Simultaneous Detection of AAV Genome, Transcript, and Protein Localization in Intact Cells and Tissues at High Resolution

Recent years have seen significant progress in AAV capsid engineering for gene delivery with increased efficiency and desired cell-type specificity to match the needs of pre-clinical research and gene therapy. The cellular uptake of AAVs, however, can stop short of the ultimate goal of cargo protein production - due to AAV silencing, insufficient nuclear transport, inefficient uncoating, failed second-strand synthesis, or other still to be discovered mechanisms. Defining the relationship between AAV genome uptake, transcription and cargo protein synthesis efficiencies in different cell types and tissues can help bypass key bottlenecks in gene delivery and guide effective AAV engineering. We adapted a recently published in-situ-transcription-based signal amplification method, the “Zombie technique” (1), to detect AAV genomes in a variety of fixed cells and tissues. Zombie involves producing 20 to 380 base-pair long barcode transcripts by exogenously-supplied T7 phage polymerase, which are then visualized at high resolution in intact cells through fluorescent in situ hybridization (FISH) by hybridization chain reaction (HCR). We demonstrate that Zombie enables simultaneous detection of AAV genomes and AAV transcripts by FISH and cargo proteins by immunofluorescence. Consequently, we can determine the rates of AAV genome nuclear uptake, cargo transcription and cargo translation at different time points post-infection. Combining the Zombie method with cell-type-specific markers (protein immunolabelling or mRNA FISH) can give insights into subcellular AAV processing in complex tissues and help pinpoint to limiting factors in the overall transduction process. Interestingly, we find that after direct brain injection of AAV6, although cargo genomes do enter the nuclei of microglia, these genomes do not result in functional cargo protein production in this cell type. Furthermore, we analyze the pattern of AAV genome nuclear uptake, transcription and translation in mice after systemic delivery of AAV9 and engineered AAV capsids (AAV.PHP.eB, AAV.CAP-B10) (2, 3) across brain, liver and spleen. In conclusion, the Zombie technique adapted to AAV vectors enables simultaneous cargo genome, mRNA and protein visualization and thus can help differentiate cellular and subcellular processing steps of engineered gene delivery vectors, facilitating their refinement for basic and translational research