Abstract
Dysferlin is a large transmembrane protein composed of a C-terminal transmembrane domain, two DysF domains, and seven C2 domains that mediate lipid- and protein-binding interactions. Recessive loss-of-function mutations in dysferlin lead to muscular dystrophies, for which no treatment is currently available. The large size of dysferlin precludes its encapsulation into an adeno-associated virus (AAV), the vector of choice for gene delivery to muscle. To design mini-dysferlin molecules suitable for AAV-mediated gene transfer, we tested internally truncated dysferlin constructs, each lacking one of the seven C2 domains, for their ability to localize to the plasma membrane and to repair laser-induced plasmalemmal wounds in dysferlin-deficient human myoblasts. We demonstrate that the dysferlin C2B, C2C, C2D, and C2E domains are dispensable for correct plasmalemmal localization. Furthermore, we show that the C2B, C2C, and C2E domains and, to a lesser extent, the C2D domain are dispensable for dysferlin membrane repair function. On the basis of these results, we designed small dysferlin molecules that can localize to the plasma membrane and reseal laser-induced plasmalemmal injuries and that are small enough to be incorporated into AAV. These results lay the groundwork for AAV-mediated gene therapy experiments in dysferlin-deficient mouse models.
Highlights
Full-length dysferlin exceeds adeno-associated virus encapsulation capacity, requiring the generation of mini-dysferlin molecules
Deletion of Exon 32 Does Not Affect Dysferlin Membraneresealing Activity—In a previous article [15], we described an atypical case of a mild dysferlinopathy phenotype in a patient harboring a lariat branch point mutation leading to in-frame skipping of exon 32
Plasma membrane protein extraction of COS-7 cells transfected with dysferlin⌬exon32 showed that 40.84 Ϯ 8.9% of transfected dysferlin⌬exon32 was targeted to the plasma membrane compared with WT dysferlin (Fig. 1, C and D)
Summary
Full-length dysferlin exceeds adeno-associated virus encapsulation capacity, requiring the generation of mini-dysferlin molecules. On the basis of these results, we designed small dysferlin mole- ping of exon 32 on her second DYSF allele This allele produced cules that can localize to the plasma membrane and reseal laser- mRNA devoid of exon 32 [15], which translates into an interinduced plasmalemmal injuries and that are small enough to be nally truncated dysferlin protein lacking part of the fourth C2 incorporated into AAV. We characterized the functionality of internally truncated dysferlin constructs, each lacking a subsequent C2 domain, and demonstrated that some C2 domains are dispensable for the correct localization of dysferlin to the plasma membrane and importantly for its ability to repair laser-induced plasmalemmal injuries in patient-derived dysferlin-deficient muscle cells. Our results lay the groundwork for AAV-mediated gene therapy experiments in dysferlin-deficient mouse models
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