Secreted acid sphingomyelinase as a potential gene therapy for limb girdle muscular dystrophy 2B
Daniel C. Bittel, Sen Chandra Sreetama, Goutam Chandra, Robin Ziegler, Kanneboyina Nagaraju, Jack H. Van der Meulen, Jyoti K. Jaiswal
Daniel C. Bittel, Sen Chandra Sreetama, Goutam Chandra, Robin Ziegler, Kanneboyina Nagaraju, Jack H. Van der Meulen, Jyoti K. Jaiswal
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Research Article Muscle biology

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

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.

Authors

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

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Figure 4

ASM produced by human liver cell–targeted hASM-AAV improves repair of patient myoblasts in culture.

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ASM produced by human liver cell–targeted hASM-AAV improves repair of pa...
(A) Western blot for hASM and (B) quantification of hASM activity in lysates from HepG2 cells transduced with either control-AAV or hASM-AAV under a liver-specific promoter (n = 3 independent replicates). (C) Quantification of hASM activity in culture supernatants from control-AAV– and hASM-AAV–infected HepG2 cells (n = 3 independent replicates). (D) Confocal images of healthy and LGMD2B patient myoblasts prior to and following focal laser injury (site marked by white arrow) showing FM 1-43 dye (green) labeling. LGMD2B patient myoblasts were treated with culture supernatants from control and hASM-AAV–infected HepG2 cells in CIM. (E) Plot showing the averaged kinetics of FM-dye entry in healthy and patient myoblasts (n > 15 cells per condition). Data are presented as mean ± SEM. *P < 0.001 (vs. control-AAV–treated cells) by independent samples t test (B and C) or mixed-model ANOVA with analyses for interaction effects between treatment condition and time was used (E, vs. control-AAV–treated cell supernatant). Scale bars: 10 μm.