AbstractThe delivery of DNA is an important method within biomedical research for both fundamental cellular studies and the generation of future therapeutics. Many of the techniques currently used however, have a common flaw: a strict limitation in the amount of DNA they can accommodate. This restriction prevents the progression of research towards numerous high-value therapeutic targets within applications such as gene therapy. Thus, the necessity for an alternative, improved delivery system is significant. Baculoviruses present as an exciting alternative, as they have a seemingly unlimited capacity for DNA accommodation due to the flexibility of the nucleocapsid surrounding their genome. This feature, along with their ease of genetic manipulation, simple production and their intrinsically episomal nature within mammalian systems, suggest that baculoviruses are ideal candidates for alternative DNA delivery tools.
The aim of this thesis is to explore the potential of baculoviral nanosystems for the delivery of large, multigene DNA circuitry into drug-targetable cells and to optimise the currently used systems. The stability of the baculovirus genome was questioned through the generation of minimised mutant genomes, namely SynBac variants. However, the removal of genes from the baculovirus genome proved to reduce the genomic stability throughout five generations in cell culture in this case. The stability of the widely used MultiBac system however, remained relatively constant throughout the experiment, deeming it an appropriate vector for use in subsequent investigatory studies. Therefore, its derivative, MultiBacMam, was used to investigate the potential of baculovirus vectors as an alternative DNA delivery tool for large DNA circuitry, namely a cellular metabolism biosensor, AMPFret. This biosensor was first optimised to enhance the cellular sensitivity through the exchange of the fluorescence proteins present, producing AMPFretCR, then successfully delivered into renal podocytes by the baculovirus, to enable real-time cellular energy sensing upon both direct and indirect stimulation. Finally, several challenges facing the manufacture of baculoviral vectors were identified. Forthcoming optimisation is therefore required to generate vectors with a high degree of purity, without sacrificing viral titre. The results presented throughout this thesis identify the baculovirus as an ideal system for the delivery of large, multigene DNA cassettes into mammalian systems and allude to its potential for use within future therapeutics with in vivo applications.
|Date of Award||23 Jan 2020|
|Supervisor||Imre Berger (Supervisor) & Ian R Collinson (Supervisor)|