1. In-depth comparison of adeno-associated virus containing fractions after CsCl ultracentrifugation gradient separationMojca Janc, Kaja Zevnik, Ana Dolinar, Tjaša Jakomin, Maja Štalekar, Katarina Bačnik, Denis Kutnjak, Magda Tušek-Žnidarič, Lorena Zentilin, Dmitri G. Fedorov, David Dobnik, 2024, original scientific article 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.
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
Published in DiRROS: 07.08.2024; Views: 39; Downloads: 31
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2. Aberrantly expressed hsa_circ_0060762 and CSE1L as potential peripheral blood biomarkers for ALSMetka Ravnik-Glavač, Massimo Mezzavilla, Ana Dolinar, Blaž Koritnik, Damjan Glavač, 2023, original scientific article Abstract: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive adult-onset neurodegenerative disease that is often diagnosed with a delay due to initial non-specific symptoms. Therefore, reliable and easy-to-obtain biomarkers are an absolute necessity for earlier and more accurate diagnostics. Circular RNAs (circRNAs) have already been proposed as potential biomarkers for several neurodegenerative diseases. In this study, we further investigated the usefulness of circRNAs as potential biomarkers for ALS. We first performed a microarray analysis of circRNAs on peripheral blood mononuclear cells of a subset of ALS patients and controls. Among the differently expressed circRNA by microarray analysis, we selected only the ones with a host gene that harbors the highest level of conservation and genetic constraints. This selection was based on the hypothesis that genes under selective pressure and genetic constraints could have a major role in determining a trait or disease. Then we performed a linear regression between ALS cases and controls using each circRNA as a predictor variable. With a False Discovery Rate (FDR) threshold of 0.1, only six circRNAs passed the filtering and only one of them remained statistically significant after Bonferroni correction: hsa_circ_0060762 and its host gene CSE1L. Finally, we observed a significant difference in expression levels between larger sets of patients and healthy controls for both hsa_circ_0060762 and CSE1L. CSE1L is a member of the importin β family and mediates inhibition of TDP-43 aggregation; the central pathogenicity in ALS and hsa_circ_0060762 has binding sites for several miRNAs that have been already proposed as biomarkers for ALS. In addition, receiver operating characteristics curve analysis showed diagnostic potential for CSE1L and hsa_circ_0060762. Hsa_circ_0060762 and CSE1L thus represent novel potential peripheral blood biomarkers and therapeutic targets for ALS.
Keywords: amyotrophic lateral sclerosis, ALS, circRNA, biomarker, peripheral blood biomarker, hsa_circ_0060762, CSE1L, biomedicine
Published in DiRROS: 12.07.2024; Views: 86; Downloads: 101
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