AAV-mediated overexpression of human α7 integrin leads to histological and functional improvement in dystrophic mice

Abstract

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by mutations in the DMD gene, with loss of its gene product, dystrophin. Dystrophin helps link integral membrane proteins to the actin cytoskeleton and stabilizes the sarcolemma during muscle activity. We investigated an alternative therapeutic approach to dystrophin replacement by overexpressing human α7 integrin (ITGA7) using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal and cardiac muscle that links the extracellular matrix (ECM) to the actin skeleton. It is modestly upregulated in DMD muscle and has been proposed to be an important modifier of dystrophic symptoms. We delivered rAAV8.MCK.ITGA7 to the lower limb of mdx mice through isolated limb perfusion (ILP) of the femoral artery. We demonstrated ~50% of fibers in the tibialis anterior (TA) and extensor digitorum longus (EDL) overexpressing α7 integrin at the sarcolemma following AAV gene transfer. The increase in ITGA7 in skeletal muscle significantly protected against loss of force following eccentric contraction-induced injury compared with untreated (contralateral) muscles while specific force following tetanic contraction was unchanged. Reversal of additional dystrophic features included reduced Evans blue dye (EBD) uptake and increased muscle fiber diameter. Taken together, this data shows that rAAV8.MCK.ITGA7 gene transfer stabilizes the sarcolemma potentially preserving mdx muscle from further damage. This therapeutic approach demonstrates promise as a viable treatment for DMD with further implications for other forms of muscular dystrophy.

Figure 1

Expression of human α7 in the mdx mouse hind limb following isolated limb perfusion of rAAV8.MCK.ITGA7. (a) Untreated muscle fibers show no α7 integrin staining in contralateral limb muscle. (b) Immunostaining with an antibody specific to ITGA7 reveals α7 integrin in rAAV8.MCK.ITGA7-treated mdx muscles; ITGA7 antibody only recognizes human ITGA7 and does not cross-react with mouse α7. (c) Quantification of the average percentage of myofibers overexpressing α7 integrin in rAAV8.MCK.ITGA7-treated mdx muscles (n = 7; error bar, SEM). (d) Western blot analysis for α7 integrin demonstrates the presence of a 26 kD cleavage fragment in mdx-treated muscle (n = 5) which is absent in control mdx samples (n = 2). AAV, adeno-associated virus.

Figure 2

rAAV8.MCK.ITGA7 treatment improves histology in mdx mice. (a) rAAV8.MCK.ITGA7 treatment in mdx mice results in a decrease in centralized nuclei, a hallmark of DMD pathology, compared with untreated mdx controls (**P < 0.01). (b) Quantification of the average fiber diameter of rAAV8.MCK.ITGA7 treated (includes transduced and untransduced fibers) versus untreated (contralateral side) mdx muscle shows no difference in fiber diameter following hematoxylin and eosin staining. Error bars, SEM for (n = 7). (c) Hematoxylin and eosin images of treated rAAV8.MCK.ITGA7 mdx muscle (right) illustrate a decrease in the number of centralized nuclei compared with untreated contralateral mdx muscle. AAV, adeno-associated virus; DMD, Duchenne muscular dystrophy.

Figure 3

rAAV8.MCK.ITGA7 treatment promotes myofiber hypertrophy. (a) Co-immunofluorescence staining shows myosin-stained (red cytoplasm) type IIb (red) fibers counterstained with α7 (green membrane) in a treated TA muscle illustrating larger fiber size diameter. (b) In this histogram, the fiber size diameter of the transduced type IIb fibers (red) can be directly compared with untransduced type IIb fibers (green) in the same treated muscle and in control muscle (black) from contralateral side (transduced (red) = 47.98 ± 1.06 versus untransduced (green) = 42.85 ± 1.79 versus control (black) = 43.35 ± 1.39; *P < 0.05). Error bars represent SEM for n = 4. AAV, adeno-associated virus; TA, tibialis anterior.

Figure 4

rAAV8.MCK.ITGA7 treatment improves muscle membrane integrity. Mdx muscles treated with 1 × 10¹¹ vg of rAAV8.MCK.ITGA7 were compared with untreated contralateral mdx muscles for Evans blue dye uptake at 6 weeks post gene transfer following downhill running. (a) ITGA7-treated mdx muscle and (b) untreated contralateral muscle stained with an antibody specific to α7 integrin (green) and Evans blue dye (orange). (c) Quantification of the percentage of Evans blue positive fibers in treated versus untreated mdx muscle. rAAV8.MCK.ITGA7 treatment significantly protected mdx muscle membranes against Evans blue dye uptake compared with untreated (contralateral) muscles (**P < 0.01). Evans blue dye fibers were quantified as a percent out of a total of 1,500 fibers counted per animal. Error bars, SEM for (n = 6). AAV, adeno-associated virus; EBD, Evans blue dye; vg, vector genome.

Figure 5

Additional α7 integrin protects mdx muscle from contraction-induced damage. Mdx muscles treated by isolated limb perfusion via the femoral artery with 1 × 10¹² vg (high dose, red) and 3 × 10¹¹ vg (low dose, green) of rAAV8.MCK.ITGA7 (mouse) were compared with untreated contralateral mdx EDL muscles (blue) and WT (C57BL/10) EDL muscles (black) 6 weeks post gene transfer. (a) Measurement of normalized specific force following tetanic contraction in rAAV8.MCK.ITGA7-treated muscles was not increased with either low or high dose compared with untreated contralateral mdx muscle. (b) Muscles were then assessed for loss of force following repetitive eccentric contractions. Both dose cohorts of rAAV8.MCK.ITGA7 (green and red) significantly protected mdx muscle from loss of force compared with untreated (contralateral) muscles (blue). Two-way analysis of variance demonstrates significance in decay curves (***P < 0.001). Moreover, Bonferroni post-hoc analysis revealed that in the high dose (red) force retention following contractions 3–10 (*P < 0.05 and **P < 0.01) showed no significant difference from WT muscles (black). Error bars, SEM for n = 9 (rAAV8.MCK.ITGA7), 5 (WT, C57BL/10), or 19 (mdx) muscles per condition. AAV, adeno-associated virus; EDL, extensor digitorum longus; vg, vector genome; WT, wild-type.