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Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for Gene Therapy Applications

ABSTRACT: Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for Gene Therapy Applications

PROVIDER: PRJNA938438 | ENA |

REPOSITORIES: ENA

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Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for Gene Therapy Applications

Project description:Human embryonal kidney cells (HEK-293) are the most common host cells used for transient recombinant adeno-associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub-cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK-293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. We found significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in-process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK-293 cells Comparison of three HEK-293 suspension cell lines transcriptomics during an AAV production process

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Project description:To determine the target genes of RBM10,we have employed microarray based gene expression profiling by knocking down RBM10 in HEK 293 cells. Microarray analysis after RBM10 knockdown on HEK 293 cells showed that over 1000 genes were down regulated while another 800 genes up regulated as a result of the knockdown. Among the down regulated genes, we found the significant presence of cardiovascular disease related genes, especially cardiac hypertrophy and heart failure.

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