes, including transcription and
the processing of DNA lesions, and is rapidly sequestered by ssDNA binding proteins (SSBs) (e.g. RPA, RAD51
and BRCA1/BRCA2) to protect and facilitate any needed repair. AD is the most common form of
neurodegeneration, with early pathogenesis / neuronal cell death due in part to the accumulation of DNA damage
as a consequence of defective repair mechanisms (particularly homologous recombination [HR], which is heavily
reliant on ssDNA signaling pathways). Improved methods for detecting and mapping ssDNA and SSB-ssDNA
complexes that accompany DNA damage repair would greatly improve our understanding of how failure of these
pathways contributes to AD, and potentially reveal novel drug targets and biomarkers. However, tools to study
ssDNA-related signaling are lacking. The first innovation of our approach is the development of a novel
immunotethering approach, wherein: 1) an antibody to an ssDNA-associated feature (e.g. SSB) is used to locally
tether an ssDNA-specific nuclease to chromatin in permeabilized nuclei; 2) next, the nuclease is activated to
selectively cleave nearby ssDNA and not dsDNA; and 3) cleaved fragments are collected and sequenced to
yield a precise ssDNA target localization profile. The development of protein A/G (pAG) fused to an ssDNA-
specific nuclease is a key innovation, as it enables the definitive identification of ssDNA associated with any
localizing factor. A second innovation of our approach is the development of nucleosome spike-in controls
containing either ssDNA or dsDNA, which will be used: 1) to confirm nuclease specificity; and 2) to enable
quantitative comparisons in disease / control samples -/+ eventual drug treatment. The goals of this Phase I
project are to develop the CUT&RUssN workflow (Aim 1) and demonstrate its ability to map SSB-ssDNA
complexes in cells, thus enabling the novel study of ssDNA repair pathways in AD models (Aim 2). In Phase II,
we will expand the CUT&RUssN platform to additional chromatin features (e.g. SSBs or histone PTMs) and their
associated cellular mechanisms (e.g. transcription, R-loops, DNA replication). In addition, we will develop robust
protocols for widely studied AD models and human post-mortem brains, including low cell input applications and
assay automation to enable large-scale clinical studies. At the end of Phase II, we will launch a CUT&RUssN
beta-kit and assay services, which will be marketed to researchers, drug developers, and clinicians to accelerate
AD drug discovery.
Terms: <AD dementia><AD model><AD pathology><Aging><Alzheimer><Alzheimer Type Dementia><Alzheimer disease><Alzheimer sclerosis><Alzheimer syndrome><Alzheimer's><Alzheimer's Disease><Alzheimer's disease dementia><Alzheimer's disease model><Alzheimer's disease pathology><Alzheimer's pathology><Alzheimers Dementia><Alzheimers disease><Antibodies><Assay><Automation><Autopsy><BRCA1><BRCA1 gene><BRCA2><BRCA2 gene><Bar Codes><Benchmarking><Best Practice Analysis><Binding Proteins><Bioassay><Biologic Assays><Biological Assay><Biological Markers><Brain><Brain Nervous System><Breast Cancer 1 Gene><Breast Cancer 2 Gene><Breast Cancer Type 1 Susceptibility Gene><Breast Cancer Type 2 Susceptibility Gene><Cause of Death><Cell Body><Cell Communication and Signaling><Cell Function><Cell Nucleus><Cell Process><Cell Signaling><Cell model><Cell physiology><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular model><ChIP Sequencing><ChIP-seq><Chromatin><Clinical Research><Clinical Study><Complex><DNA><DNA Binding><DNA Binding Interaction><DNA Repair Pathway><DNA bound><DNA mapping><Deoxyribonucleic Acid><Development><Development and Research><Disease Progression><Double-Stranded DNA><Drug Therapy><Drugs><Early Onset Gene Breast Cancer 1><Early Onset Gene Breast Cancer 2><Encephalon><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><FANCD1><Failure><G(s), alpha Subunit><G(s), α Subunit><G(s)alpha><G(s)α><GTP-Binding Protein alpha Subunits, Gs><GTP-Binding Protein α Subunits, Gs><Gene Transcription><Genetic Transcription><Genome Stability><Genomic Segment><Genomic Stability><Genomic approach><Genomics><Goals><Gs alpha Family G-Protein><Gsα><Gαs><Hereditary Breast Cancer 1><Hereditary Breast Cancer 2><Histones><Homologous Recombinational Repair><Human><Hybrids><Intervention><Intervention Strategies><Intracellular Communication and Signaling><Invaded><Investigators><Lesion><Ligand Binding Protein><Ligand Binding Protein Gene><Mammalian Cell><Maps><Measures><Mediating><Medication><Methods><Micrococcal Nuclease><Modern Man><Molecular Configuration><Molecular Conformation><Molecular Stereochemistry><Nerve Cells><Nerve Degeneration><Nerve Unit><Neural Cell><Neurocyte><Neuron Degeneration><Neurons><Non-Polyadenylated RNA><Nucleosomes><Nucleus><Pathogenesis><Pathway interactions><Performance><Pharmaceutic Preparations><Pharmaceutical Preparations><Pharmacotherapy><Phase><Play><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Primary Senile Degenerative Dementia><Process><Prognostic Marker><Protein Binding><Protein Modification><Proteins><Protocol><Protocols documentation><R & D><R&D><RAD52><RAD52 gene><RNA><RNA Expression><RNA Gene Products><RNF53><Recombination Repair><Regulatory Ns Protein><Research><Research Personnel><Researchers><Resolution><Ribonucleic Acid><Role><SS DNA BP><Sampling><Services><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Single-Stranded DNA><Single-Stranded DNA-Binding Protein><Specificity><Staphylococcal Nuclease><Stimulatory Gs G-Protein><Stretching><Subcellular Process><TNase><Technology><Thermonuclease><Thermostable Nuclease><Transcription><Yeast homolog of RAD52><Yeasts><alpha Subunit Stimulatory GTP-Binding Protein><alpha-Gs><alzheimer model><barcode><bio-markers><biologic marker><biological signal transduction><biomarker><biomarker discovery><bound protein><brca 1 gene><brca 2 gene><chromatin immunoprecipitation-sequencing><clinical applicability><clinical application><conformation><conformational state><cost><dementia of the Alzheimer type><developmental><disease control><disorder control><drug development><drug discovery><drug treatment><drug/agent><ds-DNA><dsDNA><epigenomics><genomic effort><genomic region><genomic strategy><homologous recombination><improved><innovate><innovation><innovative><interventional strategy><necropsy><nerve cell death><nerve cell loss><neural degeneration><neurodegeneration><neurodegenerative><neurological degeneration><neuron cell death><neuron cell loss><neuron death><neuron loss><neuronal><neuronal cell death><neuronal cell loss><neuronal death><neuronal degeneration><neuronal loss><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><nuclease><pathway><postmortem><primary degenerative dementia><prognostic biomarker><recombinational repair><repair><repaired><research and development><senile dementia of the Alzheimer type><social role><targeted biomarker><tool><α-Gs>