SUPERVISOR: Nicole BORTH

PROJECT ASSIGNED TO: Alexander Karl MOLIN

DNA methylation is a life-sustaining regulatory system that allows cells to precisely control gene expression and adapt to changing conditions without changing the fundamental DNA sequence. Their patterns are altered by de novo methylation or demethylation in response to stress and new environmental conditions, determining their function and responsiveness to external signals. Understanding the epigenetics and transcription of a cell line is critical for ensuring its identity, function, and stability, as well as ensuring that experimental results are legitimate, replicable, and applicable to the specific biological context.

BOKU and an industry partner are collaborating on the "Knowledge-based Production of Gene Therapy Vectors" project, which will shift recombinant adeno-associated virus (rAAV) production from the commonly used empirical systems to knowledge- and model-based systems. This will improve product and manufacturing quality while simultaneously reducing production costs. Considering the newly emerging possibilities for gene therapy, this has the potential to significantly improve the availability of promising medications for humanity.

To accomplish this, the system of rAAV production for gene therapy in HEK cell lines will be divided into work packages and thoroughly investigated. The project starts at the base with characterization of processes and product quality, followed by an understanding of cellular limitations, strategies for stable cell lines, miniaturized process development platforms, and finally, the optimization of AAV production. Cell lines for this project are made available both from industry partners and from internal sources. The inhouse cells are derived from an adapted HEK293, which have been accustomed to suspension culture and cultured in several mediums, as well as a commercially available suspension-adapted HEK293-6E line.

This PhD thesis will focus on the knowledge of cellular limitations, specifically the distinctions between cell lines, as part of a working package within this collaboration. Genome and methylome data will be used in conjunction with freshly generated transcriptional data. The methylome sequencing data will be utilized to detect differentially methylated areas within the cell line, as well as their relationship to genomic location and chromatin state. The transcriptome data will be generated throughout the bioprocess to track the cells’ response to viral infection (using a technique already utilized by the project partner). Apart from the variations in phenotype that each cell line exhibits, attention is also paid to the general behaviour of cells in the face of a viral infection and the specific responses of those to the various model-genes used. The transcriptome data as well as the genome, methylome, and chromatin-status data will be associated with process-specific differences in virus productivity and the ratio of full to empty capsids, potentially identifying growth limiting factors or specific requirements for efficient production. An open database will be created for the HEK293 genome and transcriptome information that will also provide visualizations and connections to other databases, such as GeneCards or HumanMine.

In summary, it can be said that the cellular response to various viruses and gene productions will be studied in a systematic and structured manner to understand the cellular mechanisms and requirements for an efficient production of rAAV with high quality and yield. The gathered information can be used to optimize cell lines and standards for culture conditions in industrial scale.