Does the addition of HBV Packaging Signals to therapeutic RNAs make them better assembly substrates for the HBV Cp?

An essential prerequisite for gene therapy applications is the efficient delivery of genetic material to the target cell via a vector. Specific and efficient packaging of this genetic cargo into a delivery vector is essential. This thesis describes a novel approach to producing virus-like particles (VLPs) for gene therapy applications. Previously, the Stockley and Twarock groups have successfully demonstrated that many single-stranded (ss) RNA viruses/VLPs assemble via multiple sequence-specific RNA-protein interactions. These interactions define an assembly pathway for capsid formation. The RNA secondary structures involved, known as packaging signals (PSs), vary in folding propensity and the sequences in recognition motifs presented. This results in differing affinities for the cognate coat protein. This thesis describes evidence that potentially therapeutic/diagnostic enhanced green fluorescent protein (eGFP), human ornithine transcarbamylase (hOTC) and p53 RNAs can be modified to improve packaging. The modifications build upon the knowledge of PS-mediated assembly from the pre-genomic RNA (pgRNA) of the Hepatitis B Virus (HBV). Two modifications to the RNA were employed. The first method added HBV flanking sequences containing cognate PSs derived from HBV pgRNA to the open reading frames (ORF) 5′ and 3′ ends. The second approach, also described, uses computational synonymous recoding to incorporate HBV-like PS features throughout the cargos ORF. A series of in vitro HBV viral assemblies were then undertaken, which allowed me to show that these RNAs can act as substrates for VLP assembly. Through extensive particle analysis via size exclusion chromatography, dynamic light scattering and electron microscopy, I have determined that in some cases these modifications led to cargoes being packaged into T=4 VLPs with the same/or greater efficiency than the wildtype HBV pgRNA.