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 5

Liver-targeted hASM gene therapy improves dysferlinopathic myofiber repair.

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Liver-targeted hASM gene therapy improves dysferlinopathic myofiber repa...
(A) Schematic showing the gene therapy approach used in vivo in a dysferlin-deficient mouse model for LGMD2B (B6A/J). Twelve weeks after a single i.v. dose of liver-specific hASM-AAV or control-AAV (1.1 × 1013 vg/kg), tissues were isolated for functional measurements. (B) Plot showing hASM activity in the livers isolated from hASM-AAV– and control-AAV–injected mice (n = 5 mice per condition). (C) Plot showing hASM activity in the serum of hASM-AAV and control-AAV 12 weeks after injection (expressed in U/L). (D) Images of myofibers labeled with anti-IgM antibodies (arrowheads show IgM-positive myofibers). (E) Plot showing serum alanine transaminase (ALT) concentration to assess extent of liver damage in control-AAV– and hASM-AAV–treated mice 12 weeks after injection. (F) Quantification of IgM-positive myofibers in D. (G) Images and (H) kinetics of FM-dye uptake by myofibers in freshly isolated biceps, following focal laser injury at site marked by white arrow (n = 20 myofibers per mouse). (I) Plot showing the myofibers that successfully repaired after laser injury (n > 15). (J–L) Isolated EDL muscles from control-AAV– and hASM-AAV–treated mice were injured by repeated 10% eccentric contractions and sarcolemma damage was monitored by labeling with procion orange (PO) dye. (J) Images showing the PO-labeled fibers (arrowheads) and (K) quantification of the number of PO-labeled fibers per muscle (n = 4 muscle per group due to excess damage during preparation of EDL muscle in 1 mouse in each group). (L) Change in muscle contractile force with 10 repeated eccentric contractions (n = 5 mice per group). Data are presented as mean ± SEM. *P < 0.05 vs. control-AAV. Group differences in tissue hASM activity (B), serum hASM activity (C), serum ALT activity (E), IgM+ myofibers (F), percentage of repair fibers (I), and PO+ myofibers (K), all assessed via independent samples t test. Serum hASM activity (C), repair kinetics (H), and eccentric force decrement analyses (L), were performed via mixed-model ANOVA with analyses for interaction effects between treatment condition and time. Scale bars: 50 μm (G) and 100 μm (D and J).