Investigation into the entry and intracellular trafficking of a bacteriophage-derived gene therapy vector
Author(s)
Stoneham, Charlotte Anne
Type
Thesis
Abstract
Gene therapy is a promising approach for the treatment of cancer. While most progress in gene therapy has been achieved using eukaryotic viral vectors, they have had limited success in systemic gene therapy applications due to undesired uptake in non-targeted tissues, insertional mutagenesis, and immunogenicity. These shortcomings have driven the development of gene delivery vectors derived from alternative sources.
The bacteriophage-derived Adeno-associated virus-phage (AAVP) is a novel hybrid prokaryotic-eukaryotic viral vector consisting of a mammalian transgene cassette flanked by ITRs from AAV serotype 2 inserted into an intergenomic region of an M13 bacteriophage. Anti-tumour therapy following systemic administration has previously been demonstrated in several in vivo cancer models, with tumour specificity achieved through display of an αv integrin-targeting ligand on the viral capsid. However, despite the apparent success of this novel vector in several cancer models, high titres of AAVP are required for transduction of mammalian cells.
The mechanisms of entry and intracellular trafficking of the targeted AAVP vector were therefore investigated in order to identify key barriers to transduction. Using a combination of flow cytometry, confocal, and electron microscopy techniques, together with pharmacological agents, RNAi and dominant negative mutants, it was demonstrated that receptor-mediated endocytosis of the AAVP vector is both dynamin and clathrin-dependent. Following endocytosis, the majority of AAVP particles are sequestered by the endosomal-lysosomal degradative pathway, which presents a major obstacle to gene delivery by the vector.
The feasibility of improving AAVP-mediated gene transfer through endosomal/lysosomal disruption was demonstrated using the lysosomotropic agent chloroquine. Subsequently, novel multifunctional AAVP vectors bearing endosomal-escape peptides were generated. These novel vectors demonstrated improved transgene expression and retained integrin-targeting capabilities of the RGD peptide. These studies will prove invaluable for the future development of AAVP. Furthermore, the findings presented in this thesis underline the importance of such studies for the improvement of gene therapy vectors.
The bacteriophage-derived Adeno-associated virus-phage (AAVP) is a novel hybrid prokaryotic-eukaryotic viral vector consisting of a mammalian transgene cassette flanked by ITRs from AAV serotype 2 inserted into an intergenomic region of an M13 bacteriophage. Anti-tumour therapy following systemic administration has previously been demonstrated in several in vivo cancer models, with tumour specificity achieved through display of an αv integrin-targeting ligand on the viral capsid. However, despite the apparent success of this novel vector in several cancer models, high titres of AAVP are required for transduction of mammalian cells.
The mechanisms of entry and intracellular trafficking of the targeted AAVP vector were therefore investigated in order to identify key barriers to transduction. Using a combination of flow cytometry, confocal, and electron microscopy techniques, together with pharmacological agents, RNAi and dominant negative mutants, it was demonstrated that receptor-mediated endocytosis of the AAVP vector is both dynamin and clathrin-dependent. Following endocytosis, the majority of AAVP particles are sequestered by the endosomal-lysosomal degradative pathway, which presents a major obstacle to gene delivery by the vector.
The feasibility of improving AAVP-mediated gene transfer through endosomal/lysosomal disruption was demonstrated using the lysosomotropic agent chloroquine. Subsequently, novel multifunctional AAVP vectors bearing endosomal-escape peptides were generated. These novel vectors demonstrated improved transgene expression and retained integrin-targeting capabilities of the RGD peptide. These studies will prove invaluable for the future development of AAVP. Furthermore, the findings presented in this thesis underline the importance of such studies for the improvement of gene therapy vectors.
Date Issued
2013
Date Awarded
2013-02
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Hajitou, Amin
Sponsor
Medical Research Council (Great Britain)
Publisher Department
Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)