Results
Title

In-Depth Comparison of Adeno-Associated Virus Containing Fractions after CsCl Ultracentrifugation Gradient Separation.

Authors

Janc, Mojca; Zevnik, Kaja; Dolinar, Ana; Jakomin, Tjaša; Štalekar, Maja; Bačnik, Katarina; Kutnjak, Denis; Žnidarič, Magda Tušek; Zentilin, Lorena; Fedorov, Dmitrii; Dobnik, David

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.

Subjects

ENZYME-linked immunosorbent assay; ADENO-associated virus; TRANSMISSION electron microscopy; TREATMENT effectiveness; RECOMBINANT viruses

Publication

Viruses (1999-4915), 2024, Vol 16, Issue 8, p1235

ISSN

1999-4915

Publication type

Academic Journal

DOI

10.3390/v16081235