Isolation And Characterization Of Novel Adeno-Associated Viruses For The Advancement Of Gene Therapy

Kalyani Nambiar, University of Pennsylvania


Adeno-associated viruses (AAVs) are nonpathogenic, single-stranded DNA viruses that have a broad tissue tropism, which make them effective candidates for human gene therapy. Currently available AAV vectors have been shown to be effective for certain clinical applications, but there is room for improvement of their transduction efficiency. Hence, the discovery of new, superior AAV capsids for use as vectors for gene therapy is paramount. Traditional discovery of AAV genomes from biomaterials has relied on PCR-based techniques; however, the risk of PCR-related errors in the resulting sequences using these methods is of concern. Thus, we sought to test the effect of polymerase fidelity on AAV genome recovery and developed AAV-Single Genome Amplification (AAV-SGA), a technique to accurately isolate AAV sequences from mammalian sources. Using a high fidelity polymerase and AAV-SGA, we isolated novel AAV genomes from primate tissues. These genomes displayed evidence of positive selection and recombination within the host. Additionally, we characterized the biological properties of the novel capsids as AAV vectors and observed variable cell and tissue transduction properties in vivo, with notable transduction of brain microvasculature by AAVrh92 and efficient systemic transduction of AAVrh90. We report the favorable transduction profile of AAVrh91 in the primate central nervous system after intrathecal delivery. This work describes a new method for AAV genome discovery and identification of novel vectors that have the potential to be developed into effective clinical gene therapy vectors.