In-Depth Comparison of Adeno-Associated Virus Containing Fractions after CsCl Ultracentrifugation Gradient Separation
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A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
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Authors
Janc, Mojca
Zevnik, Kaja
Dolinar, Ana
Jakomin, Tjaša
Štalekar, Maja
Bačnik, Katarina
Kutnjak, Denis
Žnidarič, Magda Tušek
Zentilin, Lorena
Fedorov, Dmitrii
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en
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23
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Viruses, Volume 16, issue 8
Abstract
Recombinant adeno-associated viruses (rAAVs) play a pivotal role in the treatment of genetic diseases. However, current production and purification processes yield AAV-based preparations that often contain unwanted empty, partially filled or damaged viral particles and impurities, including residual host cell DNA and proteins, plasmid DNA, and viral aggregates. To precisely understand the composition of AAV preparations, we systematically compared four different single-stranded AAV (ssAAV) and self-complementary (scAAV) fractions extracted from the CsCl ultracentrifugation gradient using established methods (transduction efficiency, analytical ultracentrifugation (AUC), quantitative and digital droplet PCR (qPCR and ddPCR), transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA)) alongside newer techniques (multiplex ddPCR, multi-angle light-scattering coupled to size-exclusion chromatography (SEC-MALS), multi-angle dynamic light scattering (MADLS), and high-throughput sequencing (HTS)). Suboptimal particle separation within the fractions resulted in unexpectedly similar infectivity levels. No single technique could simultaneously provide comprehensive insights in the presence of both bioactive particles and contaminants. Notably, multiplex ddPCR revealed distinct vector genome fragmentation patterns, differing between ssAAV and scAAV. This highlights the urgent need for innovative analytical and production approaches to optimize AAV vector production and enhance therapeutic outcomes.Description
Publisher Copyright: © 2024 by the authors.
Keywords
analytical methods, analytical ultracentrifugation (AUC), CsCl ultracentrifugation gradient, digital droplet PCR (ddPCR), Illumina sequencing, recombinant adeno-associated viruses (rAAVs), size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), transmission electron microscopy (TEM)
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Janc, M, Zevnik, K, Dolinar, A, Jakomin, T, Štalekar, M, Bačnik, K, Kutnjak, D, Žnidarič, M T, Zentilin, L, Fedorov, D & Dobnik, D 2024, 'In-Depth Comparison of Adeno-Associated Virus Containing Fractions after CsCl Ultracentrifugation Gradient Separation', Viruses, vol. 16, no. 8, 1235. https://doi.org/10.3390/v16081235