Biology subjects

Burns, C. H.

Publications and source records attributed to Burns, C. H..

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↗

bioRxiv2020

Pancreatic beta-cell specific deletion of VPS41 causes diabetes due to defects in insulin secretion

Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determines this molecular composition, remain poorly understood. VPS41, a component of the endo-lysosomal tethering HOPS complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic {beta}-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in {beta}-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.

physiology↗

bioRxiv2018

Programmed Delayed Splicing: A Mechanism for Timed Inflammatory Gene Expression

Inflammation involves timed gene expression, suggesting that the fine-tuned onset, amplitude, and termination of expression of hundreds of genes is of critical importance to organismal homeostasis. Recent study of post-transcriptional regulation of inflammatory gene expression led to the suggestion of a regulatory role for pre-mRNA splicing. Here, using a hybrid capture approach to purify incompletely spliced, chromatin-associated pre-mRNAs, we use deep sequencing to study pre-mRNA splicing of the NF-B transcriptome. By freezing transcription and examining subsequent splicing of complete transcripts, we find many introns splice tens to hundreds of times slower than average. Investigating the basis of these delays, we focused on evolutionarily conserved introns with suboptimal splice donor sequences and found that strengthening these donor sites by as few as two nucleotides in minigene reporter assays markedly increased gene expression for several targets. This suggests that such sites can act as timing elements that both delay mRNA production and limit expression amplitude. To broaden this mechanistic view, we applied deep learning sequence-to-function models with feature attribution to identify additional regulatory sequences--both intronic and exonic--that may contribute to delayed splicing through mechanisms independent of donor site strength. This integrated approach revealed non-canonical motifs enriched in slow-splicing introns, pointing to a broader repertoire of cis-elements that can fine-tune transcript maturation during inflammation. Together, these findings support a model in which the temporal regulation of pre-mRNA splicing serves as a layer of control in inflammatory gene expression, and raise the possibility that similar timing mechanisms operate in other rapid-response transcriptional programs.

immunology↗