Towards the establishment of a highly effective adeno-associated virus production by leveraging next generation sequencing and transcriptomics in HEK293 cells

Gene therapy is a rapidly developing field in modern medicine that offers the potential to cure diseases previously thought to be incurable. Among the available delivery methods, recombinant adeno-associated viruses (rAAV) have emerged as one of the most promising vectors due to their low immunogenicity, long-term expression even in differentiated cells, and very low rate of integration into the host genome. However, process yields remain low despite increasing demand for rAAV-based gene therapies and eligibility of more patients for treatment. Therefore, there is a critical need to increase productive capabilities. This work aimed to optimize rAAV production processes in human embryonic kidney cells (HEK293) by establishing a deeper understanding of the underlying mechanism through a holistic approach. To this end, several approaches were combined, such as the comparison of two HEK293 cell lines with different productivity profiles and their differences in nutrient consumption and cell line-specific changes in gene expression patterns, utilizing next-generation transcriptome sequencing. In this case, a negative correlation of cellular growth and rAAV productivity was observed, leading to a substantial increase in yield in the high-producer cell culture. However, the percentage of full and empty capsids, a critical quality parameter, was worse in this case. Overall, both cell lines exhibited a decreased nutrient consumption after transfection, with the exception of selected compounds such as glutamine. Among the analyzed nutrients, no obvious limitation could be identified. This suggests that rAAV productivity is not inherently linked to nutrient abundance, but rather to cell line-specific phenotypes and genetic prerequisites, e.g., the activity of biosynthetic pathways. The functional enrichment analysis of both cell lines showed a substantial disruption of cellular homeostasis at 18 h after transient transfection. This was reflected by increased transcription of central regulators of the ER stress response, DNA damage response, and apoptotic mechanisms in both cell lines. At the time of analysis, the majority of anabolic and catabolic mechanisms were downregulated, which supports the results of the nutrient analysis. The high-producer cell line exhibited greater activity in transcription and translation, as evidenced by significant changes in ribosome biogenesis and chromatin modification, among other factors. Overall, the differential expression of genes after transfection showed similar trends in both the high-producer and low-producer phenotype, but to varying degrees. Based on these results, twelve target genes were selected to be screened in a small interfering RNA-mediated gene knockdown experiment with the aim of increasing rAAV productivity in the low-producer phenotype. However, it was not possible to identify genes that were clearly beneficial for rAAV production upon knockdown. Partial depletion of catenin beta 1, ubiquitin conjugating enzyme E2 G2, and glycogen synthase kinase 3 beta transcripts showed positive trends in terms of cell health and viral productivity, warranting further research. In light of significant changes in chromatin structure and cell cycle regulation revealed by transcriptomic analysis, five small molecule enhancers were tested for their effect on rAAV productivity in HEK293 suspension cells. M344, a histone deacetylase inhibitor and flavopiridol, a cyclin-dependent kinase inhibitor, improved rAAV productivity by at least 2-fold and up to 8-fold across different processes. This opens up the possibility of screening chemically related compounds which address the same or a similar mechanism of action, e.g., the inhibition of the histone deacetylase subtype HDAC6. Taken together, this work provides a foundation for future, more detailed experiments regarding the production of rAAV in HEK293. For example, these could include specific aspects such as analyzing nucleotide and lipid metabolism during rAAV production or identifying other effective small molecule enhancers. Additionally, the transcriptome could be analyzed for single nucleotide polymorphisms or the regulatory impact of long non-coding RNA molecules, which exhibited marked upregulation upon transient transfection. Further research is necessary to explore rational genetic engineering of rAAV-producing HEK293 cell lines to increase productivity and make rAAV gene therapy more accessible by reducing manufacturing costs.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2025

Author dc:creator

• Krämer, Jan Niklas

Repository record source_url

https://pub.uni-bielefeld.de/record/3006649

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:3006649