Quantitative analysis of genome truncation patterns in oversized adeno-associated virus vectors

Adeno-associated virus (AAV), a 4.7 kb single-stranded DNA virus, is widely used as a gene therapy vector, but its limited packaging capacity poses challenges for delivering large genes, always resulting in truncation during packaging. Quantifying truncation is difficult because both strands of the plasmid can be packaged from the 3' end. In this article, we aim to first produce single-polarity AAV and then explore its truncation pattern. To address this, we modified one of the ITRs and created an oversized self-complementary AAV, which functions as a single-polarity vector to some extent. Using this modified backbone, we generated a series of oversized single-polarity AAV (spAAV) vectors of varying lengths and sequences, analyzing DNA truncation patterns via quantitative PCR (qPCR). The results show that as the distance from the 3'-ITR increased, less DNA was detected. At 3000 bp from the 3'-ITR, 70% of the genomic DNA remained; this dropped to 50% at 4000 bp, 20% at 4500 bp, and almost none beyond 5000 bp. Additionally, reporter gene expression significantly decreased when the expression cassette extended to 4.5 kb compared to 2.7 kb under identical in vitro conditions. Our results show that DNA will be truncated far earlier before 4.5 kb during the packaging of very large genomes. This study provides important insights into the truncation patterns of AAV genomes, which is crucial for optimizing AAV vector design in gene therapy.