Engineering pseudovirions for large-scale targeted gene transfer and recombinant adeno-associated virus production
Creators: name: Asavarut, Paladd
Publisher: name: Imperial College London
Year: 2018

description: Gene transfer is a technology central to the development of gene therapy and expression of proteins and biological products necessary for designing and producing drug compounds or creating new research methodologies. Consequently, the field has experienced a large bottleneck in economic and time costs when viral vectors are involved. Over the past decade, recombinant mammalian viral vectors have been exploited for these purposes; however, the limitations in their native biology and production methods warrant new vector systems to be investigated and developed. In this thesis, we reinvestigate the humble bacteriophage, a prokaryotic virus, as a potential tool to circumvent the cost limitation of eukaryotic viruses. By combining their genome with that of Adeno-associated virus (AAV), a well-characterised mammalian virus, and developing a novel method of expressing these hybird vectors, we were able to overcome many limitations that these viruses have as separate entities. The proposed vector, termed Phagemid Adeno-associated Virion (PAAV), is as efficacious as traditional vectors, while economically costing a fraction of what is demanded by current practice in the field. The PAAV, was constructed by inserting a recombinant AAV genome into a phagemid expression vector that carries no phage structural genes. The particles are packaged using a custom-designed mammalian-targeted helper virus, resulting in vectors that can be easily produced at a minimum of 2-fold higher yield than the current gold-standard phage vector. We demonstrated through transmission electron microscopy and transducing unit assays that PAAV vectors generated by our method is less than half the size of traditional full-length phage vectors, and through vesicular staining we are able to determine that the PAAV is internalised at almost 2-fold higher than the efficiencies observed in the gold-standard phage vector. We further assessed qualitative and quantitative gene expression efficacies by the PAAV bearing GFP or Luciferase transgenes in various tumour cells, which show a dramatic increase in gene expression by up to over 10-fold of the gold-standard. To demonstrate that the PAAV and its derived vectors can be used as an alternative to DNA transfection, a method central to mammalian virus production, we designed and validated two proof-of-concept methods that are able to produce rAAV using PAAV vectors. Phages are harmless viruses with a safety profile founded by their historic use as antibiotic agents. The PAAV vector system utilizes the economic advantages of phage vectors and combines them with the efficacy of mammalian viral transgenes, offering an efficacious alternative vector that is able to transduce mammalian tumour cells. Furthermore, the PAAV system has the potential to replace conventional transfection, thereby addressing a significant bottleneck in translational research in the field. Taken together, the PAAV offers a novel and advantageous alternative platform to conventional viruses for use in therapeutic and industrial applications.