Original Article | Published:

β-Sarcoglycan gene transfer decreases fibrosis and restores force in LGMD2E mice

Gene Therapy volume 23, pages 5766 (2016) | Download Citation


Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the β-sarcoglycan (SGCB) gene causing loss of functional protein and concomitant loss of dystrophin-associated proteins. The disease phenotype is characterized by muscle weakness and wasting, and dystrophic features including muscle fiber necrosis, inflammation and fibrosis. The Sgcb-null mouse recapitulates the clinical phenotype with significant endomysial fibrosis providing a relevant model to test whether gene replacement will be efficacious. We directly addressed this question using a codon optimized human β-sarcoglycan gene (hSGCB) driven by a muscle-specific tMCK promoter (scAAVrh74.tMCK.hSGCB). Following isolated limb delivery (5 × 1011 vector genome (vg)), 91.2% of muscle fibers in the lower limb expressed β-sarcoglycan, restoring assembly of the sarcoglycan complex and protecting the membrane from Evans blue dye leakage. Histological outcomes were significantly improved including decreased central nucleation, normalization of muscle fiber size, decreased macrophages and inflammatory mononuclear cells, and an average of a 43% reduction in collagen deposition in treated muscle compared with untreated muscle at end point. These measures correlated with improvement of tetanic force and resistance to eccentric contraction. In 6-month-old mice, as indicated by collagen staining, scAAVrh74.tMCK.hSGCB treatment reduced fibrosis by 42%. This study demonstrates the potential for gene replacement to reverse debilitating fibrosis, typical of muscular dystrophy, thereby providing compelling evidence for movement to clinical gene replacement for LGMD2E.


Limb-girdle muscular dystrophy (LGMD) type 2E (LGMD2E) is an autosomal recessive disorder resulting from mutations in the gene encoding β-sarcoglycan (SGCB), causing loss of functional protein.1 LGMD2E represents a relatively common and severe form of LGMD in the United States with worldwide reports of incidence of 1/200 000–1/350 000.2 The absence of β-sarcoglycan leads to a progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement and fibrosis, all resulting in deteriorating muscle strength and function.3, 4 As a complex, the sarcoglycans (α-, β, γ-, δ-), ranging in size between 35 and 50 kD,5 are all transmembrane proteins that provide stability to the sarcolemma offering protection from mechanical stress during muscle activity.3 Loss of β-sarcoglycan in LGMD2E usually results in varying degrees of concomitant loss of other sarcoglycan proteins contributing to the fragility of the muscle membrane leading to loss of myofibers.1 Although the range of clinical phenotype of LGMD2E varies, diagnosis typically occurs before age 10 and with loss of ambulation occurring by mid to late teens.1, 6, 7 Patients present with elevated serum creatine kinase (CK), proximal muscle weakness, difficulty arising from the floor and progressive loss of ambulation. Cardiac involvement occurs in as many as fifty percent of cases.8, 9, 10

Currently, there is no cure or treatment for LGMD2E,11 although, deflazacort has been reported to benefit two siblings with this condition.12 Gene replacement therapy has intuitive appeal but many questions remain unanswered (for example, delivery system, dosing and vehicle for gene transfer). A suitable model for translational studies is the murine model of LGMD2E that completely lacks β-sarcoglycan (Sgcb-null mouse), and has clinical-pathological features in skeletal and cardiac muscle that replicate the human disease.4, 13 The prominence of the endomysial fibrosis in this model is particularly attractive for testing therapeutic products since early onset fibrosis and the extent of connective tissue deposition is much greater compared with many other dystrophic animal models.14 Considering that a major question unanswered by gene replacement therapy is potential efficacy once significant degrees of connective tissue have infiltrated dystrophic muscle, the studies described here in the β-sarcoglycan knock out mouse have particular relevance for planning future clinical trials.

For this proof of principle study, we delivered the full-length β-sarcoglycan cDNA under control of a muscle-specific promoter (tMCK) using a self-complementary adeno-associated virus (scAAV).15, 16 A major rate-limiting step in the transduction efficiency of AAV is the requirement of synthesis of a complementary strand of DNA from the single-stranded AAV genome by the host-cell machinery. The self-complementary AAV vector was engineered by McCarty et al.15, 16 to bypass this synthesis step by creating a deletion of the terminal resolution site from the AAV terminal repeat sequence. This allows for the packaging of single-stranded dimeric inverted repeat DNA molecules.15, 16 Use of the scAAV vector greatly reduces the time taken to begin producing the exogenous therapeutic protein and increases efficiency of transduction with higher transgene expression at lower doses with no added difficulty in vector cloning or virus production. Our initial efforts were directed at demonstration of β-sarcoglycan by intramuscular injection to establish vector potency. More importantly, we were able to deliver 5 × 1011 vector genome (vg) of scAAVrh.74.tMCK.hSGCB, through an isolated-limb perfusion (ILP) system,17, 18, 19 achieving 90% or greater expression throughout the tibialis anterior (TA) and gastrocnemius muscles of the lower limbs. The restoration of β-sarcoglycan expression correlated with improvement in functional outcomes assessed by absolute and specific force generation and resistance to contraction induced injury. Of particular note was the reduction in endomysial fibrosis by approximately 50% following treatment. Importantly, when we extended the study to aged mice, we found β-sarcoglycan gene transfer alone was sufficient to reverse pre-existing fibrosis by 42%. These findings have particular relevance to translational considerations for scAAVrh.74.tMCK.hSGCB gene therapy for LGMD2E.


scAAVrh.74.tMCK.hSGCB construction and vector potency

We constructed a transgene cassette containing a codon-optimized full-length human SGCB cDNA (Figure 1a). The cassette includes a consensus Kozak sequence (CCACC), an SV40 chimeric intron, a synthetic polyadenylation site, and the muscle-specific tMCK promoter20 used to drive expression of the cassette. It was packaged into a self-complementary (sc) AAVrh.74 vector that is 93% homologous to AAV8. AAVrh.74 has been shown in mice and non-human primates to be safe and effective, particularly in crossing the vascular barrier when delivered to muscle through the circulation.17, 18, 21 Vector potency was established by intramuscular injection into the left TA muscle in the Sgcb-null mouse. Delivery of 3 × 1010 vg transduced 70.5±2.5% of muscle fibers and 1 × 1011 vg transduced 89.0±4.0% of muscle fibers, 3 weeks post gene transfer.

Figure 1
Figure 1

AAV mediated β-sarcoglycan expression restores dystrophin-associated proteins and protects membrane integrity. (a) Self-complementary AAV vector containing the codon-optimized human β-sarcoglycan gene (hSGCB) driven by the muscle-specific tMCK promoter. The cassette also contains a chimeric intron to augment processing and polyadenylation signal for stability. (b) Immunofluorescence staining with anti-β-SG antibody shows high levels of sarcolemmal staining of the SGCB transgene in 5-week-old mice both 6 and 12 weeks post injection. × 20 images shown. Percentage of fibers expressing beta-sarcoglycan per TA muscle averaged 88.4±4.2% after 6 weeks (n=9, 4 male, 5 female) and 76.5±5.8% after 12 weeks (n=6, 4 male, 2 female). Protein expression confirmed in the western blot with gamma-tubulin blot shown for a loading control. (c) AAV delivery of β-sarcoglycan leads to restoration of other members of the sarcoglycan complex; α-sarcoglycan, dystrophin. × 20 images. (d) scAAVrh.74.hSGCB protects sgcb−/− membranes from damage. Image showing a large area of Evans blue-positive fibers (red) juxtaposed to a cluster of β-sarcoglycan-positive fibers that have been protected from Evans blue dye incorporation. × 40 image is shown.

Intramuscular delivery of scAAVrh.74.tMCK.hSGCB

Following vector potency, studies were extended to analyze the efficacy of therapy 6 and 12 weeks post gene transfer. As a result of the high levels of expression following the short 3-week potency study, a dose of 3 × 1010 vg total was selected for subsequent studies to use the lowest effective dose. Five-week-old sgcb−/− mice were treated with 3 × 1010 vg of scAAVrh.74.tMCK.hSGCB intramuscularly to the left TA and β-sarcoglycan expression was demonstrated using immunofluorescence in 88.4±4.2% of muscle fibers 6 weeks post injection (n=9), and in 76.5±5.8% of muscle fibers 12 weeks post injection (n=6), and expression was confirmed via western blotting (Figure 1b, Supplementary Figure 1). β-Sarcoglycan expression was accompanied by restoration of components of the dystrophin-associated protein complex (α-sarcoglycan and dystrophin) (Figure 1c). Using Evans blue dye (EBD) as a marker for membrane permeability,22, 23 we found all fibers expressing exogenous β-sarcoglycan were protected from leakage and EBD inclusion (Figure 1d). Muscle from sgcb−/− mice exhibit a severe muscular dystrophy with centrally nucleated fibers, frequent muscle fiber necrosis, fibrotic tissue and significant fiber size variability represented by both atrophic and hypertrophic fibers.3, 4 As seen in Figure 2a, hematoxylin & eosin staining shows an overall improvement in the dystrophic phenotype of diseased muscle including a reduction in central nuclei (sgcb−/− untreated—76.8±2.3% vs AAV.hSGCB treated—38.86±3.5%; P<0.0001) (Figure 2c). We also saw normalization of fiber size distribution, with an increase in the average fiber diameter following treatment (sgcb−/− untreated—32.6±0.31 μm vs AAV.hSGCB treated—35.56±0.22 μm; P<0.0001) (Figure 2d).

Figure 2
Figure 2

Histological analysis of β-SG-deficient treated skeletal muscle. scAAVrh.74.hSGCB treatment normalizes histological parameters of sgcb−/− mice. Hematoxylin & Eosin staining and Picrosirius Red staining were performed on TA muscle from sgcb−/− mice along with normal control C57/BL6 mice and scAAVrh.74.hSGCB-treated mice followed by quantification of histological parameters and % collagen staining. (a) H&E staining shows the presence of centrally nucleated fibers, inflammatory cells and large fiber diameter distribution in β-SG-deficient muscle and an improvement in histopathology following gene transfer. (b) Pircrosirius Red staining shows a decrease in red collagen staining in treated muscle. (c) Quantification of centrally nucleated fibers showing a decrease following treatment (P<0.0005, one-way ANOVA) and (d) representation of fiber size distribution and increase in average fiber size of TA muscle from C57/BL6 controls and sgcb−/− mice compared with treated mice (P<0.0001, one-way ANOVA). (e) Quantification of % collagen in TA muscle from C57/BL6 controls and sgcb−/− mice compared with sgcb−/− treated mice (P<0.0001, one-way ANOVA). 100 μm scale bar shown for × 20 images. ***P<0.001; ****P<0.0001.

The histopathological hallmark of the scgb−/− mouse is fibrosis characterized by widespread replacement of muscle tissue primarily with collagens along with other extracellular matrix components such as fibronectin, elastin, laminin and decorin.14 This replacement of muscle tissue by connective tissue challenges the potential value of gene replacement and may limit the degree of improvement.24 To test this, we assayed the mice treated for 12 weeks for reduction in fibrosis. We specifically assessed the TA muscle since we established its inherent degree of fibrosis in the KO model and because it represents a potential target following vascular ILP gene delivery. Picrosirius red staining for collagen, types I and III, of TA muscles showed a significant reduction (52.74%) in the amount of collagen present within scAAVrh.74.tMCK.hSGCB-treated muscle compared with untreated sgcb−/− mouse muscle (20.7±0.57% vs 43.8±2.3%, AAV.hSGCB treated vs sgcb−/− untreated, respectively; P<0.0001) (Figures 2b and e). Untreated sgcb−/− muscle from 5-week-old mice at the age of injection had 24.05±1.5% collagen deposition, indicating there was a slight (14.0%) reduction in the amount of collagen following the 12 weeks of treatment.

Functional correction in skeletal muscle following scAAVrh.74.tMCK.hSGCB gene transfer

To determine whether hSGCB gene transfer can improve muscle function, we assessed the functional properties of the TA muscle from sgcb−/− mice treated with scAAVrh.74.tMCK.hSCGB. Following intramuscular delivery of 3 × 1010 vg of scAAVrh.74.tMCK.hSCGB to the TA of 4-week-old sgcb−/− mice, 6 weeks post treatment the TA muscles were subjected to in situ force measurements (n=4). Treated muscles were compared with untreated contralateral muscles and those from C57BL/6 WT mice. scAAVrh.74.tMCK.hSCGB-treated muscle showed significant improvement in both absolute tetanic force and normalized specific force (Figures 3a and b). Treated muscles had an average absolute force of 1436.9±199.5 mN compared with 770.9±118.3 mN for untreated sgcb−/− controls (P<0.01). Similarly, treated TA muscles produced an average specific force of 254.01±6.9 mN/mm2 and untreated muscles produced 124.2±13.9 mN/mm2 of force (P<0.01). Finally, muscles treated with scAAVrh.74.tMCK.hSCGB showed greater resistance to contraction-induced injury compared with the untreated control muscles (Figure 3c). Treated TA muscles lost 34.0±5.1% of force from that produced after the first contraction whereas untreated diseased muscle lost 54.1±3.8% (P<0.01) of force following the eccentric contraction protocol. These data show that hSGCB gene transfer does provide a functional benefit to diseased muscle deficient for β-sarcoglycan.

Figure 3
Figure 3

scAAVrh.74.hSGCB intramuscular delivery corrects tetanic force and resistance to contraction-induced injury. The TA muscle of sgcb−/− mice treated with 3 × 1010 vg of scAAVrh.74.hSGCB via an IM injection was harvested 6 weeks post gene transfer, and subjected to a protocol to assess tetanic force and an eccentric contraction protocol to assess resistance to contraction-induced injury. (a) AAVrh.74.hSGCB-treated TA’s demonstrated significant improvement in both absolute tetanic force (P<0.01, paired t-test) and (b) normalized specific force (P<0.05, paired t-test), which was not different from wild-type force (C57/BL6). (c) AAVrh.74.hSGCB treated TA’s exhibited significant improvement in resistance to contraction-induced injury compared with untreated sgcb−/− controls (P<0.01, two-way ANOVA). Force retention following 10 contractions is shown. *P<0.05; **P<0.01.

Treatment of aged muscle with scAAVrh.74.tMCK.hSGCB

Studies of disease progression in this mouse model of LGMD2E have shown that although the most severe tissue remodeling in muscle occurs between 6 and 20 weeks, the histopathology of the muscle continues to worsen with age, resembling the disease progression in patients.3, 4, 14 Consequently, to mimic a clinical setting where treatment would occur at an older age with more advanced muscle deterioration and endomysial fibrosis, we treated 6-month-old sgcb−/− mice (n=5) intramuscularly in the TA with 3 × 1010 vg of scAAVrh.74.tMCK.hSCGB. Following 12 weeks of treatment, at 9 months of age, 80.1±4.8% of muscle fibers were transduced (Figure 4a). Picrosirius red stain for collagen types I and III showed a 42.2% reduction in the amount of collagen present in treated mice compared with untreated sgcb−/− mouse muscle (AAV.hSGCB treated—20.0±0.80% vs sgcb−/− untreated—34.6±1.4%, P<0.0001) (Figures 4b and c). At the age of treatment, 6-month-old sgcb−/− mice have 30.8±2.0% collagen deposition (n=4, 4 male); thus, these results indicate that scAAVrh.74.tMCK.hSCGB treatment not only prevents, but also has the potential to reverse existing fibrosis.

Figure 4
Figure 4

Analysis of aged mice treated intramuscularly with scAAVrh.74.tMCK.hSGCB. (a) Immunofluorescence staining of TA muscle from 6-month-old treated sgcb−/− mice 12 weeks post injection (n=5, 5 male) shows sarcolemmal expression of the SGCB transgene at levels averaging 80% in injected mice compared with untreated (n=4, 4 male). (b) Picrosirius red staining of the treated and untreated TA muscle. (c) Quantitation of collagen present in the Picrosirius red stained tissue shows a significant reduction in the amount of collagen following treatment with rAAVrh.74.tMCK.hSGCB (P<0.0001, one-way ANOVA). 100 μm scale bar shown for × 20 images. ****P<0.0001.

ILP of scAAVrh.74.tMCK.hSGCB in sgcb −/− mice

The ability to target multiple muscles in one limb allows for a more clinically relevant delivery method for translation to LGMD2E patients. Delivery of 5 × 1011 vg of scAAVrh.74.tMCK.hSGCB by ILP in 4- to 6-week-old sgcb−/− mice (n=9, 7 male, 2 female) was analyzed 2 months post gene transfer. β-Sarcoglycan expression reached 91.8±4.7% of fibers in the gastrocnemius (GAS) muscle and 90.6±2.8% in TA (Figure 5a). ILP delivery of scAAVrh.74.tMCK.hSGCB resulted in significant protection from eccentric contraction-induced injury (P<0.05), that was not different from WT, compared with untreated contralateral muscles (Figure 5c). Vascular delivery also restored muscle histopathological parameters (Figure 5b). Central nuclei were decreased in the TA (sgcb−/− untreated—76.9±2.8% vs AAV.hSGCB treated—23.2±5.7%, P<0.001) and GAS (sgcb−/− untreated—78.2±2.4% vs AAV.hSGCB treated—16.8±6.6%, P<0.001) (Supplementary Figure 2). Gene transfer also led to an increase in the average fiber size in the TA (sgcb−/− untreated—30.53±0.52 μm vs AAV.hSGCB treated—41.9±0.46 μm; P<0.0001) and GAS (sgcb−/− untreated—38.9±0.37 μm vs AAV.hSGCB treated—33.3±0.44 μm; P<0.0001), with normalization of fiber diameter distribution (Supplementary Figure 2). It is also important to note that through the use of immunohistochemical staining for immune cells, we found a substantial decrease (~60%) in the number of CD3 cells, CD4 cells and macrophages (Table 1).

Figure 5
Figure 5

Vascular delivery of scAAVrh.74.hSGCB. Four (n=5, 5 male) and five (n=4, 2 male, 2 female) weeks β-SG-deficient mice were treated with vector via the femoral artery to deliver the vector to the lower limb muscles. At a dose of 5 × 1011 vg, β-SG expression was 90.6±2.8% in the TA and 91.8±4.7% in the GAS of treated mice accompanied by improvements in histopathology that resulted in significant improvement in specific force compared with untreated animals even following an injury paradigm. (a) β-SG protein expression from three representative mice. Muscle from a β-SG KO untreated mouse is shown for comparison in the inset (lower right). × 20 Images are shown. Expression in treated muscles confirmed via western blot and gamma-tubulin is shown as a loading control. (b) Histopathology is significantly improved following high dose treatment. Upper panels-treated TA and gastrocnemius muscles. Bottom panels-untreated β-SG-deficient control muscle. 100 μm scale bar shown for × 20 images. (c) Percentage of specific force retained in EDL muscle following 10 cycles of eccentric contraction-induced injury. Treatment with 5 × 1011 vg of AAVrh.74.hSGCB led to significant improvement in force that was equivalent to WT (normal) control muscle (P<0.05, one-way ANOVA). *P<0.05.

Table 1: Immune response in scAAVrh.74.tMCK.hSGCB ILP-treated mice

Picrosirius red staining of TA and GAS muscles also showed a significant reduction in the amount of collagen compared with untreated sgcb−/− muscle following vascular delivery (Figure 6a). Collagen levels in the TA were reduced to 21.6±1.3% in treated muscle compared with 40.2±1.5% in untreated sgcb−/− mice at the age of end point (P<0.0001). As indicated previously, sgcb−/− mice at the age of injection presented with 24.1±1.5% collagen in TA muscle, indicating again a slight reduction (10.0%) in collagen deposition following 8 weeks of treatment. Similarly, staining of the GAS muscle showed that treated mice had 22.9±0.99% collagen compared with 37.9±1.3% in untreated sgcb−/− mice at the end point (P<0.0001). Qualitative PCR was performed to detect collagen transcript levels in muscle, which correlate with the results of the Sirius red staining (Supplementary Figure 3). Taken together, these data show that AAV-mediated delivery of human β-sarcoglycan reduces muscle fibrosis, improves muscle function and reverses dystrophic pathology of sgcb−/− diseased muscle.

Figure 6
Figure 6

Reduction of fibrosis in ILP-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. (b) Quantification of collagen levels in the TA and GAS muscles from BL6 WT, untreated sgcb−/− mice, and treated mice confirm reduction in collagen levels in treated mice (P<0.001, one-way ANOVA). 100 μm scale bar shown for × 20 images. ***P<0.001.

Safety and biodistribution of rAAVrh.74.tMCK.hSGCB

Initially, normal WT mice injected with 3 × 1010vg of scAAVrh.74.tMCK.hSGCB intramuscularly into the TA showed no signs of toxicity by H&E stain indicating no adverse effects due to the virus (Supplementary Figure 4). Following the ILP vascular delivery of 5 × 1011vg total dose of scAAVrh.74.tMCK.hSGCB as described in the previous section, we next assessed the safety in a small group of mice in this cohort (n=4). We first analyzed the targeted muscles with significant gene expression, as well as off target organs including heart, lung, liver, kidney, spleen, gonads and diaphragm histologically. Paraffin sections were formally reviewed by a veterinary pathologist and there was no evidence of toxicity in any organ noted (Supplementary Table 1). Protein expression and vector biodistribution were also assessed in all of the above tissues and organs with western blotting and qPCR, respectively. Vector genome copies were detected in all organs tested; however, no protein expression was detected in any sample other than treated muscle (Figure 7; Supplementary Table 2). Finally, an analysis of wet weights of treated and untreated muscle shows no significant difference or trend when comparing the average weights from either cohort (Supplementary Table 3). These data provide evidence that the muscle-specific tMCK promoter restricted expression to skeletal muscle and the vector is non-toxic.

Figure 7
Figure 7

Vector biodistribution and protein expression. (a) Histogram of average distribution of vector in harvested tissues from ILP-treated mice given in copies of transcript per microgram of DNA added to qPCR. Left limb was treated. (b) No protein expression via western blot seen in off target organs.


An emerging form of therapy for LGMD2E is viral-mediated gene delivery to restore WT protein to affected muscle resulting in restoration of muscle function. Considering that a subset of patients can develop cardiomyopathy,8, 9, 10, 13 this would have to be considered in the long-term care of these patients. In previous reports, the Sgcb-null mouse was well characterized. Araishi et al.3 developed the β-sarcoglycan-deficient mouse with accompanying loss of all of the sarcoglycans as well as sarcospan, with at least minor preservation of merosin, the dystroglycans and dystrophin, reproducing the clinical picture seen in LGMD2E. The histological changes in this animal model were also a prototype for the clinical counterpart, including the prominence of skeletal muscle fibrosis.14 In a later publication, Dressman et al.25 injected the TA muscle using rAAV2.CMV.SGCB. Expression persisted for 21 months and muscle fibers were protected from recurrent necrosis. This was a singular study showing the potential for gene therapy in LGMD2E. This important study provided the foundation for moving forward with additional serotypes, like AAVrh.74 that are less likely to induce an immune response based on prior environmental exposure. Other additions to the gene therapy repertoire since the original report include the use of self-complementary AAV to enhance transgene expression,16 a muscle-specific promoter to better target skeletal muscle,20, 26 and the optimization of a human β-sarcoglycan gene (hSGCB) as an advantage for clinical trial. In the current study, delivery of scAAVrh74.tMCK.hSGCB provided the tools to address multiple issues and whether gene replacement alone could reverse muscle fiber fibrosis. The AAVrh.74 serotype was chosen to enhance clinical efficacy through vascular delivery using ILP.17, 18, 19 After the initial demonstration of biopotency by intramuscular gene delivery, we showed that comparable or even better restoration of β-sarcoglycan could be achieved with vascular delivery by ILP. The efficacy was also striking with the reversal of dystrophic features including fewer degenerating fibers, reduced inflammation and improved functional recovery by protection against eccentric contraction with increased force generation. From a clinical perspective, the most important novel finding in this study was the reversal of fibrosis from gene replacement alone. At the time of clinical gene transfer fibrosis could be a major obstacle to success. We previously reported that the degree of fibrosis can block efficacy following follistatin gene therapy and correlated with lack of improvement in the distance walked in the 6MWT.24 Successful gene transfer to Sgcb-null mouse resulting in correction of the disease state with reduction in fibrosis provides a pathway to the clinic for AAV-mediated hSGCB transfer for LGMD2E patients.

Materials and methods

Animal models

All procedures were approved by The Research Institute at Nationwide Children’s Hospital Institutional Animal Care and Use Committee (protocol AR12-00040). B6.129-Sgcbtm1Kcam/1J heterozygous mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA; Strain # 006832). Sgcb−/− mice were generated by breeding heterozygous mice. KO mice were bred and maintained as homozygous animals in standardized conditions in the Animal Resources Core at the Research Institute at Nationwide Children’s Hospital. Mice were maintained on Teklad Global Rodent Diet (3.8z5 fiber, 18.8% protein, 5% fat chow) with a 12:12-h dark:light cycle. Identification of SGCB−/− mice was performed by genotyping using PCR. All animals were housed in standard mouse cages with food and water ad libitum.

Beta-sarcoglycan gene construction

The full-length human beta-sarcoglycan cDNA (GenBank Accession No. NM_0034994.3) was codon optimized and synthesized by GenScript Inc, Piscataway, NJ, USA. Codon optimization through GenScript uses an algorithm that takes into account parameters that include transcription, mRNA processing and stability, translation and protein folding to design a cDNA sequence that results in maximum expression in muscle tissue (www.genscript.com). The cDNA was then cloned into a plasmid containing AAV2 ITRs and the cassette included a consensus Kozak sequence (CCACC), an SV40 chimeric intron and a synthetic polyadenylation site (53 bp). The recombinant tMCK promoter was a gift from Dr Xiao Xiao (University of North Carolina). It is a modification of the previously described CK6 promoter27 and includes a modification in the enhancer upstream of the promoter region containing transcription factor binding sites. The enhancer is composed of two E-boxes (right and left). The tMCK promoter modification includes a mutation converting the left E-box to a right E-box (2R modification) and a 6-bp insertion (S5 modification). The pAAV.tMCK.hSGCB vector was constructed by ligation of 1040 bp KpnI/XbaI fragment from pUC57-BSG (Genscript Inc.) into the KpnI/XbaI sites of pAAV.tMCK.hSGCA.26

rAAV production

A modified cross-packaging approach which we have previously reported19 was used to produce the rAAV vector. Here, a triple transfection method with CaPO4 precipitation in HEK293 cells allows for AAV2 ITRs to be packaged into a different AAV capsid serotype.28, 29 The production plasmids were (i) pAAV.tMCK.hSGCB, (ii) rep2-caprh.74 modified AAV helper plasmids encoding cap serotype 8-like isolate rh.74 and (iii) an adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6 and VA I/II RNA genes. Vectors were purified and encapsidated vg titer (utilizing a Prism 7500 Taqman detector system; PE Applied Biosystems, Carlsbad, CA, USA) was determined as previously described.30 The primer and fluorescent probe targeted the tMCK promoter and were as follows: tMCK forward primer, 5′-ACC CGA GAT GCC TGG TTA TAA TT-3′; tMCK reverse primer, 5′-TCC ATG GTG TAC AGA GCC TAA GAC-3′; and tMCK probe, 5′-FAM-CTG CTG CCT GAG CCT GAG CGG TTA C-TAMRA-3′.

Gene delivery

For intramuscular injection, mice were anesthetized and maintained under 1–4% isoflurane (in O2). The anterior compartment of the lower left limb of 4- to 6-week-old SGCB−/− mice was cleaned with 95% EtOH then the TA was injected with 3 × 1011 vg of scAAVrh.74.tMCK.hSGCB diluted in saline in a 30-μl volume using a 30 gauge ultra-fine insulin syringe. The contralateral muscle was left untreated to serve as a control. TA muscle from both limbs was removed at either 6 (n=9, 4 male, 5 female) or 12 (n=6, 4 male, 2 female) weeks post injection to assess gene transfer efficiency. In experiments involving 6-month-old mice (n=5, 5 male), treatment consisted of intramuscular injection into the left TA with 3 × 1011 vg scAAVrh.74.tMCK.hSGCB. For isolated limb perfusion experiments, sgcb−/− mice were perfused at 4 (n=5, 5 male) and 5 (n=4, 2 male, 2 female) weeks of age with 5 × 1011 vg of scAAVrh.74.tMCK.hSGCB by injection into the femoral artery as previously described.19 Animals were euthanized and muscles were analyzed 8 weeks post gene transfer.

EDL force generation and protection from eccentric contractions

A physiological analysis of the EDL muscles from mice treated by ILP was performed. The EDL muscle from both lower hind limbs of treated mice was dissected at the tendons and subjected to a physiology protocol to assess function that was previously described by our laboratory and others19, 31 with some adaptations. During the eccentric contraction protocol, a 5% stretch-re-lengthening procedure executed between 500 and 700 ms (5% stretch over 100 ms, followed by return to optimal length in 100 ms). Following the tetanus and eccentric contraction protocol, the muscle was removed, wet-weighed, mounted on chuck using gum tragacanth, and then frozen in methyl-butane cooled in liquid nitrogen.

TA force generation and protection from eccentric contractions

A protocol to assess functional outcomes in the TA muscle was performed on muscles extracted from mice treated by IM injection. This TA procedure is outlined in several previous studies.32, 33 After the eccentric contractions, the mice were then euthanized and the TA muscle was dissected out, weighed and frozen for analysis. Analysis of the data was performed blindly but not randomly.


Cryostat sections (12 μm) were incubated with a monoclonal human beta-sarcoglycan primary antibody (Leica Biosystems, New Castle, UK; Cat. No. NCL-L-b-SARC) at a dilution of 1:50 in a block buffer (1 × TBS, 10% Goat Serum, 0.1% Tween) for 1 h at room temperature in a wet chamber. Sections were then washed with TBS three times, each for 20 min and re-blocked for 30 min. AlexaFluor 594 conjugated goat anti-mouse secondary IgG1 antibody (Life Technologies, Grand Island, NY, USA; Cat. No. A21125) was applied at a 1:250 dilution for 45 min. Sections were washed in TBS three times for 20 min and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Four random × 20 images covering the four different quadrants of the muscle section were taken using a Zeiss AxioCam MRC5 camera. Percentage of fibers positive for beta-sarcoglycan staining (>50% of muscle membrane staining intensity) was determined for each image and averaged for each muscle.

Western blot analysis

Tissue sections from the left treated TA muscle and the right contralateral TA muscle (20–20 micron thick) were collected into a micro-centrifuge and homogenized with 100 μl homogenization buffer (125 mM Tris-HCl, 4% SDS, 4 M urea) in the presence of 1 protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA). After homogenization, the samples were centrifuged at 10 000 rpm for 10 min at 4 °C. Protein was quantified on NanoDrop (Thermo Scientific, Waltham, MA, USA). Protein samples (20 μg) were electrophoresed on a 3–8% polyacrylamide Tris-acetate gel (NuPage, Invitrogen, Carlsbad, CA, USA) for 1 h 5 min at 150 V and then transferred onto a PVDF membrane (Amersham Biosciences, Piscataway, NJ, USA) for 1 h 15 min at 35 V. The membrane was blocked in 5% non-fat dry milk in TBST for 1 h, and then incubated in a 1:100 dilution of a polyclonal human beta-sarcoglycan antibody (Novus Biologicals, Littleton, CO, USA; Cat. No. NBP-1–90300) and a 1:5000 of a monoclonal mouse gamma-tubulin antibody (Sigma-Aldrich, St Louis, MO, USA; Cat. No. T6557). Anti-mouse (Millipore, Billerica, MA, USA; Cat. No. AP308P) and anti-rabbit (Life Technologies; Cat. No. 656120) secondary-HRP antibodies were used for ECL immunodetection.

EBD assay

A dose of 3 × 1010 vg of scAAVrh.74.tMCK.hSGCB was delivered to 4-week-old sgcb−/− mice to the left TA through an intramuscular injection. Four weeks post injection, mice were injected in the intraperitoneal cavity on the right side at 5 μl/g body weight of a filter sterilized 10 mg/ml EBD in 1x phosphate buffer solution. Mice were then killed 24 h post injection and tissues were harvested and sectioned. Sections were fixed in cold acetone for 10 min and then the immunofluorescence protocol was used to stain for human beta-sarcoglycan.

Morphometric analysis

Muscle fiber diameters and percentage of myofibers with centrally located nuclei were determined from TA and GAS muscles stained with hematoxylin and eosin (H&E). Four random × 20 images per section per animal were taken with a Zeiss AxioCam MRC5 camera. Centrally nucleated fibers were quantified using the NIH ImageJ software (Bethesda, MD, USA). Fiber diameters were measured as the shortest diameter through the muscle fiber using Zeiss Axiovision LE4 software (Carl Zeiss Microscopy, Munich, Germany).

Biodistribution qPCR analysis

Taqman quantitative PCR was performed to quantify the number of vector genome copies present in targeted and untargeted contralateral muscle as well as non-targeted organs as previously described.18, 30 A vector-specific primer probe set was used to amplify a sequence of the intronic region directly downstream from the tMCK promoter that is unique and located within the scAAVrh.74.tMCK.hSGCB transgene cassette. The following primers and probe were used in this study: tMCK intron Forward Primer 5′-GTG AGG CAC TGG GCA GGT AA -3′; tMCK intron Reverse Primer 5′-ACC TGT GGA GAG AAA GGC AAA G -3′; and tMCK intron Probe 5′-6FAM-ATC AAG GTT ACA AGA CAG-GTT TAA GGA GAC CAA TAG AAA-tamra-3′ (IDT). Copy number is reported as vector genomes per microgram of genomic DNA.

Immunohistochemistry for immune cell staining

Immunohistochemistry was used to identify immune cells. Frozen tissue sections on Fisherbrand Superfrost charged microscope slides were incubated with rat anti-mouse monoclonal antibodies using an anti-rat Ig HRP Detection kit (BD Pharmagen, San Jose, CA, USA; Cat: 551013): CD3 (Cat: 555273), CD4 (Cat: 550280), CD8 (Cat: 550281) and Mac-3 for macrophages (Cat: 550292). All primary antibodies were diluted at 1:20 with phosphate-buffered saline. Positive immune staining was visualized using DAB chromagen diluted in DAB buffer with Streptavidin-HRP peroxidase ectastain ABC Peroxidase. Ten random × 40 images were taken for each muscle and each corresponding stain. The number of mononuclear cells was counted and expressed as total number per mm2.

Picrosirius red stain and collagen quantification

Frozen sections placed onto Fisherbrand Superfrost charged microscope slides were fixed in 10% Neutral Buffered Formalin for 5 min, then rinsed in distilled water. Slides were then incubated in Solution A (Phosphomolydbic acid) from the Picrosirius Red Stain Kit (Polysciences Inc., Warrington, PA, USA; Catalog # 24901) for 2 min. After a thorough rinse in distilled water, the slides were placed in Solution B (Direct Red 80/2 4 6-Trinitrophenol) for 15 min, followed by an additional rinse in distilled water and then incubation in Solution C (0.1 N hydrochloride acid) for 2 min. Slides were counterstained for 2.5 min with 1% Fast Green in 1% Glacial Acetic Acid from Poly Scientific (Catalog #S2114) using a 1:10 dilution in DI water. Finally, the slides were rinsed again in distilled water, dehydrated in graded ethanol, cleared in xylene and mounted with coverslips using Cytoseal 60 media from Thermo-Scientific (Waltham, MA, USA; Cat#8310). Images were taken using the AxioVision 4.9.1 software (Carl Zeiss Microscopy). For analysis of Sirius red staining and % collagen quantification, the contrast between the red and the green colors was enhanced using Adobe Photoshop. The color deconvolution plugin in the ImageJ software program was selected and the RGB color deconvolution option was used. The Red image includes all connective tissue from the Sirius Red stain. The Green image includes all muscle from the Fast Green counterstain. Only the Red image and the original image were used. A threshold was then applied to the images to obtain black and white images with areas positive for collagen in black and negative areas in white. Using the measure function, the area of collagen was calculated. The total tissue area was then determined by converting the originally image to ‘8-bit’ and adjusting the threshold to 254, which will be one unit below completely saturating the image. The total tissue area was then measured as done previously and total area was recorded. The percentage of collagen was then calculated by dividing the area of collagen by the total tissue area. The mean percentage for each individual was then calculated.


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We thank the Nationwide Children’s Viral Vector Core for Vector Production. This work has been supported by Families belonging to the GFB Italian Onlus (non-profit organization), Nationwide Children’s Hospital Foundation to LRR-K, and a T32 Graduate Student Training Fellowship from NINDS (T32 NS077984) to ERP.

Author information


  1. Biomedical Sciences Graduate Program, The Ohio State University, Columbus, OH, USA

    • E R Pozsgai
    • , J R Mendell
    •  & L R Rodino-Klapac
  2. Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA

    • E R Pozsgai
    • , D A Griffin
    • , K N Heller
    • , J R Mendell
    •  & L R Rodino-Klapac
  3. Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, USA

    • K N Heller
    • , J R Mendell
    •  & L R Rodino-Klapac
  4. Department of Pediatrics and Neurology, The Ohio State University, Columbus, OH, USA

    • J R Mendell
    •  & L R Rodino-Klapac


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The authors declare no conflict of interest.

Corresponding author

Correspondence to L R Rodino-Klapac.

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