Simultaneous detection of the AAV genomes and their transcripts in situ at the single-cell level

Gene therapy using recombinant adeno-associated viral (AAV) vectors is a promising approach for treating inherited diseases. Precise characterization of AAV vector genomes and transcripts is essential for further optimization of this technique. Current visualization methods require multiple assays for detecting DNA and RNA, often involving mRNA-to-cDNA conversion. This can obscure insights into spatial distributions, particularly when AAV DNA and mRNA exhibit divergent trends. To address this challenge, we developed a padlock probe (PLP)-based rolling-circle amplification (RCA) technique. Using SplintR DNA ligase, which ligates single-stranded DNA splinted by complementary RNA sequences, enabled our method to directly target AAV mRNA without requiring conversion to cDNA, as well as genomic DNA. Incorporation of an intron within the transgene sequence allows probe designs that distinguish transgene DNA from its mRNA. This strategy enables specific detection of AAV single-stranded DNA (+), single-stranded DNA (-), and mRNA, each effectively amplified by PLP-RCA. Furthermore, this approach enables us to differentiate AAV single-stranded from double-stranded DNA by a combined treatment of lambda exonuclease and restriction enzyme digestion, providing the possibility of tracking the AAV genome processing following transduction. In transduced HeLa cells and liver tissues from AAV-injected mice, PLP-RCA revealed distinct temporal patterns of AAV DNA and mRNA localization, uncovering early DNA instability that influences transduction efficiency. This technique provides a robust and versatile platform for spatially resolved, single-cell analysis of AAV genome and transcript dynamics, facilitating a deeper understanding of AAV biology and aiding the optimization of vector-based gene therapies.