C2 Domain Therapeutics for Muscular Dystrophy

Abstract Mutations in the dysferlin gene (DYSF) cause Limb Girdle Muscular Dystrophy Type 2B (LGMD 2B) and Miyoshi Myopathy (MMD1) and affect ~1:100,000 individuals worldwide. Dysferlin is comprised of ferlin and dysferlin domains flanked by C2 domains and it accumulates in t-tubule membranes (TTs) at triad junctions (TJs). Our labs have been collaborating to learn how DYSF fulfills its two best understood functions in muscle, repair of the sarcolemmal membrane and stabilization of the Ca2+ transient. We recently showed that DYSF’s most N-terminal C2 domain, C2A, plays unique roles in both (Muriel et al., J Physiol. 2022): like WT DYSF, DYSF missing the C2A domain accumulates in TTs at TJs but it fails to support DYSF’s repair or Ca2+ signaling activities. In isolation, C2A has novel activities, including binding of phospholipids, Ca2+ and other proteins. Here we show that, expressed alone as a soluble protein, the DYSF-C2A domain can support both membrane repair and normal Ca2+ signaling, although unlike full length DYSF it does not concentrate at TJs. This effect is specific, as other DYSF C2 domains and pathogenic mutants in C2A are inactive. Surprisingly, the C2 domain of PKCα (C2-PKCα), which shares 58% homology with C2A, concentrates at TJs very efficiently but has no repair or Ca2+ signaling activity. We adapted this TJ-targeting activity of C2-PKCα in a chimeric construct with C2-PKCα to restore the localization of the DYSF-C2A domain localization to TJs. The chimera increases the activity of C2A, especially at low expression levels. These and other data suggest: (i) C2-PKCα interacts with proteins at TJs to promote its accumulation there; (ii) targeting C2-PKCα-DYSF-C2A to TJs, where it can bind Ca2+ that leaks into the junctional cleft, may be sufficient to support Ca2+ signaling and membrane repair; and (iii) AAV encoding an optimized chimeric construct of PKCα-C2 and DYSF-C2A may offer a therapy for dysferlinopathies. We will test these ideas in 4 specific aims: (1) optimize the efficacy and specificity of engineered constructs of Dysf-C2A to rescue the deficits in Ca2+ signaling and membrane repair in DYSF-null muscle in vitro; (2) identify the mechanism of targeting of the effective chimeric constructs to TJs; (3) test if Ca2+ binding at TJs is sufficient to account for the activities of these constructs, and to use other chimeric constructs to measure the [Ca2+] in the TJ cleft under a range of conditions; and (4) determine if AAV encoding the most potent of our constructs can rescue the phenotypes of dysferlinopathic mice in vitro and in vivo. Our hypothesis is that the C2A domain of DYSF, targeted to TJs, is a potent, efficient and stable replacement for WT DYSF in dysferlinopathic muscle. The goal of this multi-PI application is to critically test this hypothesis and each of the concepts on which it is based. If we are successful, our approach will facilitate AAV-mediated gene therapy by allowing effective packaging (our ORFs are all <1.8 kb) and thus improving transduction efficiency. As dysregulation of Ca2+ and faulty membrane repair can underlie other diseases of striated muscle, our approach may ultimately prove applicable to other muscular dystrophies as well.