| Naslov: | In-depth comparison of adeno-associated virus containing fractions after CsCl ultracentrifugation gradient separation |
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| Avtorji: | ID Janc, Mojca (Avtor)
ID Zevnik, Kaja (Avtor)
ID Dolinar, Ana (Avtor)
ID Jakomin, Tjaša (Avtor)
ID Štalekar, Maja (Avtor)
ID Bačnik, Katarina (Avtor)
ID Kutnjak, Denis (Avtor)
ID Tušek-Žnidarič, Magda (Avtor)
ID Zentilin, Lorena (Avtor)
ID Fedorov, Dmitri G. (Avtor)
ID Dobnik, David (Avtor) |
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| Datoteke: |  URL - Izvorni URL, za dostop obiščite https://doi.org/10.3390/v16081235
 PDF - Predstavitvena datoteka, prenos (8,47 MB)
MD5: CDB8CCAAB1750BA1DA6796CF3DA6E6E8
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| Jezik: | Angleški jezik |
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| Tipologija: | 1.01 - Izvirni znanstveni članek |
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| Organizacija: |  NIB - Nacionalni inštitut za biologijo
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| Povzetek: | 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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| Ključne besede: | 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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| Status publikacije: | Objavljeno |
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| Verzija publikacije: | Objavljena publikacija |
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| Datum objave: | 01.08.2024 |
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| Leto izida: | 2024 |
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| Št. strani: | str. 1-23 |
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| Številčenje: | Vol. 16, iss. 8, [article no.] 1235 |
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| PID: | 20.500.12556/DiRROS-20182  |
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| UDK: | 578 |
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| ISSN pri članku: | 1999-4915 |
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| DOI: | 10.3390/v16081235 |
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| COBISS.SI-ID: | 203835907 |
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| Opomba: | 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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| Datum objave v DiRROS: | 07.08.2024 |
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| Število ogledov: | 359 |
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| Število prenosov: | 304 |
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| Metapodatki: |    |
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