Systemic AAV-Mediated β-Sarcoglycan Delivery Targeting Cardiac and Skeletal Muscle Ameliorates Histological and Functional Deficits in LGMD2E Mice

Abstract

Limb-girdle muscular dystrophy type 2E (LGMD2E), resulting from mutations in β-sarcoglycan (SGCB), is a progressive dystrophy with deteriorating muscle function, respiratory failure, and cardiomyopathy in 50% or more of LGMD2E patients. SGCB knockout mice share many of the phenotypic deficiencies of LGMD2E patients. To investigate systemic SGCB gene transfer to treat skeletal and cardiac muscle deficits, we designed a self-complementary AAVrh74 vector containing a codon-optimized human SGCB transgene driven by a muscle-specific promoter. We delivered scAAV.MHCK7.hSGCB through the tail vein of SGCB^-/- mice to provide a rationale for a clinical trial that would lead to clinically meaningful results. This led to 98.1% transgene expression across all muscles that was accompanied by improvements in histopathology. Serum creatine kinase (CK) levels were reduced following treatment by 85.5%. Diaphragm force production increased by 94.4%, kyphoscoliosis of the spine was significantly reduced by 48.1%, overall ambulation increased by 57%, and vertical rearing increased dramatically by 132% following treatment. Importantly, no adverse effects were seen in muscle of wild-type mice injected systemically with scAAV.hSGCB. In this well-defined model of LGMD2E, we have demonstrated the efficacy and safety of systemic scAAV.hSGCB delivery, and these findings have established a path for clinically beneficial AAV-mediated gene therapy for LGMD2E.

Keywords: AAV; LGMD2E; gene therapy; limb-girdle muscular dystrophy; muscular dystrophy; β-sarcoglycan.

Figure 1

Restoration of SGCB Expression following Intravenous Delivery of scAAVrh.74.MHCK7.hSGCB (A) scAAVrh.74.MHCK7.hSGCB cassette identical to our original cassette with the exception of the inclusion of the MHCK7 promoter instead of tMCK for expression in cardiac muscle. (B) Immunofluorescence imaging 6 months post-injection of skeletal muscles, diaphragm, and heart from sgcb^−/− mice intravenously injected with a 1e−12 vg total dose of scAAVrh.74.MHCK7.hSGCB. Shown are representative images of skeletal muscles displaying a mean of 98.13% ± 0.31% transduction. Also shown are representative images of heart tissue displaying high levels of hSGCB transgene expression. 20× images are shown for all immunofluorescence (IF) images. Scale bar, 100 μm. (C) Western blotting of all muscles from one treated sgcb^−/− mouse confirming hSGCB transgene expression. (D) Western blotting for hSGCB expression in the hearts of five sgcb^−/− treated mice, with densitometry quantification showing overexpression of hSGCB up to 72.0% of BL6 WT levels.

Figure 2

Effect of Systemic Treatment with scAAVrh74.MHCK7.hSGCB on Muscle Pathology (A) H&E stain of diaphragm and QUAD muscle from C57BL/6 WT, sgcb^−/−, and scAAVrh.74.MHCK7.hSGCB-treated mice showing normalized histopathology. Representative 20× images are shown. Scale bar, 100 μm. (B) Quantification of reduction in centrally nucleated fibers in sgcb^−/− treated muscle compared with untreated sgcb^−/− muscle (TA, GAS, GLUT, and diaphragm, p < 0.0001; QUAD, PSOAS, and TRI, p < 0.05). (C and D) Normalization of fiber distribution in GAS, PSOAS, and TRI (C) and increase in mean fiber size in treated muscles compared to untreated sgcb^−/− muscles (D) (p < 0.001, (n = 5/group).

Figure 3

Reduced Collagen Deposition in Intravenously Treated β-SG KO Mice (A) Picrosirius red staining shows reduced fibrosis in treated mice, indicated by a decrease in collagen deposition compared with untreated sgcb^−/− mice in diaphragm and GAS. Representative 20× images are shown. Scale bar, 100 μm. (B) Quantification of collagen levels in the diaphragm and GAS muscles from C57BL/6 WT mice (n = 4), untreated sgcb^−/− mice (n = 4), and treated sgcb^−/− mice (n = 5) confirm reduction in collagen levels in both treated muscles (p < 0.0001).

Figure 4

Correction of Kyphoscoliosis in Thoracic Spine (A) Kyphoscoliosis in sgcb^−/− mice as evident by X-ray radiography. (B) The KI score of sgcb^−/−mice (3.69) is low compared with C57BL/6 WT mice (6.01) (p < 0.01) but increases upon treatment with scAAVrh.74.MHCK7.hSGCB (5.39) (p < 0.05 compared with sgcb^−/−) (n = 6/group).

Figure 5

Assessment of Cardiomyopathy in Heart Muscle (A) H&E and picrosirius red stains of 7-month-old BL6 WT, sgcb^−/−, and AAV.MHCK7.hSGCB-treated sgcb^−/− hearts 6 months post-treatment, indicating myocardial degeneration in untreated sgcb^−/− muscle and improvement following treatment. Representative 10× images are shown. Scale bar, 200 μm. (B) Cardiac MRI analysis showing reduction in sgcb^−/− hearts in SV (p < 0.01), CO, and EF (p < 0.05) and improvements 6 months after treatment (n = 6/group). (C) Western blotting of two C57BL/6 WT hearts, two sgcb^−/− hearts, and five AAV.MHCK7.hSGCB-treated sgcb^−/− hearts showing decreased cardiac troponin I levels in diseased mice. (D) Densitometry quantification showing reduction of cTrpI to 60.38% of BL6 WT levels and an overexpression of up to 135.8% of BL6 WT levels.

Figure 6

Diaphragm Function Correction and Increased Open-Field Cage Activity (A) Diaphragm muscle strips were harvested to measure force and resistance to fatigue in BL6 WT mice (n = 5), sgcb^−/− mice (n = 4), and AAV.MHCK7.hSGCB-treated sgcb^−/− mice (n = 5), all at 7 months of age. Six months of treatment restored force to WT levels (p < 0.01 compared with sgcb^−/−) and improved resistance to fatigue. (B) Overall ambulation in the x and y planes is significantly decreased in sgcb^−/− mice (p < 0.0001) and slightly improved in MCHK7-treated mice (p < 0.05). Vertical activity rearing onto the hindlimbs also decreased in sgcb^−/− mice (p < 0.01) and significantly increased in MCHK7-treated mice (p < 0.05) (n = 6/group).

Figure 7

Biodistribution and Off-Target Transgene Expression Analysis of Systemic scAAVrh.74.MHCK7.hSGCB Delivery (A) Distribution histogram of mean vg copies of transcript per microgram DNA added to qPCR reaction in various tissues from two sgcb^−/− mice after intravenous (i.v.) delivery of scAAVrh.74.MHCK7.hSGCB at a 1e−12 vg total dose. (B) Biodistribution western blots on muscles and organs from scAAVrh.74.MHCK7.hSGCB systemically injected sgcb^−/− mice, indicating no expression of the hSGCB transgene in any non-muscle samples.