Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Kyle Moss,Mehmet E. Yalvac,Alicia Ridgley,Jerry R. Mendell,Lei Chen,Darren Murrey,Burcak Ozes,Zarife Sahenk,Morgan Myers

doi:10.1016/j.omtm.2021.06.010

Abstract

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

Highlights

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is the most common Limb girdle muscular dystrophy (LGMD) subtype worldwide and is caused by mutations in the CAPN3 gene, which encodes a skeletal muscle-specific, Ca2+-activated, nonlysosomal cysteine protease, calpain 3 (CAPN3).[1]
It has been proposed that the dysregulation of skeletal muscle functions in LGMDR1 is, at least in part, a consequence of the lack of RyR stabilization by CAPN3.13–16 The absence of functional CAPN3 has been shown to result in reduced levels of several important Ca2+-handling proteins, such as ryanodine receptor 1 (RyR1) and sarcoplasmic reticulum (SR)/endoplasmic reticulum Ca2+-ATPase, SERCA17, and impaired Ca2+/calmodulindependent protein kinase type II (CaMKII) signaling, affecting Ca2+-dependent transcriptional pathways that control muscle growth, fiber transition, or mitochondrial biogenesis.[18,19,20,21]
We assessed the biopotency of the vector following systemic delivery via the tail vein of CAPN3 KO mice at low and high doses and generated biodistribution and short-term treatment efficacy data 4 weeks after injection for both cohorts. hCAPN3 expression in CAPN3 KO muscle following systemic delivery was variable among different muscles and especially in the LD cohort, which was significantly lower compared with IM delivery at 1 Â 1011 vg (

Summary

Introduction

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A (calpainopathy), is the most common LGMD subtype worldwide and is caused by mutations in the CAPN3 gene, which encodes a skeletal muscle-specific, Ca2+-activated, nonlysosomal cysteine protease, calpain 3 (CAPN3).[1]. The full length of CAPN3 is expressed in skeletal muscle,[4] and the enzyme, present in its inactive form, is capable of self-activation by autolysis, which exposes the catalytic site for substrate accessibility.[5,6] CAPN3 is involved in cleavage and/or breakdown of multiple key skeletal muscle proteins, those involved in assembly and scaffolding of myofibrillar organization, such as titin, vinculin, C-protein, and others.[5,7,8] Loss of this activity, which is presumably involved in sarcomere maintenance and turnover, has been implicated in the pathogenesis of LGMDR1.7,9–11 In addition, CAPN3 possesses thiol-dependent proteolytic activity directed against the skeletal muscle ryanodine receptor 1 (RyR1), a Ca2+ release channel localized at the sarcoplasmic reticulum (SR) terminal cisternae.[12,13] It has been proposed that the dysregulation of skeletal muscle functions in LGMDR1 is, at least in part, a consequence of the lack of RyR stabilization by CAPN3.13–16 The absence of functional CAPN3 has been shown to result in reduced levels of several important Ca2+-handling proteins, such as RyR1 and SR/endoplasmic reticulum Ca2+-ATPase, SERCA17, and impaired Ca2+/calmodulindependent protein kinase type II (CaMKII) signaling, affecting Ca2+-dependent transcriptional pathways that control muscle growth, fiber transition, or mitochondrial biogenesis.[18,19,20,21] Accumulating evidence suggests that dysregulation of Ca2+ homeostasis in skeletal muscle may be a significant underlying event in LGMDR1,22 leading to mitochondrial abnormalities/dysfunction,[18,21,23,24] increased oxidative stress,[23,25] reduced energy production, and impaired muscle regeneration and adaptation.[19,21,26]

Methods

Results

Conclusion