Background: Strategies for dystrophin restoration, such as AAV-based microdystrophin therapy, are promising approaches for treating Duchenne muscular dystrophy (DMD). These strategies have demonstrated efficacy in mouse DMD models, specifically mdx mice. However, the introduction of microdystrophin to dystrophin-null mice can trigger immune responses with drug-related emergence of anti-dystrophin antibodies. This immune reaction has the potential to hinder the effectiveness of gene therapy.
Methods: AAV9 vector expressing microdystrophin was administered to mdx mice through retro-orbital injection. Concurrently, subsets of AAV-treated mdx mice also received IP injection with 1. anti-CD11b + antiCR1/2; 2. TLR7/8/9 antagonist; 3. combination of anti-Cd11b + anti-CR1/2 + TLR7/8/9 antagonist. Serum samples from treated mdx mice were analyzed for anti-dystrophin antibodies using the Luciferase Immunoprecipitation System (LIPS), a sensitive and specific method for anti-drug antibody (ADA) detection and analysis. The LIPS assay utilized two dystrophin-luciferase fusion proteins representing the amino terminus 33% and carboxy terminus 33% of human dystrophin as antigens.
Results: The upper 95th percentile of LIPS readings (Relative Light Unit) from 40 treatment-naïve mouse sera samples determined the cut-point, allowing for a 5% false-positive ratio. Samples exhibiting RLU value above the cut-point were considered as dystrophin antibody positive. AAV9 microdystrophin administration induced anti-dystrophin antibodies in 71% of tested mdx mice (12/17). Of the anti-dystrophin antibody-positive mice, 92% (11/12) showed reactivity to amino-terminus, and 50% (6/12) against carboxyl-terminus. All 3 co-treatment groups showed some reduction in dystrophin antibody production: 50% with anti-CD11b + anti-CR1/2 (6/12), 40% with TLR7/8/9 antagonist (4/10), and 44% with the combined treatment (8/18).
Conclusion: The administration of anti-complement receptors and/or TLR7/8/9 antagonist may diminish AAV microdystrophin-induced anti-dystrophin antibody production in mdx mice.