| Title: | In-depth comparison of adeno-associated virus containing fractions after CsCl ultracentrifugation gradient separation |
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| Authors: | ID Janc, Mojca (Author)
ID Zevnik, Kaja (Author)
ID Dolinar, Ana (Author)
ID Jakomin, Tjaša (Author)
ID Štalekar, Maja (Author)
ID Bačnik, Katarina (Author)
ID Kutnjak, Denis (Author)
ID Tušek-Žnidarič, Magda (Author)
ID Zentilin, Lorena (Author)
ID Fedorov, Dmitri G. (Author)
ID Dobnik, David (Author) |
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| Files: |  URL - Source URL, visit https://doi.org/10.3390/v16081235
 PDF - Presentation file, download (8,47 MB)
MD5: CDB8CCAAB1750BA1DA6796CF3DA6E6E8
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| Language: | English |
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| Typology: | 1.01 - Original Scientific Article |
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| Organization: |  NIB - National Institute of Biology
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| 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. |
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| Keywords: | 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 |
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| Publication status: | Published |
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| Publication version: | Version of Record |
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| Publication date: | 01.08.2024 |
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| Year of publishing: | 2024 |
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| Number of pages: | str. 1-23 |
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| Numbering: | Vol. 16, iss. 8, [article no.] 1235 |
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| PID: | 20.500.12556/DiRROS-20182  |
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| UDC: | 578 |
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| ISSN on article: | 1999-4915 |
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| DOI: | 10.3390/v16081235 |
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| COBISS.SI-ID: | 203835907 |
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| Note: | 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;
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| Publication date in DiRROS: | 07.08.2024 |
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| Views: | 307 |
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| Downloads: | 280 |
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