A reduced Complexity Cross in BALB/c substrains to identify the genetic basis of oxycodone dependence phenotypes

PROJECT SUMMARY
Substance use disorders (SUDs) are heritable psychiatric disorders with a significant genetic component. Opioid
dependence, one of the most heritable SUDS, has reached epidemic proportions in the United States. Human
genome-wide association studies (GWAS) are statistically underpowered to detect the majority of common
genetic variation that contributes to opioid dependence. Discovery-based genetics in mammalian model
organisms is a powerful complement to human GWAS and can uncover novel genetic factors, biological
pathways, and gene networks underlying addiction traits. Mouse models are advantageous because they enable
collection of the relevant brain tissue at the appropriate time points under controlled opioid dosing. Furthermore,
gene editing permits the validation of functional variants in vivo within the same species on a controlled, genetic
background. Reduced Complexity Crosses (RCCs) are genetic crosses between inbred mouse substrains that
are nearly genetically identical and can vastly improve the speed at which causal genetic factors can be
identified. Our primary objective is to use an RCC between BALB/c substrains to discover the genetic and
molecular basis of opioid addiction-relevant traits at two stages of opioid dependence following repeated
administration of the mu opioid receptor agonist oxycodone (OXY; the active ingredient of Oxycontin®). We
found robust differences between BALB/c substrains in opioid adaptive behaviors, including state-dependent
learning of OXY-induced locomotor stimulation and reward following limited, low-dose administration (1.25
mg/kg, IP) as well as the emotional-affective component of opioid withdrawal and weight loss following repeated
high-dose administration (40 mg/kg, IP). In Aim 1, we will map quantitative trait loci (QTLs) underlying these
OXY phenotypes in an RCC F2 cross. In Aim 2, we will map QTLs controlling gene expression (eQTLs) in the
relevant brain tissues of control F2 mice and in OXY-trained F2 mice. We will then nominate candidate causal
genes and nucleotides underlying behavior by integrating eQTL with behavioral QTL analysis. To increase
precision in assigning candidate variants with the regulation of gene expression and behavior and to identify
biological pathways and opioid-adaptive gene networks in specific cell types, we will use single nucleus RNA-
seq (snRNA-seq) of brain tissue following limited, low-dose OXY and repeated high-dose OXY. In Aim 3, we
will validate candidate functional variants underlying OXY phenotypes using CRISPR/Cas9 gene editing of each
of the two alternate alleles onto each reciprocal substrain background. This approach will allow us to demonstrate
both necessity and sufficiency of the quantitative trait nucleotides. The proposed studies will identify the genetic
basis of unique opioid phenotypes across two stages of opioid dependence. Independent from gene discovery,
these studies have broader application in revealing novel, actionable insight toward cellular adaptations at
progressive stages of the opioid addiction process and potentially improving behavioral outcomes.

Grant Number: 7U01DA050243-05
NIH Institute/Center: NIH
Principal Investigator: CAMRON BRYANT