Characterisation of adeno-associated virus DNA contaminants and development of a P5 promoter replacement, high purity adeno-associated virus production system

Gene therapy is the transfer of nucleic acids for therapeutic benefit, a process potentially achieved through several delivery methods. One of the most common gene therapy delivery modalities is adeno associated virus (AAV). The availability of a system to produce large quantities of AAV, and of diverse AAV serotypes, that can infect different cell types makes AAV very attractive for widespread clinical implementation. Viruses like AAV that are manipulated to become highly useful delivery tools rely on complex biological systems for production. It is imperative that these systems are well characterised and designed to be highly safe and efficacious. This PhD thesis investigated nucleic acid contamination of AAV preparations, looking at the production system as a source, and the problems that this may pose for AAV infected cells. Specific regions of DNA from AAV producer plasmids were incorporated into virions more abundantly. These were found adjacent to the outside of the inverted terminal repeats (ITRs) that flank the expression cassette, and on the plasmid containing the replication and capsid genes, directly upstream of the P5 promoter which drives expression of the replication proteins, REP78 and REP68. Contaminant sequences were found to be transferred into transduced cells during AAV infection, persistent within cells and transcriptionally active. Investigation of P5 upstream contaminants showed potential for protein to be produced persistently from contaminant sequences both in vitro and in vivo. Finally, precise sequence regions causing DNA incorporation upstream of the P5 promoter were examined. This information was used to produce alternative production 3 reagents that, in their first iteration, provided proof of principle of P5-related contaminant removal from the final AAV product, and in additional iterations resulted in a modified P5 promoter (P5-HS). This promoter limited upstream contamination thereby resulting in higher purity AAV, whilst retaining vector yields equivalent to the original AAV production system.