Strategies for monitoring host factors associated with Adeno-Associated Virus vector stability and persistence.

Description

This is a three year funded PhD position associate with an EU IMI funded programme “ARDAT” aimed at studying Adeno-Associated Virus (AAV) vector development and usage. AAV vectors are routinely used in gene therapy and in delivering genes of interest to human cells. In order to efficiently monitor AAV stability and persistence the aim will be to develop a number of RNA/DNA quantitative assays that will enable for the investigation of the variant AAV forms, transgene expression and vector integration. These assays will monitor vector stability over time following infection/transduction of variant in vitro cell-lines. The assays to be designed will be based upon the numerous AAV replication stages of vector as well as episomal DNA constructs. This will include ssRNA, dsDNA as well as as well as other forms of AAV genetic material. Quantification assays will be designed based on the transgene of interest being expressed from the vector, therefore allowing for comparison of different vector constructs as well as those varying in the size or molecular form of the transgene. Human cell lines will be tested as will human hepatocyte organoid culture system for vector maintenance and propagation. All AAV RNA/DNA forms will be quantitated and compared longitudinally in TZM-bl, huh-7, Hep-RG, THP-1, THP-1 differentiated into macrophage and DC lineages as well as muscle cell lines (as yet to be determined). Hep-RG cells can additionally form small liver organoid cultures which can be monitored longitudinally for vector stability and propagation.    Minion NGS technologies will also be utilised to monitor for i) AAV vector mRNA expression over time and ii) correlation between AAV stability in relation to host factor gene expression patterns. Such an analysis will allow for the comparison of AAV stability to expression of host factors that are predicted or found to influence vector stability.