Biology subjects

Kay, M. A.

Publications and source records attributed to Kay, M. A..

7 recordsLinked to original sources

bioRxiv2024

AAV-mediated genome editing is influenced by the formation of R-loops

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3 end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop- deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

molecular biology↗

bioRxiv2022

Selection of rAAV Vectors that Cross the Human Blood-Brain Barrier 1 and Target the Central Nervous System Using a Transwell Model

A limitation for rAAV-mediated gene transfer into the central nervous system (CNS) is the low penetration of vectors across the human blood-brain barrier (BBB). High doses of intravenously-delivered vector are required to reach the CNS, which has resulted in varying adverse effects. Moreover, selective transduction of various cell types might be important depending on the disorder being treated. To enhance BBB penetration and improve CNS cell selectivity, we screened an AAV capsid-shuffled library using an in vitro transwell BBB system with separate layers of human endothelial cells, primary astrocytes and/or hiPSC-derived cortical neurons. After multiple passages through the transwell, we identified chimeric AAV capsids with enhanced penetration and improved transduction of astrocytes and/or neurons compared to wild-type capsids. We identified the amino acid (aa) from region 451-470 of AAV2 associated with the capsids selected for neurons, and a combination of aa from regions 413-496 of AAV-rh10 and 538-598 of AAV3B/LK03 associated with capsids selected for astrocytes. An siRNA screen identified several genes that affect transcytosis of AAV across the BBB. Our work supports the use of a human transwell system for selecting enhanced AAV capsids targeting the CNS and may allow for unraveling the underlying molecular mechanisms of BBB penetration.

molecular biology↗

bioRxiv2022

A Standardized Ontology for Naming tRNA-derived RNAs Based on Molecular Origin

Small RNAs processed from transfer RNA are recognized to have regulatory functions distinct from protein synthesis. This rapidly advancing field has led to a constellation of transcript nomenclatures. Building upon the accepted tRNA naming system and linking tRNA-derived small RNAs to their molecular sources, we propose an improved nomenclature. Facilitated by the tDRnamer system, it will promote inter-study comparisons leveraging a biologically-rooted nomenclature for this emerging class of intriguing regulators.

bioinformatics↗

bioRxiv2020

The 3'tsRNAs are aminoacylated: Implications for their biogenesis

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG tsRNA from 3 end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3-tsRNAs remains elusive and it is unclear if the 3-ends of 3-tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG 3-tsRNA is fully charged and originated solely from charged mature tRNA. When the leucyl-tRNA synthetase was knocked down, less tsRNA was generated while the mature tRNA was not reduced further supporting that tsRNA generation is regulated. The fact that the 3-tsRNA is aminoacylated has implications for their biogenesis and provides additional insights into their biological role in post-transcriptional gene regulation.

molecular biology↗

bioRxiv2020

Tyrosine kinase inhibitors induce mitochondrial dysfunction during cardiomyocyte differentiation through alteration of GATA4-mediated networks

Maternal drug exposure during pregnancy increases the risks of developmental cardiotoxicity, leading to congenital heart defects (CHDs). In this study, we used human stem cells as an in-vitro system to interrogate the mechanisms underlying drug-induced toxicity during cardiomyocyte differentiation, including anticancer tyrosine kinase inhibitor (TKI) drugs (imatinib, sunitinib, and vandetanib). H1-ESCs were treated with these drugs at sublethal levels during cardiomyocyte differentiation. We found that early exposure to TKIs during differentiation induced obvious toxic effects in differentiated cardiomyocytes, including disarranged sarcomere structure, interrupted Ca2+-handling, and impaired mitochondrial function. As sunitinib exposure showed the most significant developmental cardiotoxicity of all TKIs, we further examine its effect with in-vivo experiments. Maternal sunitinib exposure caused fetal death, bioaccumulation, and histopathologic changes in the neonatal mice. Integrative analysis of both transcriptomic and chromatin accessibility landscapes revealed that TKI-exposure altered GATA4-mediated regulatory network, which included key mitochondrial genes. Overexpression of GATA4 with CRISPR-activation restored morphologies, contraction, and mitochondria function in cardiomyocytes upon TKI exposure early during differentiation. Altogether, our study identified a novel crosstalk mechanism between GATA4 activity and mitochondrial function during cardiomyocyte differentiation, and revealed potential therapeutic approaches for reducing TKI-induced developmental cardiotoxicity for human health. HighlightsO_LIEarly-stage exposure to TKIs induced cardiotoxicity and mitochondrial dysfunction C_LIO_LIGATA4 transcriptional activity is inhibited by TKIs C_LIO_LINetwork analysis reveals interactions between GATA4 and mitochondrial genes C_LIO_LIGATA4-overexpression rescues cardiomyocytes and mitochondria from TKI exposure C_LI

systems biology↗

bioRxiv2019

Using a bar-coded AAV capsid library to select for novel clinically relevant gene therapy vectors

ABSTRACTWhile gene transfer using recombinant adeno-associated viral (rAAV) vectors have shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet derived cells into functional {beta}-cells in animal models, we constructed two highly complex barcoded replication competent capsid shuffled libraries and selected for high transducing variants on primary human islets. We describe a chimeric capsid (AAV-KP1) that penetrated and transduced primary human islet cells and human embryonic stem cell derived {beta}-cells with up to 10-fold higher efficiency compared to previously studied best in class AAV vectors. Remarkably, this chimeric capsid was also able to transduce both mouse and human hepatocytes at very high levels in a humanized-chimeric mouse model, thus providing a versatile vector which has the potential to be used in both preclinical testing and human clinical trials for both liver-based diseases and diabetes.

genetics↗

bioRxiv2019

Allele-specific silencing ameliorates restrictive cardiomyopathy due to a human myosin regulatory light chain mutation

BackgroundRestrictive cardiomyopathy (RCM) is a rare heart disease associated with mutations in sarcomeric genes and with phenotypic overlap with hypertrophic cardiomyopathy. There is no approved therapy. Here, we explore the potential of an interfering RNA (RNAi) therapeutic for a human sarcomeric mutation in MYL2 causative of restrictive cardiomyopathy in a mouse model. MethodsAAV9-M7.8L shRNA was selected from a pool of RNAi oligonucleotides containing the SNV in different positions to specifically target the mutated allele causative of RCM by FACS screening. Two groups of RLC-N47K transgenic mice were injected with a single dose of AAV9-M7.8L shRNA at 3 days of age and at 60 days of age. Mice were subjected to treadmill exercise and echocardiography after treatment to determine VO2max and left ventricular mass. At the end of treatment, heart, lung, liver and kidney tissue was harvested to determine viral tropism and for transcriptome and proteomic analysis. Cardiomyocytes were isolated for single cell studies. ResultsOne time injection of AAV9-M7.8L RNAi in 3-day-old humanized RLC mutant transgenic mice silenced the mutated allele (RLC-47K) with minimal effects on the normal allele (RLC-47N) assayed 16 weeks post-injection. AAV9-M7.8L RNAi suppressed the expression of hypertrophic biomarkers, reduced heart weight and attenuated a pathological increase in left ventricular mass (LVM). Single adult cardiac myocytes from mice treated with AAV9-M7.8L showed partial restoration of the maximal contraction velocity with marked reduction in hypercontractility as well as relaxation kinetics and improved time to maximal calcium reuptake velocity. In addition, cardiac stress protein biomarkers, such as calmodulin-dependent protein kinase II (CAMKII) and the transcription activator Brg1 were reduced suggesting recovery towards a healthy myocardium. Transcriptome analyses further revealed no significant changes of argonaute (AGO1, AGO2) and endoribonuclease dicer (DICER1) transcripts while endogenous microRNAs were preserved suggesting the RNAi pathway was not saturated. ConclusionsOur results show the feasibility, efficacy, and safety of RNAi therapeutics directed at human restrictive cardiomyopathy. This is a promising step towards targeted therapy for a prevalent human disease. Clinical PerspectiveWhat is new? O_LIRestrictive cardiomyopathy due to a mutation of human MYL2 modeled in cells and mice can be treated with RNA interference C_LIO_LIReduction of disease-causing allele improves function at the cellular and organ level C_LIO_LIOff target effects evaluated and not found to be significant C_LI What are the clinical implications? O_LIAllele-specific RNA silencing of human alleles may be effective in treating inherited restrictive cardiomyopathy C_LIO_LIRNA therapies targeting individual mutations may need to be developed prior to consideration of clinical translation C_LI

molecular biology↗