OPTIMIZING SPLICE ISOFORMS AS A GENOME EDITING STRATEGY FOR <EM>CACNA1A</EM>-RELATED DISORDERS

Loss-of-function mutations in CACNA1A, encoding the Ca_V2.1 P/Q-type calcium channel, cause severe neurological disorders, including episodic ataxia and absence epilepsy. Although Ca_V2.1 deficiency leads to compensatory upregulation of the related Ca_V2.2 N-type calcium channel, this endogenous response is insufficient to prevent neurological dysfunction, in part because Ca_V2.2 is less efficient than Ca_V2.1 at supporting neuronal communication.
Both Ca_V2.1 and Ca_V2.2 undergo a conserved alternative splicing event that produces two major isoforms, EFa and EFb, which differ in their ability to support synaptic transmission. However, in the brain, only Ca_V2.1 maintains sufficiently high levels of the more effective EFa isoform for optimal neuronal function. We therefore hypothesized that increasing the levels of the EFa isoform of Ca_V2.2 could compensate for Ca_V2.1 deficiency.
To test this hypothesis, we have developed a CRISPR/Cas9-based approach to regulate the expression of these splice variants. Our results demonstrate that enhancing the levels of the EFa isoform of Ca_V2.2 corrects motor coordination deficits and resolves absence epilepsy in Ca_V2.1-deficient mice, as well as rescues electrophysiological defects in human iPSC-derived neurons with CACNA1A mutations.
Together, these findings demonstrate that targeted modulation of calcium channel splice variants can effectively correct functional abnormalities due to Ca_V2.1 loss. By focusing on the upregulation of Ca_V2.2, our strategy has the potential to counteract the effects of a wide range of CACNA1A mutations.