Standardized Nomenclature and Reporting for PacBio HiFi Sequencing and Analysis of rAAV Gene Therapy Vectors

Talevich, E.; Tseng, E.; Diallo, A.; Sellami, N.; Elliott, A.; Cantarel, B. L.; Tonthat, N.; Chatterjee, P.; Tai, P. W. L.; Aldridge, C.

doi:10.1101/2024.05.07.592296

bioRxiv · 10.1101/2024.05.07.592296

Standardized Nomenclature and Reporting for PacBio HiFi Sequencing and Analysis of rAAV Gene Therapy Vectors

Talevich, E.·Tseng, E.·Diallo, A.·Sellami, N.·Elliott, A.·Cantarel, B. L.·Tonthat, N.·Chatterjee, P.·Tai, P. W. L.·Aldridge, C.

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Abstract

Despite recombinant adeno-associated viruses (rAAVs) being the leading platform for gene therapy, there is a lack of standardized computational analysis methods and reporting to assess the contents of each capsid through long-read sequencing. PacBios highly accurate long-read HiFi sequencing enables comprehensive characterization of AAV genomes but requires bioinformatics expertise for analyzing, interpreting and comparing the results. To address this need and improve the understanding of functional viral payloads, our working group established standardized nomenclature and reporting for long-read sequencing data of rAAV vectors. The working group recommendations cover critical quality attributes (CQAs) related to vector purity (full-length vs. fragmented genomes) and identification of contaminants (host DNA, plasmid DNA). Our data analyses of de novo manufacturing runs by the recommended protocol revealed specificity of full and partially filled capsids and high-resolution characterization of partial/truncated vector species. Finally, we provide an open-source software implementing this standardized AAV analysis and reporting to promote transparency, facilitate data comparability, and improve rAAV vector design and quality control.

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Talevich, E., Tseng, E., Diallo, A., Sellami, N., Elliott, A., Cantarel, B. L., Tonthat, N., Chatterjee, P., Tai, P. W. L., Aldridge, C.. 2024-05-12. Standardized Nomenclature and Reporting for PacBio HiFi Sequencing and Analysis of rAAV Gene Therapy Vectors. https://doi.org/10.1101/2024.05.07.592296

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