Establishing Oprm1 exon 7-associated variants as novel therapeutic targets for mitigating adverse effects of clinically used mu opioids without altering analgesia in pain management - Project Summary/Abstract Mu opioids, such as fentanyl and oxycodone, remain in the mainstream for moderate-to-severe pain management. However, they also produce many side-effects, leading to the development of opioid use disorder, a main cause of the opioid epidemic and climbing opioid overdose death. The actions of mu opioids are primarily mediated through the mu opioid receptors encoded by the single-copy mu opioid receptor gene, OPRM1, that undergoes extensive alternative splicing, creating multiple splice variants. One set of OPRM1 variants, exon 7- associated full-length 7TM C-terminal variants (E7 variants), contain a unique intracellular C-terminal tail sequence with 30 amino acids encoded by E7 that are abundant and highly conserved from rodents to humans. Culminative evidence has indicated that E7 variants contribute to several mu opioid-induced adverse side-effects. First, truncating E7-enocded C-terminal tails in mice (mE7M-B6) attenuated mu opioid-induced tolerance, reward, and respiratory depression without effect on analgesia. Second, similarities and dissimilarities in several phenotypes between mE7M-B6 and β-arrestin2-KO mice suggest that E7 variants involve β-arrestin2-dependent and β-arrestin2-independent mechanisms. Third, a mouse model in which only a single E7 variant, mMOR-1O, is expressed (mMOR-1O-KI) showed enhanced morphine tolerance and reward, complementing those from mE7M-B6 mice. Finally, a vivo-morpholino antisense oligonucleotide (ASO) and a newly developed rabbit monoclonal antibody (RabmAb) targeting E7 variants diminished morphine tolerance and reward in mice. Together, these observations strongly support our central hypothesis that Oprm1 E7 variants mediate several adverse effects associated with clinically used mu opioids, such as tolerance, reward, and respiratory depression, and targeting E7 variants can diminish these adverse effects but maintain mu opioid analgesic potency via other Oprm1 7TM variants. These observations also provide compelling rationales for further investigating the role of Oprm1 E7 variants in various actions of mu opioids using ASOs and antibodies as proposed in this application with four independent, but integrated specific aims. Aim 1 is to characterize ASOs and RabmAbs that target mouse Oprm1 E7 variants in both in vitro cell models and in vivo C57BL/6J (B6) mice, providing the necessary information for designing experiments in Aims 2 & 3. Aims 2 & 3 are to investigate the effects of the selected ASO and RabmAb on mu opioid-induced tolerance, reward, addiction, and respiratory depression in B6 mice under naive (Aim 2) and chronic pain conditions (Aim 3), respectively. Aim 4 is to develop and characterize a nanobody or an ASO targeting human/monkey exon O (E7 homolog) sequences and investigate the effects of the nanobody or ASO on mu opioid abuse potential in rhesus monkeys. The proposed studies promise to yield significant findings in establishing the E7 variants as therapeutic targets and provide the groundwork for developing a therapeutic medication that combines clinically used mu opioids with an inhibitor (nanobody or ASO) to mitigate the adverse effects of mu opioids in pain management.
