Orthogonal characterization of rAAV reveals vector attributes that drive ITR repair, self-complementary genome formation, and transgene expression

Abstract

Recombinant adeno-associated virus (rAAVs) vectors are the flagship vehicles for delivering DNA payloads for human gene therapy. However, only a few outstanding therapies have reached the market in the past decade. One reason for the slow development of rAAV-based gene therapies has been our limited knowledge of basic AAV biology. Therefore, the goal of this work was to investigate the influence of rAAV inverted terminal repeat (ITR) design, promoter use, and serotype selection on vector genome heterogeneity and transduction efficiency. Our analyses revealed additional, larger and smaller, vector genome species that were identified as unresolved and bound by the ITRs. Alkaline gel analysis and long-read single-molecule sequencing confirmed the presence of double-stranded genomes with self-complementary (sc) configurations for most of the serotypes and ITR designs tested. sc genome formation and ITR repair were favored under specific capsid-genome feature combinations, resulting in genomes larger than 5 kb in some cases. Furthermore, we found that vector genome length had an impact on ITR repair. Finally, transduction studies confirmed that the AAV capsid plays a role in functional ITR repair and potency. In conclusion, sc genome packaging is favored under specific ITR designs, and their stability is influenced by the AAV capsid.