CaV1.2 Ca2+ channels are critical conduits for Ca2+ entry into a diverse array of excitable cells. As such, these channels must be precisely tuned to function appropriately for each cell type, and in response to varying physiological cues. To this end, the channels employ multiple mechanisms of regulation, including alternative splicing, voltage dependent inactivation and calcium dependent inactivation. However, a growing number of mutations have been identified in CaV1.2, leading to severe phenotypes including neurological deficits, long-QT syndrome (LQTS), and death. Timothy Syndrome (TS) represents one such class of mutations, in which a single point mutation within CaV1.2 leads to a severe multisystem disorder characterized by developmental delays, autism and profound LQTS. Many of the known TS mutations have been shown to decrease channel inactivation, implicating Ca2+ channel blockers (CCBs) as a promising treatment option. However, despite some modest success with verapamil, CCBs have had limited efficacy in treating TS. Here, we postulate that this lack of efficacy may be due to a differential effect of CCBs on mutant versus wild type CaV1.2, necessitating the exploration of alternative therapeutic options for these patients. We propose that manipulating CaV1.2 splicing represents just such an alternative strategy, with significant promise for the treatment of these patients. Specifically, as the majority of TS patients harbor a mutation within a mutually exclusive exon, we will design antisense oligonucleotides (AONs) targeted to the exon containing the mutation. As such, we expect to force the exclusion of the deleterious exon, induce inclusion of the unaffected alternate exon, and produce a fully functional alternate channel splice variant. Importantly, this strategy will bypass the current limitations of conventional therapies, providing significant clinical benefit for TS patients. Moreover, application of this technique to WT CaV1.2 channels may lead to promising new therapeutic insights within a broader population of patients. In particular, we will apply our splice modulating AONs to test the hypothesis that atypical splice patterns in some patients may render them more susceptible to detrimental cardiac effects of DHP treatment. Finally, as numerous genetic mutations occur within mutually exclusive exons, our application of AONs to TS will serve as a generalizable strategy applicable to numerous genetic mutations in a multitude of proteins. Overall, targeted manipulation of protein splicing represents a large and untapped opportunity, providing a path forward for treatment of a diverse array of diseases.
Calcium channel splice variation plays a vital role in normal human physiology, individual responses to drugs, and human disease. However, the tools for studying the functional effects of altered splicing are limited. Creation of a new tool for targeted splice manipulation will enable new understanding of the limitations of currently available drugs and will serve as a promising new treatment strategy for devastating genetic diseases such as Timothy Syndrome.