20.500.12556/DiRROS-20182 In-depth comparison of adeno-associated virus containing fractions after CsCl ultracentrifugation gradient separation 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. recombinant adeno-associated viruses (rAAVs) CsCl ultracentrifugation gradient analytical methods digital droplet PCR (ddPCR) transmission electron microscopy (TEM) analytical ultracentrifugation (AUC) size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) Illumina sequencing virology rekombinantni z adenovirusi povezani virusi centrifugiranje v CsCl gradientu analitične metode digitalni kapljični PCR virologija true false true Angleški jezik Slovenski jezik Neznano 2024-08-07 12:45:13 2024-08-07 12:45:13 2024-08-08 04:07:53 0000-00-00 00:00:00 2024 0 0 Nasl. z nasl. zaslona; Soavtorji: Kaja Zevnik, Ana Dolinar, Tjaša Jakomin, Maja Štalekar, Katarina Bačnik, Denis Kutnjak, Magda Tušek Žnidarič, Lorena Zentilin, Dmitrii Fedorov, and David Dobnik; Opis vira z dne 7. 8. 2024; str. 1-23 iss. 8, [article no.] 1235 Vol. 16 Avg. 2024 0000-00-00 Zaloznikova Objavljeno apc 0000-00-00 0000-00-00 2024-08-01 578 1999-4915 10.3390/v16081235 203835907 https://doi.org/10.3390/v16081235 1 5ea55045-5499-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=28228 RAZ_Janc_Mojca_2024.pdf RAZ_Janc_Mojca_2024.pdf 1 CDB8CCAAB1750BA1DA6796CF3DA6E6E8 a486896cf528bee4f303099a0adbe1c92348ff174a1cb27c4219389a3156ed3f c04ff225-5499-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=28229 Nacionalni inštitut za biologijo 0 0 0