DNA Methylation Profiling of rAAVs Reveals Hypomethylated Genomes and Evidence for Predominant Packaging of Replicated Genomes

Immune responses to recombinant adeno-associated viral vectors (rAAVs) impair the therapeutic potential of gene therapies. One trigger for these responses upon systemic administration is the activation of Toll-like receptor 9 by unmethylated cytosine–phosphate–guanine motifs within the vector genome. To better understand the epigenetic profile of rAAVs, we performed bisulfite sequencing on vector genomes produced either via standard triple transfection or via a stable cell line. Furthermore, we investigated whether the epigenetic state of the template DNA influences the encapsidated genome by using in vitro-methylated plasmids during triple transfection. Our results demonstrate that, regardless of the production platform, rAAV genomes lack substantial CpG methylation. Notably, even when a fully CpG-methylated template plasmid was used, the resulting vector DNA exhibited only 4.56% methylation, indicating that alternative strategies—beyond modifying the template DNA—are required to reduce CpG-associated immunogenicity. Additionally, comparing the methylation patterns of rAAVs generated from GpC- and CpG-methylated plasmids revealed that vector genomes were predominantly packaged following genome replication rather than directly from the plasmid DNA. These findings provide critical insights into rAAV packaging mechanisms and inform future strategies for vector engineering.