Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, and is amenable to gene-editing and gene replacement therapies. Serious adverse events have been reported in patients receiving AAV-microdystrophin during Phase I/II clinical trials due to immune responses against the AAV capsid. Here, we comprehensively characterize AAV-induced immune responses in vivo. Mice were systemically dosed and re-dosed with AAV9 at 1.16E14vg/kg carrying either micro-dystrophin, Cas9, or Cas9 with a frameshift mutation (Cas9-FS), disrupting Cas9 protein expression. Peripheral blood mononuclear cells (PBMCs) were collected at baseline and after the first and second administrations of AAV (five hours, two weeks and four weeks post dosing). TLR signaling was first investigated, since AAV genomes contain unmethylated CpG motifs. We calculated the risk potential for TLR9 activation for each transgene: Cas9 (KTLR9=20), Cas9-FS vectors (KTLR9=20) and micro-dystrophin (KTLR9=13). All three vectors are above the speculated threshold (KTLR9>10) to activate TLR9. Accordingly, Luminex assays revealed that all three vectors increased TNF?, IP-10, MCP-1, and MIP-1? following the second dose of AAV at the five-hour time point, strongly suggestive of TLR9:MyD88 activation. Moreover, we probed for complement levels, as consumption of C3 and C4 were reported in ongoing DMD clinical trials and observed significant consumption of C3 and C4 across all three vectors after the second dose at the five-hour time point, suggesting activation of the classical pathway, which relies on antibody production. Our preliminary findings support this hypothesis, as we observed peak anti-AAV IgM and IgG responses after the second dose. Finally, we performed single cell RNA-sequencing to analyze immune cell phenotypes and our initial findings suggest pronounced activation of all main immune types following the second administration of AAV. Ongoing studies are needed to identify critical immune cell types that could be targeted to improve the safety and efficacy of AAV-based therapies.