High-resolution histological landscape of AAV DNA distribution in cellular compartments and tissues following intramuscular and intravenous injection

Abstract Adeno-associated virus (AAV) is one of the most important gene delivery vehicles for in vivo gene therapy. Intramuscular (IM) and intravascular (IV) injection are commonly used for AAV gene transfer. Unfortunately, the fate of AAV vectors following administration remains unclear at the histological level. Taking advantage of RNAscope, a recently developed in situ hybridization technique that can reveal high-resolution viral DNA localization information, here we evaluated bodywide distribution of an AAV9 vector in the context of cell and tissue microenvironment. We observed distinctive kinetics of cell and nuclear entry of the AAV DNA in striated muscle and liver following IM and IV injection. We also found characteristic distribution patterns of the AAV DNA in various histological structures in internal organs, including gonads and lymph nodes, following IV injection. Finally, we showed significantly bodywide spreading of the AAV DNA following IM injection. These results add a new dimension to our understanding of AAV transduction biology and provide a basis for assessing the full impact of AAV gene therapy.