Enhancing AAV-microdystrophin gene therapy after repeat dosing by blocking phagocytosis.

Background: Inefficient transduction is a major limitation in achieving therapeutic levels of AAV-delivered microdystrophin capable of improving muscle function in patients with Duchenne muscular dystrophy. Additionally, some patients experience acute complications due to activation of innate immune pathways, such as complement. We propose that inhibiting complement receptor 1/2/3 (CR 1/2/3)-mediated phagocytosis and endosomal TLR 7/8/9 signaling pathways may decrease immune and inflammatory responses while simultaneously increasing the availability of AAV virus for muscle transduction. Methods: Results: Viral load was significantly increased by 77-fold in white blood cells after two rAAV9-microdystrophin doses compared to mice receiving a single dose. Repeated gene therapy resulted in a lower viral load and microdystrophin expression in muscle compared to a single rAAV dose. 63% of mice treated with two rAAV9-microdystrophin doses produced antibodies to dystrophin, which was less in mice treated with two rAAV9-microdystrophin doses and combination therapy (25%). Likewise, AAV capsid specific antibody levels were reduced in mice receiving combination therapy. Microdystrophin expression in skeletal muscle evaluated by mass spectrometry, immunofluorescence, and western blotting showed significantly higher levels in combination-treated mice compared to rAAV9-microdystrophin alone. Conclusions: Our results demonstrate that combination treatment with complement receptor 1/2/3 antibodies and a TLR 7/8/9 antagonist enhances rAAV9-microdystrophin gene therapy in