In this section
@misc{Boi:2025,
author = {Boi, S and Mathias, S and Huang, Y-H and Zauner, M and Ali, S and Kraemer, N and Thiele, K and Jung, U},
title = {Optimizing rep proteins expression balance for enhanced production of recombinant AAV vectors},
type = {Poster},
year = {2025}
}
TY - GEN
AB - Recombinant adeno-associated viruses (rAAV) are the main viral vectors used in clinical trials and are generally produced using transiently transfected HEK293 cells. A crucial aspect of optimizing rAAV production involves developing genetic constructs and methodologies tailored to maximize functional titers in these cell lines. Previous data from our group highlighted the significant impact of the interaction between specific HEK cell lines and AAV plasmids on titer yields. Therefore, understanding the mechanistic effects of Rep protein expression is essential. We conducted a detailed analysis of the Rep wildtype system to understand baseline expression levels, the dynamics of Rep protein ratios, and the separation of Rep from Cap proteins. Utilizing Western Blot and mRNA ddPCR techniques, we characterized the expression profiles of the four Rep proteins across various setups, including wildtype, split construct, and co-transfection, correlating these with viral genomes (vg), capsid particles (cp), and transduction units (TU) yields. This enabled us to formulate a hypothesis for optimal Rep expression strategies. Additionally, luciferase assays and tests with diverse transcriptional activities from established and novel promoters, beside varied Rep ratios, enriched our expression analysis. Our findings led to the development of a model for an optimal long Rep (lRep) to short Rep (sRep) ratio, allowing us to rescue combinations that initially resulted in low rAAV titers. In conclusion, we simplified the regulatory feedback loops of Rep expression by identifying optimal baseline levels for specific long-Rep and short-Rep isoforms, thereby enhancing the efficiency of rAAV production processes. Notably, the wildtype results contrasted with rAAV data, underscoring the intricate nature of the rAAV replication mechanism.
AU - Boi,S
AU - Mathias,S
AU - Huang,Y-H
AU - Zauner,M
AU - Ali,S
AU - Kraemer,N
AU - Thiele,K
AU - Jung,U
PY - 2025///
TI - Optimizing rep proteins expression balance for enhanced production of recombinant AAV vectors
ER -
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