Kuntz, S. | Search bioRxiv

bioRxiv2024

A Dose-Response Model for Accurate Detection and Quantification of Transcriptome-Wide Gene Knockdown for Oligonucleotide-Based Medicines

Synthetic antisense oligonucleotides and siRNAs are a class of Oligonucleotide-Based Medicines (OBMs) that can hybridize with pre-mRNA and mRNA, recruit a mechanism-of-action specific enzymatic complex, and knockdown target gene expression. This class of molecules provides an excellent substrate for designing precision gene-modulatory therapeutics; however, quantifying on- and off-target dose response as measured by next-generation sequencing for this class of therapeutics has remained under-powered and ambiguous. Often in silico predictions of off-targets (ranked by edit tolerance) are used as putative off-target analysis in ASO and siRNA drug design. We construct a simple, effective theory of transcriptional dynamics and enzymatic activity in order to describe the transcriptome-wide response to these oligonucleotides. We establish rigorous quantification methods of off-target analysis in oligonucleotide drug design. We also extend the DESeq work [1, 2] of Negative Binomial noise in gene expression measurements to describe noise, including outliers, in OBM-dose response NGS experiments. We demonstrate the performance of our model on both synthetic and experimental Digital Gene Expression (DGE) data of dose response in ASO-treated cells. We present our analysis package, DoReSeq, as a freely available resource for the community. We hope this will elevate the standards of off-target analysis for such an important class of precision therapeutics.

pharmacology and toxicology↗

bioRxiv2023

Developmental Glucocorticoid exposure in zebrafish primes expression of genes linked to human psychiatric disorders

Early life stress (ELS) is one of the strongest risk factors for developing psychiatric disorders in humans. As conserved key stress hormones of vertebrates, glucocorticoids (GCs) are thought to play an important role in mediating the effects of ELS exposure in shaping adult phenotypes. In this process, early exposure to high level of GCs may induce molecular changes that alter developmental trajectory of an animal and primes differential adult responses. However, comprehensive characterization of identities of molecules that are targeted by developmental GC exposure is currently lacking. In our study, we describe lifelong molecular consequences of high level of developmental GC exposure using an optogenetic zebrafish model. First, we developed a new double-hit stress model using zebrafish by combining exposure to a high endogenous GC level during development and acute adulthood stress exposure. Our results establish that similar to ELS-exposed humans and rodents, developmental GC exposed zebrafish model shows altered behavior and stress hypersensitivity in adulthood. Second, we generated time-series gene expression profiles of the brains in larvae, in adult, and upon stress exposure to identify molecular alterations induced by high developmental GC exposure at different developmental stages. Third, we identify a set of GC-primed genes that show altered expression upon acute stress exposure only in animals exposed to a high developmental GC. Interestingly, our datasets of GC primed genes are enriched in risk factors identified for human psychiatric disorders. Lastly, we identify potential epigenetic regulatory elements and associated post-transcriptional modifications following high developmental GC exposure. Thus, we present a translationally relevant zebrafish model for studying stress hypersensitivity and alteration of behavior induced by exposure to elevated GC levels during development. Our study provides comprehensive datasets delineating potential molecular targets underlying the impact of developmental high GC exposure on adult responses.

neuroscience↗