The transfer of a desired gene to several types of target tissues has been accomplished successfully in the past using existing Adeno-associated viruses (AAVs). Also, it has recently been shown that AAV can stimulate robust antibody responses due to long-term transgene expression or abolishment of transgene product by cell-mediated immune responses, suggesting the potential use of AAVs as vaccines. Most humans already have pre-existing immunity to common AAV serotypes making novel AAVs of low seroprevalence attractive as gene transfer or vaccine vehicles. This thesis describes my primary research objectives that included the isolation of novel AAV serotypes based on AAV DNA sequences from porcine tissues, novel AAV vector production, and biological characterization of porcine AAVs in vitro and in vivo. This was followed by evaluating immune responses in mice vaccinated with porcine AAV vectors expressing the hemagglutinin (HA) from the avian influenza A/Hanoi/30408/2005 (H5N1) strain. These findings show that low seroprevalence porcine AAV vectors were able to efficiently transduce a wide range of cells and tissues. The porcine vectors also performed well as vaccine candidates and were efficient at stimulating host immune responses. Although porcine vectors were successful as vaccines, further studies involving long term gene expression by porcine AAVs is still necessary to confirm their role as gene therapy vehicles.

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The use of a bacterial expression system allowed the production and purification of PDX1 for <I>in vitro</I> phosphorylation analysis. This procedure allowed the purification of several forms of PDX1, some of which have never been reported previously. This study reiterates the fact that the activation of PDX1 and insulin gene transcription arises by a complex series of events, which are rapidly being unravelled, to provide greater insight as to how to obtain gene therapy treatments for diabetes mellitus.

Gene therapy, where non-islet cells are transduced to express insulin, encapsulated and implanted into the diabetic patient, is a possible alternative therapy for type I diabetes, which may alleviate some of the long-term complications of the disease. Insulin, however, is synthesised as a precursor, proinsulin, which is proteolytically modified by two endocrine-cell specific proteases, PC2 and PC3. Non-neuroendocrine cells do not express PC2 and PC3, but express a related protease, furin. Furin is unable to process proinsulin to insulin efficiently, so we employed PCR mutagenesis to alter the human preproinsulin cDNA around the normal processing sites, which when transcribed and translated yield cleavage sites recognised by furin. Wild type and mutant preproinsulin cDNAs were cloned into a mammalian expression vector under the control of the CMV promoter, and were expressed in the C2C12 and L6 myoblast cell lines and also the HepG2 liver cell line. Radioimmunoassays using an insulin specific antibody and an antibody that recognises proinsulin and insulin equally revealed that cells transfected with the wild type cDNA secreted 90% proinsulin whereas cells expressing the mutant cDNA secreted 75-90% processed insulin, suggesting more efficient processing of the mutant proinsulin by the endogenous furin. Further manipulation of the cDNAs linking them to the neomycin selection gene via an internal ribosome entry sequence (IRES), gave rise to stably transfected L6ins cell lines, expressing 2-10 ng/ml/24h total insulin-like immunoreactivity (ILI). With stable HepG2ins cells expressing in excess of 2.5 μg/5x10⁷ cells/24h mature, biologically active human insulin. These high expressing HepG2ins cells were implanted into BB/Edinburgh rates and strepozotocin treated nude mice.

The high gene knockdown specificity and efficiency of RNA interference (RNAi) provides a potentially viable avenue for the development of a new class of nucleic acid therapeutics for gene-based disease conditions. However, serum instability, inefficient cellular trafficking and non-specific effects of small interfering RNAs (siRNAs), one of the functional mediators of RNAi, has necessitated the development of carriers to facilitate targeted cell delivery. The decline of viral vectors in human gene therapy as a consequence of safety issues has intensified the importance of non-viral vector development. Among the non-viral vectors available for siRNA delivery, cationic liposomes have emerged as an attractive option owing to their simplicity, versatility, relatively low toxicity and potential for cell-specific targeting. Although existing cationic lipids and liposomes traditionally used for DNA delivery have also been used for siRNAs, there still exists a need to develop cationic lipids tailored towards siRNA transfection for improved gene silencing efficiency. Among the cell specific targets available for RNAi therapeutics, hepatocytes expressing the asialoglycoprotein receptor (ASGP-R) are an ideal choice due to the large number of disease targets present for treatment. In this investigation four novel cationic liposome formulations were prepared from equi-molar quantities of either the cationic cholesterol derivative 3β [N-(N’,N’- dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or 3β [N-(N’, N’, N’- trimethylammoniumpropyl)-carbamoyl] cholesterol iodide (Chol-Q) and DOPE, with and without the hepatotropic ligand, cholesteryl-β-D-galactopyranoside. Electrophoretic gel analysis and SYBR®green displacement assays were employed to determine siRNA binding and condensation efficiencies for all cationic liposomes; while liposome and lipoplex size measurements were made by cryoTEM. SiRNAlipoplex stability in serum was determined by the nuclease protection assay. Cell studies performed on the ASGP-R+ human hepatoma cells, HepG2 and the ASGP-Rembryonic kidney cells, HEK293, to determine lipoplex toxicity and transfection efficiencies were also undertaken. We show that the cationic liposomes formulated for this investigation were able to bind synthetic siRNA optimally at a positive to negative charge ratio of ± 1 : 6. In addition, the cationic liposomes were able to afford siRNA duplexes substantial protection from ribonuclease digestion in serum. From the results obtained in this study, it appears that the cationic liposomes are well tolerated by both the HEK293 and HepG2 cells in vitro. More importantly, the results obtained demonstrated higher transfection efficiencies for the targeted lipoplexes compared with the untargeted controls, strongly supporting the notion that incorporation of the cholestryl-β-D-galactopyranoside into the liposome structure increases transfection efficiency to the targeted HepG2 cells in culture via ASGP receptor mediation. Comparative studies in the HEK293 cell line yielded low transfection effeciences in the order of 20%, with no significant difference being recorded between galactosylated and non-galactosylated lipoplexes. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2011.

Glaucoma is a group of optic nerve degenerative diseases, which leads to gradual and permanent vision loss. Recent developments in the field of gene therapy have proposed increasingly promising treatments for glaucoma, in the form of delivery of neuro-protective or neuro-regenerative genes to the retina. Despite these developments, there are concerns related to the biocompatibility and invasiveness of common gene delivery systems, since they are commonly mediated by viral gene carriers and invasive administration methods. Non-viral gene delivery systems offer a safe and increasingly efficient alternative to deliver therapeutic genes to the retina. An example of these systems is gemini-phospholipid nanoparticles (GL-NPs), which have been successfully used to deliver genes in similarly challenging anatomical settings, such as the skin. The objective of this thesis is to demonstrate the potential of GL-NPs, as candidate gene delivery vehicles for topically administered genes, targeted to the retina. The dicationic gemini surfactant, 12-7NH-12 was used, along with the helper lipids, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), to prepare various types of GL-NPs, and assess their transfection efficiency in the rat retinal ganglion cell line (RGC-5). The transfection efficiency was evaluated using flow cytometry, as a function of several physical and chemical parameters of GL-NPs. These include a range of charge ratios (5:1 to 15:1 ????), helper lipid composition (several DOPE: DPPC ratios), order of assembly (plasmid-gemini + lipid versus gemini-lipid + plasmid), and manufacturing method of helper lipid vesicles (thin film versus high pressure homogenization method). Size and zeta (??) potential characterization of GL-NPs was carried out in parallel, using dynamic light scattering, to relate the physical parameters of GL-NPs to their respective transfection efficiency. A comprehensive toxicological evaluation was undertaken to assess the extent of GL-NP???s toxicity in RGC-5 cells, using the resazurin-based PrestoblueTM cell toxicity assay. Optimized GL-NPs were used to induce expression of the brain derived neurotrophic factor (BDNF) in RGC-5 cells, and were assessed in terms of their capacity to induce neurite outgrowth. Quantification of neurite outgrowth was carried out by measuring average neurite length in RGC-5 cells, by confocal microscopic imaging of immunostained neurites. Furthermore, confocal microscopic studies were carried out to assess the extent of GL-NP???s corneal permeation in a 3-D human corneal epithelial (HCE) model. A parallel toxicological evaluation was completed to ensure GL-NP???s biocompatibility with the corneal epithelial cells. Finally, GL-NP biodistribution pattern and gene transfer capacity was assessed in a mouse model, following topical and intravitreal administration. The transfection efficiency in RGC-5 cells, which ranged between 2.1 ?? 0.3% and 14.5 ?? 1.4%, was highly dependent on GL-NP???s charge ratio, helper lipid composition, order of assembly, and manufacturing technique. GL-NPs at 10:1 ???? charge ratio, assembled with homogenized DOPE (25%)-DPPC (75%) helper lipid vesicles, in the plasmid-gemini + lipid order, mediated the highest transfection efficiency in RGC-5 cells. These GL-NPs had a size of 222.8 ?? 4.2 nm and a ?? potential of +33.5??2.9 mV. Optimized GL-NPs were highly biocompatible with both RGC-5 and HCE model cells, with viability values ranging between 94.8 ?? 6 % to 100 ?? 3.4 %. Assessment of corneal permeation showed that GL-NPs were able to bind to the corneal epithelial surface and achieve a moderate permeation depth (35-40 ??m), following topical application in the HCE model. Intravitreal injection of the non-viral GL-NPs in mice has successfully led to their localization within the nerve fiber layer (NFL) of the retina. Finally, GL-NPs were non-invasively delivered to several anterior chamber tissues, including the limbus, the iris and conjunctiva, following topical administration. GL-NPs offer several advantageous features critical to topical and intravitreal ocular administration of gene carriers, including in vitro corneal binding and effective biodistribution following in vivo topical and intravitreal administration, high biocompatibility, and a highly tunable transfection efficiency. The current data presents 12-7NH-12 GL-NPs as a promising candidate for ocular gene therapy applications.

Spinal muscular atrophy (SMA) is the most commonly inherited neurodegenerative disease that leads to infant mortality worldwide. There are no known cures for SMA, but small increase in protein levels of SMN can be beneficial. We have developed adenoviral (Ad) vectors that express a human transgene of SMN and have tested their safety in vitro. We have demonstrated that these viruses can effectively express the transgene following cell entry and that the levels are relative to the virus dose. The viruses do not appear to impact the health and function of the cells, and are capable of increasing the number of Gems. We also attempted to change the tropism of the viruses through fiber protein modifications in order to target muscles and motor neurons. Our results suggest that a therapy based on an Ad-SMN fiber-modified vector may ultimately be successful in treating patients of SMA.

The transfer of a desired gene to several types of target tissues has been accomplished successfully in the past using existing Adeno-associated viruses (AAVs). Also, it has recently been shown that AAV can stimulate robust antibody responses due to long-term transgene expression or abolishment of transgene product by cell-mediated immune responses, suggesting the potential use of AAVs as vaccines. Most humans already have pre-existing immunity to common AAV serotypes making novel AAVs of low seroprevalence attractive as gene transfer or vaccine vehicles. This thesis describes my primary research objectives that included the isolation of novel AAV serotypes based on AAV DNA sequences from porcine tissues, novel AAV vector production, and biological characterization of porcine AAVs in vitro and in vivo. This was followed by evaluating immune responses in mice vaccinated with porcine AAV vectors expressing the hemagglutinin (HA) from the avian influenza A/Hanoi/30408/2005 (H5N1) strain. These findings show that low seroprevalence porcine AAV vectors were able to efficiently transduce a wide range of cells and tissues. The porcine vectors also performed well as vaccine candidates and were efficient at stimulating host immune responses. Although porcine vectors were successful as vaccines, further studies involving long term gene expression by porcine AAVs is still necessary to confirm their role as gene therapy vehicles.

Duchenne muscular dystrophy (DMD) is characterized by necrosis and progressive loss of muscle fibers. DMD patients have a mutation in the gene encoding dystrophin, a large membrane-associated cytoskeletal protein on the cytoplasmic side of the sarcolemma. Gene therapy using fully deleted adenoviral vectors shows great potential for the eventual treatment of DMD and other genetic diseases. These vectors are less immunogenic than their predecessors and have the capacity to carry large DNA inserts such as the full-length dystrophin (12 kb). However, the lack of viral genes results in a weakened and subsiding (short) transgene expression in muscle. Findings in the lung and liver have shown the adenoviral E4 region, in particular E4 open reading frame 3 (ORF3) to contribute to the maintenance of transgene expression. We constructed an adenovirus in which E4 ORF3 was reintroduced into a fully-deleted adenovirus along with full-length dystrophin (AdCBDysORF3). Dystrophin levels produced by AdCBDysORF3 were found to be not sustained in mdx mice, dropping significantly by day 90. However, expression levels did increase when AdCBDysORF3 was complemented with other viral proteins such as EIB. Likewise, increasing the expression of the primary adenovirus receptor (CAR) in muscle also resulted in a higher initial dystrophin expression in myofibers.

Prostate cancer (CaP) is the 2nd most common cancer in North American men. Tumour management strategies are appropriate for early stage disease, but advanced disease has a poor prognosis and requires prompt treatment. Therefore, research into delay of tumour progression and efficacious treatment of aggressive cancer are of interest. Ganglioside was assessed for its role in altering markers of metastatic potential and susceptibility of CaP to adenovirus-mediated gene therapy. Healthy (RWPE-1) and malignant (DU-145, PC-3) prostate cells were cultured with or without mixed ganglioside. Differences in growth, ganglioside and integrin densities, and adenoviral infectivity were assessed between treatment and control groups. Ganglioside decreased (p<0.01) growth of PC-3 cells relative to untreated control. Ganglioside decreased (p<0.01) GD1a and increased (p<0.04) integrin densities in malignant prostate cells, suggesting ganglioside may increase metastatic potential of CaP. Ganglioside significantly increased adenovirus entry in PC-3 cells, thereby improving susceptibility of CaP to adenovirus-mediated gene therapy. / Nutrition and Metabolism