Volume 16, Issue 4, Pages 657-664 (April 2008) Systemic Microdystrophin Gene Delivery Improves Skeletal Muscle Structure and Function in Old Dystrophic mdx Mice  Paul Gregorevic, Michael J Blankinship, James M Allen, Jeffrey S Chamberlain  Molecular Therapy  Volume 16, Issue 4, Pages 657-664 (April 2008) DOI: 10.1038/mt.2008.28 Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 1 Systemic microdystrophin gene delivery improves whole-body indices of disease state by restoring dystrophin expression in the striated musculature of aged dystrophic mice. Untreated 24-month-old dystrophic, mdx mice (mdx, gray columns) showed (a) reduced body mass (P = 0.057) when compared with age-matched wild-type mice (WT, black columns), (b) reduced lean mass, and (c) reduced fat mass. By comparison, treated mdx mice (Tmdx, red columns) that had received ∼1 × 1013 vector genomes of rAAV6:microdystrophin through tail vein injection at 20 months of age, when analyzed 4 months after the injection, exhibited increased body mass and lean mass when compared with untreated dystrophic mice. (d) Untreated dystrophic mice demonstrated elevated serum levels of creatine kinase (CK) when compared with WT mice, while treated animals demonstrated CK levels >90% reduced compared with the untreated cohort. (e) Cryosections of striated muscles (also see Figures 2a and 4a) that were examined for dystrophin expression (green) demonstrate that the muscles of untreated dystrophic animals lack the uniform protein expression that is observed in WT mice, and which is restored by treatment with systemic administration of rAAV6:microdystrophin. *P < 0.05 for mdx versus WT. **P < 0.05 for Tmdx versus mdx. Scale bar = 100 μm. rAAV6, recombinant adeno-associated virus serotype 6. Molecular Therapy 2008 16, 657-664DOI: (10.1038/mt.2008.28) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 2 Systemic microdystrophin gene delivery improves the morphological properties of the limb muscles of aged dystrophic mice. (a) Cryosections of tibialis anterior (TA) hindlimb muscles from untreated dystrophic mice (mdx) lack the uniform dystrophin expression that is observed in wild-type mice (green), and instead demonstrate substantial evidence of myofiber degeneration and remodeling. (b) In contrast, dystrophin expression (and, consequently membrane localization of α-dystrobrevin-2 and β-dystroglycan) is restored to the majority of myofibers in the TA muscles of treated mice. (c) Expression of microdystrophin did not appear to significantly reduce the proportion of unusually small myofibers in the limb muscles of treated dystrophic animals although (d) the incidence of centrally located myonuclei (a measure of myofiber remodeling and incomplete regeneration) was reduced in the transduced muscle fibers of treated mouse TA muscles compared with the muscles of untreated dystrophic mice [Tmdx dys+ve, yellow line, and in d, yellow column]. (e) The TA muscles of treated animals displayed a trend (P = 0.089) toward an increased mass when compared with the muscles of untreated dystrophic animals, even surpassing that of muscles from age-matched wild-type mice. *P < 0.05 for mdx versus wild type. **P < 0.05 for Tmdx versus mdx. Scale bars = 100 μm. Molecular Therapy 2008 16, 657-664DOI: (10.1038/mt.2008.28) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 3 Systemic microdystrophin gene delivery improves the functional properties of the limb muscles of aged dystrophic mice. The tibialis anterior muscles of untreated dystrophic mdx mice (mdx, gray) displayed reduced (a) absolute and (b) normalized maximal force-producing capacity when compared with the muscles of age-matched nondystrophic mice [wild type (WT), black]. In contrast, the muscles of mice systemically treated with rAAV6:microdystrophin (Tmdx, red) demonstrated a significantly greater capacity to generate force when compared with the muscles of untreated dystrophic mice. The absence of dystrophin in the muscles of untreated dystrophic animals contributes to an increased susceptibility to contraction-induced injury when repeatedly subjected to strain of (c) 20% or (d) 40% beyond the optimum length during maximal contraction. LC1, LC2, and LC3 denote force outputs after 1, 2, and 3 “lengthening contractions” of this nature. The muscles of dystrophic mice that had received rAAV6:microdystrophin 4 months prior to evaluation exhibit (c and d top panels) improved force output when compared with the muscles of untreated animals because of greater initial capacity, but (c and d bottom panels) showed little change with respect to relative resistance to contraction-induced injury. *P < 0.05 for mdx versus WT. **P < 0.05 for Tmdx versus mdx. rAAV, recombinant adeno-associated virus. Molecular Therapy 2008 16, 657-664DOI: (10.1038/mt.2008.28) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 4 Systemic microdystrophin gene delivery improves the morphological and functional properties of the respiratory musculature of aged dystrophic mice. Aged dystrophic mice that had received a systemic administration of rAAV6:microdystrophin 4 months prior to evaluation demonstrated considerably improved morphology of the diaphragm musculature when compared with untreated dystrophic mice (a) despite sustaining less than complete restoration of dystrophin expression (Tmdx, red, b). The diaphragm musculature of untreated dystrophic mice (mdx, gray) was comprised of an increased proportion of small myofibers (c), and muscle cells with centrally located nuclei (d) when compared with the musculature of age-matched nondystrophic mice [wild type (WT), black]. By comparison, the dystrophin-postive myofibers within the muscles of treated animals (Tmdx dys+ve, yellow) were on average larger, and considerably less frequently centrally nucleated, corresponding to the reduced incidence of myofiber turnover (c,d). Treatment had little effect on restoring the depressed normalized force-producing capacity observed as a consequence of dystrophic pathology in the diaphragm musculature (e), but dramatically enhanced the resistance of muscles to mild (20% strain, f) and severe (40% strain, g) contraction-induced injury, when compared with the muscles of untreated dystrophic mice. *P < 0.05 for mdx versus WT. **P < 0.05 for Tmdx versus mdx. Scale bar = 400 μm. rAAV, recombinant adeno-associated virus. Molecular Therapy 2008 16, 657-664DOI: (10.1038/mt.2008.28) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions