Genetic Variation of Ultra-Potent Synthetic Opioid Sensitivity in Mice

PROJECT SUMMARY/ABSTRACT
The weaponization of Ultrapotent synthetic opioids (UPS) has made finding a novel reversal agent a priority. The
current opioid response agent, Naloxone, is not as effective against UPS opioids and does not reverse one of
its known effects, wooden chest syndrome. Our long-term goal is to define the biological basis of opioid
overdose risk and promote the discovery of safe and effective agents that reverse fentanyl lethality. A key
objective is determining the molecular mechanisms underlying individual variability to fentanyl toxicity using
genetically diverse mice. In aim 1 we plan to identify genes and variants that modify the influence of Mcoln1 on
acute UPS opioid toxicity. Mcoln1 was identified in a GWAS study of overdose risk, and preliminary data support
a genetic knockout of Mcoln1 resulting in death more rapidly from morphine or fentanyl. We will create an
additional CRISPR knockout of Mcoln1 on a more sensitive genetic background, NOD/ShiLtJ and compare it
with the C57BL/6 knockout we have for response to the UPS opioid fentanyl. The LD50 will be determined for
these strains using our piezoelectric respiratory depression detection system. We will also study respiratory
mechanics and pulmonary and chest wall impedance in response to three doses of fentanyl. In another cohort
of mice, they will be tested by plethysmography to acquire respiratory metrics such as tidal volume and minute
ventilation. We will also collect arterial blood to measure blood gases of oxygen and carbon dioxide during the
plethysmography session to monitor the response to fentanyl at that level. Finally, another cohort of naïve and
fentanyl-treated mice will be dissected for brain stems. The pre-bötzinger complex will be identified and analyzed
by single-nucleus RNA-Seq, comparing the cellular populations and differential gene expression across
genotypes, sexes and treatments. In aim 2 we plan to identify the physiological, neural, and molecular
mechanisms of variable fentanyl-induced toxicity and lethality among eight inbred mouse strains. These eight
strains, which served as the foundation for the advanced mouse populations of the Collaborative Cross and
Diversity Outbred mice, contain approximately 45 million SNPs segregating between them. We have determined
that the LD50 for fentanyl varies > 150-fold across both sexes of the eight strains. As in aim 1, in aim 2 we will
phenotype cohorts of mice to detect the diverse phenomena associated with UPS opioids, including Opioid-
Induced Respiratory Depression (OIRD), Opioid-Induced Persistent Apnea (OIPA), Wooden Chest Syndrome
(WCS), closure/collapse of the upper and cardiovascular/hemodynamic disturbances. This phenotyping will be
coupled to identifying the cellular populations, through single nucleus RNA-Seq, within the brainstem pre-
bötzinger region that varies across naïve and fentanyl-treated strains of both sexes of mice. The differentially
expressed genes that define these populations will help us identify targets for therapeutic development
associated with the different fentanyl lethality phenotypes.

Grant Number: 5R01DA059060-03
NIH Institute/Center: NIH
Principal Investigator: Jason Bubier