Secreted acid sphingomyelinase as a potential gene therapy for limb girdle muscular dystrophy 2B
Daniel C. Bittel, … , Jack H. Van der Meulen, Jyoti K. Jaiswal
Daniel C. Bittel, … , Jack H. Van der Meulen, Jyoti K. Jaiswal
Published January 4, 2022
Citation Information: J Clin Invest. 2022;132(1):e141295. https://doi.org/10.1172/JCI141295.
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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 2

hASM activates endocytosis via the CLIC/GEEC pathway.

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hASM activates endocytosis via the CLIC/GEEC pathway. (A) Confocal image...
(A) Confocal images of the bottom surface (cell-coverslip interface) of mouse myoblasts expressing mRFP-tagged caveolin-1 either untreated (top) or treated with 6 U/L purified hASM (bottom). Grayscale image shows the whole cell at the start of imaging (time point 0). The red line on the cell marks the pixels shown in the grayscale kymograph demonstrating caveolin-1 mobility, presented as images acquired at 1 frame per second, for a 3-minute period (kymograph y axis = total acquisition time of 180 seconds). Note the broken tracks of pixels, indicating movement of caveolae present at the cell membrane. The red arrow in the hASM-treated kymograph indicates the time of hASM addition at the 60-second mark. (B) Plot showing quantification of caveolin-1 puncta in each condition (n = 50 puncta from 10 cells). (C) To track cell membrane shedding, live cells were labeled with FITC-PEG-cholesterol prior to imaging. Grayscale images show confocal image of the cell membrane at the coverslip surface at the start of imaging (time point 0), and the white box marks the extracellular space on the coverslip adjacent to the cell used to monitor the cholesterol-labeled vesicles shed by the cell. The zoom of this region is shown in the pseudocolored panels on the right, where red color indicates vesicles present at the onset of imaging (baseline), and green color indicates vesicles present 2 minutes after mock (untreated) treatment or treatment with 6 U/L hASM (hASM-treated). (D) Quantification of FITC-PEG-cholesterol–enriched particles shed by cells treated or not treated with 6 U/L hASM (n = 10 cells per condition). (E) Quantification of the rate of loss of cell-associated FITC-PEG-cholesterol fluorescence by the cells imaged in C and D (n = 10 cells per condition). (F) Images showing an optical section through the middle of mouse myoblasts expressing the CLIC/GEEC reporter GPI-GFP before and 4 minutes after treatment with 6 U/L hASM. (G–J) Plots showing (G and I) kinetics and (H and J) rate of internalization of GPI-GFP in (G and H) C2C12 myoblasts and (I and J) healthy and patient myoblasts. Data represent mean ± SEM. *P < 0.05 (vs. untreated cells) via independent samples t test (B, D, and E); kinetics and rate-analyses were performed via mixed-model ANOVA, with α set at P < 0.05 (G–J). Scale bars: 10 μm and 5 μm (zoomed images).