Sequential VEGF-A165 plasmid and AAV-follistatin gene therapy enhances muscle hypertrophy and capillarisation in C57BL/6 mice

Introduction. Skeletal muscle loss in sarcopenia and neuromuscular disorders remains a major unmet medical need. AAV9-delivered follistatin (FST), a myostatin/activin antagonist, induces muscle hypertrophy; however, fibre growth without adequate vascular adaptation may limit therapeutic efficacy. We evaluated whether co-administration of a VEGF-A165 plasmid enhances the hypertrophic and angiogenic effects of intramuscular AAV-FST gene transfer in C57BL/6 mice. Methods. Thirty-six C57BL/6 mice (18 males, 18 females) were assigned to PBS vehicle (n=10), AAV-FST (1 x 10^11 vg; n=10), VEGF plasmid (100 ug; n=6), or combination treatment (VEGF plus AAV-FST; n=10). The contralateral hindlimb served as an internal control. Endpoints at Day 115 included hindlimb muscle mass ratio (R/L), transgene expression, FST protein levels, muscle fibre morphometry, capillary density, and safety assessments. Results. Combination therapy produced the highest R/L ratio (1.176 +/- 0.091; p=0.004; d=2.04), whereas AAV-FST alone showed a borderline effect (R/L=1.113; p=0.050). Compared with AAV-FST monotherapy, combination treatment increased muscle FST mRNA approximately 2.1-fold, protein levels approximately 2.0-fold, and the muscle-to-liver expression ratio 2.6-fold. It also induced larger muscle fibres and doubled CD31+ vessel counts versus AAV-FST alone, indicating simultaneous hypertrophy and angiogenesis. No adverse haematological, biochemical, or histopathological findings were observed. Discussion. Combined AAV-FST and VEGF therapy enhanced local muscle hypertrophy, increased capillary density, and improved the muscle-to-liver transgene expression profile compared with AAV-FST monotherapy. The regimen was well tolerated and supports further evaluation of angiogenic preconditioning as a strategy to improve muscle-directed gene therapy for muscle-wasting disorders.