Secreted acid sphingomyelinase as a potential gene therapy for limb girdle muscular dystrophy 2B.

Daniel C Bittel,Robin Ziegler,Jyoti K Jaiswal,Jack H Van Der Meulen,Kanneboyina Nagaraju,Sen Chandra Sreetama,Goutam Chandra

doi:10.1172/jci141295

Abstract

Efficient sarcolemmal repair is required for muscle cell survival, with deficits in this process leading to muscle degeneration. Lack of the sarcolemmal protein dysferlin impairs sarcolemmal repair by reducing secretion of the enzyme acid sphingomyelinase (ASM), and causes limb girdle muscular dystrophy 2B (LGMD2B). The large size of the dysferlin gene poses a challenge for LGMD2B gene therapy efforts aimed at restoring dysferlin expression in skeletal muscle fibers. Here, we present an alternative gene therapy approach targeting reduced ASM secretion, the consequence of dysferlin deficit. We showed that the bulk endocytic ability is compromised in LGMD2B patient cells, which was addressed by extracellularly treating cells with ASM. Expression of secreted human ASM (hASM) using a liver-specific adeno-associated virus (AAV) vector restored membrane repair capacity of patient cells to healthy levels. A single in vivo dose of hASM-AAV in the LGMD2B mouse model restored myofiber repair capacity, enabling efficient recovery of myofibers from focal or lengthening contraction–induced injury. hASM-AAV treatment was safe, attenuated fibro-fatty muscle degeneration, increased myofiber size, and restored muscle strength, similar to dysferlin gene therapy. These findings elucidate the role of ASM in dysferlin-mediated plasma membrane repair and to our knowledge offer the first non–muscle-targeted gene therapy for LGMD2B.

Highlights

Skeletal muscle cells enable physical movement and are frequently damaged by strenuous activity, overload, and eccentric contractions [1, 2]
To test the effect of human ASM (hASM) on plasma membrane repair, primary human myoblasts from limb girdle muscular dystrophy 2B (LGMD2B) patients were treated with purified hASM protein
With the role of clathrin-independent carriers (CLICs) in bulk membrane removal, we examined the role of bulk membrane endocytosis in repair by using the lectin wheat germ agglutinin (WGA) to label the plasma membrane and assess its endocytic removal in response to different doses of hASM (Figure 3A)

Summary

Introduction

Skeletal muscle cells (myofibers) enable physical movement and are frequently damaged by strenuous activity, overload, and eccentric contractions [1, 2]. Miyoshi myopathy (MM) and limb girdle muscular dystrophy 2B (LGMD2B) are two such autosomal recessive muscular dystrophies that manifest in early adulthood and lead to progressive skeletal muscle weakness and wasting [4] These diseases (collectively called dysferlinopathy) are caused by mutations in the DYSF gene, which encodes a large (237 kDa) muscle membrane protein — dysferlin [5, 6]. Even prior to overt muscle degeneration, dysferlinopathic patient myofibers exhibit plasma membrane (sarcolemma) defects, including membrane tears, extrusions, subsarcolemmal accumulation of vesicles and vacuoles, and thickening of the basal lamina [7]. These early abnormalities are suggested to be caused by poor repair of sarcolemmal injury [7, 8]. Failed or deficient myofiber repair activates chronic inflammatory responses and leads to muscle degeneration — a notable feature of dysferlinopathic skeletal muscle [11,12,13]

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Results

Conclusion