The infantile GM2 gangliosidoses are severe neurodegenerative disorders, caused by a defect in the β-hexosaminidase system. They are characterized by lysosomal accumulation of the substrate, GM2 ganglioside, which results in severe neuronal damage and death in the early years of life. Sandhoff mice deficient in both major hexosaminidase isozymes, Hex A and Hex B, mimic the disease severity in the human condition including the motor deterioration, histopathological findings, and premature death. To investigate the utility of systemic adeno-associated virus 9 (AAV9)-based gene delivery in treating GM2 gangliosidoses, we evaluated the therapeutic outcome of a single intravenous injection of recombinant AAV9 encoding the complementing Hexb gene in a Sandhoff mouse model. We showed prolonged survival, preserved motor function, and reduced GM2 ganglioside accumulation as well as inflammation when systemic AAV9 therapy was administered to 1-2 days old mice. However, the formation of liver or lung tumours accompanied the positive therapeutic effect.

Non-viral delivery vectors have potential advantages over the viral systems that currently are used extensively for delivering therapeutic genes of interest. However, non-viral gene therapy has low efficiencies in vivo, in part due to the aggregation of the particles in the delivery system associated with serum proteins and other components of the blood. An effective technique for overcoming this problem to use PluronicTM block copolymers to cover the surfaces of the particles in the delivery system with polyethylene oxide, which decreases their charge density and reduces their interactions with the serum proteins. The objectives of this project were to characterize a Pluronic-gemini surfactant system to be used as non-viral vectors for gene therapy. Five Pluronics (L44, F68, F87, F108, and F127) were evaluated by studying their physiochemical properties, including particle size and zeta potential. Also, these systems were evaluated in OVCAR-3 cell culture for gene expression and cell viability. The in vitro systems showed small particle sizes (approximately 200 nm) for all Pluronics. The particle sizes in the systems were increased dramatically (up to 2000 nm) by adding dioleylphosphatidylethanolamine (DOPE) to the systems. The zeta potential of these systems shifted the negative zeta potential of DNA (-43 mV) to a positive value (+35 mV). The addition of DOPE had very little effect on zeta potential. The in vitro transfection efficiency in OVCAR-3 showed that all of the Pluronics were able to transfect OVCAR-3 at various DNA/gemini surfactant ratios. The highest transfection efficiency was obtained with Pluronics L44, F87 and F108. PluronicF127 demonstrated the lowest transfection efficiency among the five Pluronics. Adding DOPE did not improve the transfection efficiency in any of the pluronic-gemini surfactant systems. The viabilities of the cells in these systems were high, and there were greater than the positive control (Lipofectamine 2000). The greatest cell viability (about 60%) was observed when the DNA to gemini surfactant ratio was 1:2. After adding DOPE, the cell viability decreased in all of the Pluronics except for Pluronic F68. The results of this investigation indicated that Pluronic block copolymers can transfect OVCAR-3 cell cultures in vitro and that they had a low level of cytotoxicity.

Currently, the HIV pandemic remains a major global health challenge. In order to effectively control and cure HIV-1 infection, it is necessary to perform greater research on host-HIV interactions and develop novel preventive and therapeutic approaches. The human cytidine deaminase APOBEC3G (A3G) is the first identified host restriction factor, which can serve as an initial line of defense against HIV-1 by inducing lethal mutations on proviral DNA and disrupting viral reverse transcription and integration. In order to better understand the action of A3G on HIV-1 replication, my study was focused on characterizing the interplay between A3G and HIV-1 reverse transcriptase (RT). The results indicated that A3G directly bound to RT, which contributed to A3G-mediated inhibition of viral reverse transcription. Overexpression of the RT-binding polypeptide A3G65-132 was able to disrupt wild-type A3G and RT interaction, consequently attenuating the anti-HIV effect of A3G on HIV replication. While the potent antiviral activities of A3G make it an attractive candidate for gene therapy, the actions of A3G can be counteracted by HIV-1 Vif during wild-type HIV infection. In order to overcome Vif-mediated blockage and maximize the antiviral activity of A3G, this protein was fused with a virus-targeting polypeptide (R88) derived from HIV-1 Vpr, and various mutations were then introduced into R88-A3G fusion protein. Results showed that Vif binding mutants R88-A3GD128K and R88-A3GP129A exhibited very potent antiviral activity, and blocked HIV-1 replication in a CD4+ T lymphocyte cell line as well as human primary cells. In an attempt to further determine their potential against drug resistant viruses and viruses produced from latently infected cells, R88-A3GD128K was chosen and delivered by an inducible lentiviral vector system. Expression of R88-A3GD128K in actively and latently HIV-1 infected cells was shown to be able to inhibit the replication of both drug sensitive and resistant strains of HIV-1. In conclusion, this thesis has demonstrated one of the mechanisms that how A3G can disrupt HIV-1 reverse transcription. Meanwhile, an A3G-based anti-HIV-1 strategy has been developed, which provides a proof-of-principle for a new gene therapy approach against this deadly virus.

Lentivirus-mediated gene therapy has curative potential for a variety of disorders, however, insertional oncogenesis still remains a concern. One approach to increase safety of such treatment modalities is to include a ‘cell fate control safety cassette’ in lentiviral vectors (LVs), enabling pharmacological control over the survival of gene-modified cells (GMCs). Two novel LVs with engineered expression of truncated cell surface molecules (CD19 or LNGFR) fused to a ‘cell fate control’ gene (TmpkF105YR200A) were constructed. Results demonstrated these safety cassettes could be used to control the survival of GMCs in a murine xenogeneic leukemia models. For treatment of Fabry disease, a bicistronic LV containing the fusion safety element and therapeutic α-galactosidase A was constructed. Transduction with this vector restored enzyme activity in Fabry patient’s fibroblasts. These collective results demonstrate that this approach is sufficient to eradicate GMCs, and when combined with a corrective cDNA can provide therapeutic benefit for Fabry disease.

Lentivirus-mediated gene therapy has curative potential for a variety of disorders, however, insertional oncogenesis still remains a concern. One approach to increase safety of such treatment modalities is to include a ‘cell fate control safety cassette’ in lentiviral vectors (LVs), enabling pharmacological control over the survival of gene-modified cells (GMCs). Two novel LVs with engineered expression of truncated cell surface molecules (CD19 or LNGFR) fused to a ‘cell fate control’ gene (TmpkF105YR200A) were constructed. Results demonstrated these safety cassettes could be used to control the survival of GMCs in a murine xenogeneic leukemia models. For treatment of Fabry disease, a bicistronic LV containing the fusion safety element and therapeutic α-galactosidase A was constructed. Transduction with this vector restored enzyme activity in Fabry patient’s fibroblasts. These collective results demonstrate that this approach is sufficient to eradicate GMCs, and when combined with a corrective cDNA can provide therapeutic benefit for Fabry disease.

Thesis (Ph.D.)--Murdoch University, 2002. / Thesis submitted to the Division of Health Sciences. Bibliography: leaves 210-245.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Joseph M. Le Doux; Committee Member: Andrés J. Garcia; Committee Member: Cheng Zhu; Committee Member: Nael McCarty; Committee Member: Richard Compans. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--University of Wisconsin--Madison, 2003. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (p. 103-116).

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Huntington's disease (HD) can be regarded as a model neurodegenerative disorder to screen potential genetic based therapeutics and their carriers. It is an autosomal dominant disorder caused by a mutation in a single gene that leads to progressive neurodegeneration caused in part by protein misfolding. The mutation codes for an expanded polyglutamine tract within the Huntingtin gene (HTT) which leads to neuronal loss through a pathological cascade of events. Current treatment strategies include symptom management but no disease-modifying therapies exist. Research has shown that nucleic acid based therapeutics aimed at decreasing HTT expression can prevent, or reverse, the phenotype. Translating such therapies to the clinic is hindered by the blood brain barrier (BBB) and the lack of an easily administrable, non-toxic, immunologically inert delivery vehicle capable of bypassing the BBB. This study examines a range of nucleic acid based therapeutics for their potency and toxicity, and evaluates extracellular vesicles (EVs) as a delivery vehicle through investigation of the biodistribution of brain targeted EVs and an analysis of EV loading. A small interfering RNA (siRNA) targeting a region upstream of the repeat induced potent non-toxic silencing of HTT. In examining EVs as a carrier for therapeutics for neurological diseases including HD, it was found that targeting can increase brain accumulation of EVs but that the physiological characteristics of EVs which make them susceptible to clearance by the reticuloendothelial system (RES) must be further evaluated to bioengineer modified EVs to avoid fast clearance. Lastly, loading by electroporation was found to induce siRNA aggregation which can lead to overestimation of loading by increasing siRNA content in an ultracentrifugation pellet, but cholesterol-conjugated siRNA mixed with EVs was capable of generating silencing in vitro. This thesis examined EV based treatment for HD through the selection of a nucleic acid cargo to silence HTT, and examination of EVs as a delivery vehicle via biodistribution and loading studies. If the loading can be optimised and fast clearance avoided, there is promise in the use of EVs as a carrier of siRNA for HD.