A multiomic single nucleus RNA-SEQ and atac-SEQ examination of key brainstem respiratory control regions from fentanyl overdose decedents and matched controls - over 81,000 individuals died of an…

The National Institute on Drug Abuse awarded The Washington University $1,155,062 on March 15, 2026, under the Drug Use and Addiction Research Programs (CFDA 93.279) to generate multiomics data characterizing cellular mechanisms underlying fentanyl-induced respiratory depression in opioid overdose decedents.

The award funds single-nucleus RNA-seq and ATAC-seq (snRNAseq and snATACseq) paired multiomics analysis of brainstem respiratory control regions—the prebotzinger complex and ventral respiratory group, and parabrachial and kolliker-fuse nuclei—in 40 diverse fentanyl overdose decedents and 40 matched controls. The research will characterize cell populations, cell states, and cell-type-specific differential gene expression and chromatin accessibility to implicate open chromatin regions as drivers of transcriptional alterations in regions pivotal to opioid-induced respiratory depression. Results will be integrated with genotyping, genome-wide association study, and cross-species data to identify therapeutically targetable genes and genetic mechanisms of risk underlying opioid overdose death.

Performance extends from March 15, 2026, through November 30, 2030, in Missouri. Two sub-recipients deliver specialized services: the St. Louis Integrated Health Network performs human postmortem brain dissection and anatomical isolation of specific brainstem nuclei; Icahn School of Medicine at Mount Sinai generates snRNAseq and snATACseq libraries from up to 100 brain specimens annually using 10X Genomics multiome assays and established tissue preparation protocols.

A multiomic single nucleus RNA-SEQ and atac-SEQ examination of key brainstem respiratory control regions from fentanyl overdose decedents and matched controls - over 81,000 individuals died of an opioid overdose (OD) in 2023, and >92% of those cases involved fentanyl. Fentanyl has very potent opioid-induced respiratory depression (OIRD) effects, the primary cause of opioid OD death. Key brainstem regions, the prebötzinger complex and ventral respiratory group (PREBOT/VRG) and parabrachial and pontine kölliker-fuse nuclei (PB/KFN), play critical roles in respiration, and have been implicated in OIRD. Therefore, IT is likely that the cellular etiology of opioid OD, notably the sites of action of fentanyl, involves these understudied brain regions. Despite evidence for individual differences including heritable variation in opioid OD (e.g., our gwas of opioid OD death), most biologically mechanistic studies have focused on addiction-centric brain regions. Consistent with the likelihood of differential cellular signatures across these brain regions, our preliminary data suggest pervasive bulk tissue expression differences in the PREBOT (i.e., genes distinct from those implicated in regions relevant to addiction). In addition, prior studies have primarily relied on bulk tissue or single omics approaches to characterize the cellular etiology of opioid OD death. New multiome methods allow US to deeply characterize how the partnership between chromatin accessibility and gene expression in these brain regions drives opioid OD involving fentanyl, thus offering insights into biology and implicating genes that could be therapeutically targeted. Against the backdrop of unabated fentanyl-driven OD deaths, our proposal will: (a) generate the first single-nucleus (SN) paired rnaseq and snatacseq (i.e., multiome) data from the PREBOT/VRG and PB/KFN, from diverse human fentanyl OD decedents (N=40) and matched controls (N=40). (B) characterize cell populations, states and cell-type specific relationships between differential gene expression and chromatin accessibility in each brain region across groups. (C) estimate enrichment of opioid gwas variants and genes, yielding mechanistic insights to large-scale population-scale genetic discovery. (D) combine existing opioid-related expression differences in the same and other brain regions to delineate a brain-wide network of transcriptional variability associated with OUD and opioid OD, and produce in-silico prioritization of gene-drug pairs that are viable for preclinical follow-up.