Biology subjects

Stoyanova, E.

Publications and source records attributed to Stoyanova, E..

3 recordsLinked to original sources

bioRxiv2026

Ultra-large targeted DNA integrations in primary human cells

Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cells genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond [~]5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large--up to 10 kb DNA--integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo. Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.

genetics↗

bioRxiv2021

5-hydroxymethylcytosine mediated active demethylation is required for neuronal differentiation and function

Although high levels of 5-hydroxymethylcytosine (5hmC) accumulate in neurons, it is not known whether 5hmC can serve as an intermediate in DNA demethylation in postmitotic neurons. We report high resolution mapping of DNA methylation and hydroxymethylation, chromatin accessibility, and histone marks in developing postmitotic Purkinje cells (PCs). Our data reveal new relationships between PC transcriptional and epigenetic programs, and identify a class of genes that lose both 5mC and 5hmC during terminal differentiation. Deletion of the 5hmC writers Tet1, Tet2, and Tet3 from postmitotic PCs prevents loss of 5mC and 5hmC in regulatory domains and gene bodies and hinders transcriptional and epigenetic developmental transitions, resulting in hyper-excitability and increased susceptibility to excitotoxic drugs. Our data demonstrate that Tet-mediated active DNA demethylation occurs in vivo, and that acquisition of the precise molecular and electrophysiological properties of adult PCs requires continued oxidation of 5mC to 5hmC during the final phases of differentiation.

neuroscience↗

bioRxiv2017

Species and Cell-Type Properties of Classically Defined Human and Rodent Neurons and Glia

Determination of the molecular properties of genetically targeted cell types has led to fundamental insights into mouse brain function and dysfunction. Here, we report an efficient strategy for precise exploration of gene expression events in specific cell types in a broad range of species, including postmortem human brain. We demonstrate that classically defined, homologous neuronal and glial cell types differ between rodent and human by the expression of hundreds of orthologous, cell specific genes. Confirmation that these genes are differentially active was obtained using epigenetic mapping and immunofluorescence localization. Studies of sixteen human postmortem brains revealed cell-specific molecular responses to aging, and the induction of a shared, robust response to an unknown external event experienced by three donors. Our data establish a comprehensive approach for analysis of unique molecular events associated with specific circuits and cell types in a wide variety of human conditions.

neuroscience↗