Gene therapy has shown potential in alleviating a wide range of diseases, ranging from viral infections to autosomal diseases. One of the obstacles to gene therapy reaching its full potential is the inadequacy of methods to deliver therapeutic nucleic sequences. Current delivery of gene therapy entails use of viral and non-viral vectors. Viral vectors are however associated with drawbacks such as potential toxicity, high cost and labour-intensive production. Thus non-viral delivery alternatives are being developed in an attempt to overcome difficulties associated with nucleic acid delivery for gene therapy. Virus-like particles are potentially very useful delivery vehicles as their production is simple and cost effective, the particle surface is amenable to modification and the capsid interior can be altered to accommodate a variety of cargoes. One such particle is the recombinant HBV capsid, which comprises a single species of protein and is tolerant of amino acid substitutions on the surface exposed immunodominant loops. This study aimed to enhance the vector-like properties of the HBV virus-like particle by including amino acid substitutions on the particle surface. These substituted residues in turn provided a conjugation site for tropic and immuno evasive moieties. It was found that lysine substitutions resulted in poor conjugation to the capsid surface, whereas substituted cysteine residues resulted in almost all protein-moiety conjugates forming. In order to introduce lysine and cysteine substitutions, a novel method of cloning into the HBV was generated. In doing so, complicated procedures associated with cloning into the immunodominant loop of the HBV capsid have been alleviated. Ligands containing galactose were utilised on the surface of both the HBV capsid and liposomes to confer hepatotropism. The presence of the galactose moieties on the surface of the HBV capsid prevented indiscriminate cellular uptake in cultured cells, however did not improve hepatotropism. Galactose ligands on the surface of liposomes did improve transfection efficiency, however they required a short linker distance between liposome surface and galactose group. The inclusion of galactose in liposome formulations also provided a means to deliver siRNA to the liver of transgenic HBV mice. It is believed that with alterations to the ligand structure, it is possible to provide HBV capsids with hepatotropism in future experimentation. This study demonstrated that the exposed external surface of the HBV capsid is amenable to convenient conjugation, which potentially facilitates immune evasion and conferring of defined tropism.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/15428 |
| Date | 08 September 2014 |
| Creators | Hean, Justin |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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