Percutaneous coronary intervention with stent deployment is the dominant form of revascularisation for patients with coronary artery disease. Although drug-eluting stents have reduced the incidence of instent restenosis, they are associated with late problems related to delayed vascular healing including late stent thrombosis. The use of gene-eluting stents offers the potential to deliver localised gene therapy to the vascular wall with the aim of both reducing restenosis and promoting endothelialisation. Two candidate genes were investigated. Connective tissue growth factor (CTGF) promotes smooth muscle cell apoptosis and stimulates endothelial growth in vitro, and has an integral role in wound healing. Fibromodulin (FMOD) is involved in collagen metabolism and is a key mediator of scarless wound healing. Both genes have previously been shown to suppress restenosis in an ex vivo vein graft model. Plasmids containing these two genes were constructed with an expression cassette specially designed to maximise transgene expression in vascular smooth muscle cells. These plasmids were coated onto coronary stents with a polymer and the effects of these gene-eluting stents were investigated in an in vivo pig coronary artery model. Previous work by our group has suggested that systemic -blockade can affect the degree of transgene expression from viral vectors, and experiments were also performed to investigate the effect of β-blockers on plasmid-mediated gene expression. At 28 days there was no significant difference in angiographic late loss or neointimal hyperplasia between the groups treated with stents coated with FMOD or CTGF and the group treated with stents coated with the marker gene lacZ. This lack of efficacy appeared to be as a result of extremely poor transgene expression rather than due to a genuine failure of the transgenes to elicit a relevant biological effect. There was no difference in in vivo gene expression demonstrated as a result of β-blockade, but again this result was probably due to limited transgene expression. The potential causes of poor transgene expression in this study are reviewed and future directions for research on plasmid-mediated gene therapy are considered.

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.

Both proliferative diabetic retinopathy and exudative age-related macular degeneration are major causes of blindness which are caused by growth of defective, leaky and tortuous blood vessels in the retina. Hypoxia is implicated in triggering both of these diseases and results in induction of HIF-1alpha transcription factor in addition to the angiogenic factor VEGF. Müller cells are the major glial cell in the retina and they contribute to neovascularization in hypoxic regions of the retina through eliciting secretion of growth factors, cytokines and angiogenic factors. As Müller cells span the breadth of the retina they can secrete angiostatic factors as well as neuroprotective trophic factors, the Müller cell is a valuable cell type for targeting by potential new gene therapies. The current investigation tests the hypoxia responsiveness of an AAV vector containing a hybrid hypoxia response element together with a GFAP promoter, and this vector encodes the angiostatic protein decorin, a well characterized multi-receptor tyrosine kinase inhibitor. Decorin may have advantages over other key angiostatic factors such as endostatin or angiostatin by virtue of its multiple anti-angiogenic signaling modalities. We employed Q-RT-PCR to evaluate the cell specificity and hypoxia responsiveness of an AAV-Vector termed AAV-REG-Decorin containing a hybrid HRE and GFAP promoter driving expression of the decorin transgene. The vector also contains a silencer element between the HRE and the GFAP domains to enable low basal expression in normoxia as well as high level inducibility in hypoxia. AAV-REGDecorin was found to elicit high level expression of decorin mRNA in hypoxia with greater than 9 – fold induction of the transgene in hypoxic conditions in astrocytes by comparison to normoxic astrocytes. AAV-REG-Decorin showed low levels of transgene expression by comparison to the positive control vector AAV-CMV -decorin containing the ubiquitously active CMV-promoter. The expression levels of decorin mRNA from AAV-REG-Decorin and from AAV-GFAP-Decorin were low in the PC12 neuronal cell model and in the ARPE19 line of retinal pigment epithelial cells with respect to those of AAV-CMV-decorin and no induction of Decorin mRNA was found with AAV-REGDecorin in these two control cell lines. Our novel gene therapy vector will serve as a platform for testing efficacy in rodent disease models (OIR and laser induced choroidal neovascularization) for assessment of the benefits of tightly regulated antiangiogenic gene therapy eliciting decorin transgene expression, both in terms of timing and the cellular source of production, during the progression of the retinal pathophysiology. / Includes bibliography. / Dissertation (PhD)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Since its identification as a member of the tumour necrosis factor (TNF) family, TRAIL (TNF-related apoptosis-inducing ligand) has emerged as a new avenue in apoptosis-inducing cancer therapies. Its ability to circumvent the chemoresistance of conventional therapeutics and to interact with cancer stem cells (CSCs) self-renewal pathways, amplified its potential as a cancer apoptotic agent. Many recombinant preparations of this death ligand and monoclonal antibodies targeting its death receptors have been tested in monotherapy and combinational clinical trials. Gene therapy is a new approach for cancer treatment which implies viral or non-viral functional transgene induction of apoptosis in cancer cells or repair of the underlying genetic abnormality on a molecular level. The role of this approach in overcoming the traditional barriers of radiation and chemotherapeutics systemic toxicity, risk of recurrence, and metastasis made it a promising platform for cancer treatment. The recent first Food Drug Administration (FDA) approved oncolytic herpes virus for melanoma treatment brings forth the potency of the cancer gene therapy approach in the future. Many gene delivery systems have been studied for intratumoural TRAIL gene delivery alone or in combination with chemotherapeutic agents to produce synergistic cancer cytotoxicity. However, there still remain many obstacles to be conquered for this different gene delivery systems. Nanomedicine on the other hand offers a new frontier for clinical trials and biomedical research. The FDA approved nanodrugs motivates horizon exploration for other nanoscale designed particles' implications in gene delivery. In this review we aim to highlight the molecular role of TRAIL in apoptosis and interaction with cancer stem cells (CSCs) self-renewal pathways. Finally, we also aim to discuss the different roles of gene delivery systems, mesenchymal cells, and nanotechnology designs in TRAIL gene delivery.

No description available.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder for which no disease modifying treatment exists. Inheritance of the apolipoprotein E (APOE) ε4 allele strongly increases the risk of developing the sporadic form of AD, whereas the ε2 allele is protective. Though the precise role of the different APOE alleles remains unclear, in vitro and in vivo studies have demonstrated that each isoform differentially modulates the deposition, clearance, and degradation of amyloid-beta (Aβ) peptides that form the extracellular plaques of AD. Strategies directed toward increasing the levels of APOE2 could be useful in reducing plaques and alleviating disease progression. Additionally, advances in gene transfer using adeno-associated vectors (AAV) enable efficient and safe expression of therapeutic genes like APOE2. This dissertation, describes experiments that (1) test if intracerebroventricular gene delivery of APOE isoform can modulate amyloid pathology in transgenic animals that accumulate beta-amyloid, (2) evaluate the efficacy of a novel approach by intravenous infusion of AAV, and (3) characterize oligomeric Aβ (oAβ) in rhesus monkeys as a potential translational target. Experiment 1 explored how each APOE isoform impacts the progression of AD pathology using intracerebroventricular injection of AAV4. This demonstrated isoform-dependent effects on Aβ-related neuropathology using multi-photon imaging, in vivo microdialysis, post-mortem immunohistochemistry, and array tomography. Experiment 2 demonstrated the efficacy of intravenous delivery of AAV9 and increased efficiency of self-complementary AAV9 vectors compared to single-stranded AAV9. Furthermore, expression of transgene was restricted to CNS astrocytes by utilizing a restrictive promoter. Finally, the presence of oAβ in rhesus macaque monkeys and its possible association with age-related cognitive decline was explored using fresh samples of monkey cortex. Soluble oAβ was detected in multiple cortical areas but was not significantly associated with age. Nevertheless, a significant positive correlation was observed between oAβ in frontal pole cortex and impairment on a behavioral test of executive function. Taken together, these data suggest that gene transfer of APOEε2 may be useful for modulation APOE expression and beta-amyloid accumulations for the treatment of AD. Furthermore, the rhesus monkey may serve as a model system for future preclinical studies.

No description available.

No description available.

No description available.

The Adeno-Associated Virus(AAV) is a small, single stranded DNA virus that has been developed as a gene transfer vector. Early clinical trials using recombinant AAV vectors (rAAV) have identified the following concerns that need to be addressed in order to increase efficiency of these vectors. It has since been determined that AAV vector efficiency decreases due to the following mechanisms: ineffective endocytosis, endosomal degradation, inefficient trafficking, and the need to convert from a single-stranded AAV genome to a transcriptional active double-stranded form. The purpose of this study is to elucidate mechanisms that help stabilize the AAV genome in order to make it a more efficient vector for gene therapy. Previously, there have been studies using fluorescent labeling to track the movement of AAV into the nucleus. An integral part of AAV genomic stability may be the obstacles it encounters in the cytoplasm prior to entering the nucleus. Previous studies on improving AAV transduction have focused primarily on the nucleus. The study will hopefully shed light on the hurdles AAV encounters as it moves through the cytoplasm. Thus, this project has designed and utilized a new system for specifically tracking the status of AAV genomes in the cytoplasm as well as in the nucleus. This project utilizes a novel dual luciferase reporter system to track the movement of AAV particles from the cytoplasm into the nucleus to elucidate mechanisms that could contribute to the stabilization of the AAV genome. The novel dual reporter system is comprised of a single-stranded vector containing two different types of secreted luciferases: Cypridina Luciferase and Gaussia Luciferase. Cypridina Luciferase is placed under the control of a nuclear promoter and therefore it is expressed only in the nucleus. The second, Gaussia Luciferase, is under the control of a cytoplasmic promoter that will only be expressed in the cytoplasm upon the presence of T7 RNA polymerase. Using this dual reporter luciferase system along with RT qPCR quantification in a Hek293 cell line expressing the T7 RNA polymerase, demonstrated that genomes are present in the cytoplasm at 18 and 24 hours post infection. The second part of this study is using the dual reporter system to understand the trafficking patterns of rAAV and looking at ways to enhance transduction. One method could be administering rAAV vectors in conjunction with a drug; this approach may help overcome some of these cellular barriers encountered during infection as well as help stabilize the genome. Cidofovir (CDV) is a monophosphate nucleotide analogue that competitively inhibits the incorporation of deoxycytidine triphosphate into viral DNA via viral DNA polymerase. In vitro, CDV actively inhibits a number of DNA viruses including herpes viruses, adenovirus, polyomavirus, papillomavirus, and poxviruses. Cidofovir has already been approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus (CMV) retinitis in patients with acquired immunodeficiency syndrome (AIDS). The effects of CDV on small DNA viruses that lack their own viral DNA polymerase, like AAV, has not been documented. Results have demonstrated that CDV is able to increase single-stranded rAAV transgene expression in the nucleus by 2 to 5 fold, depending on the cell type and concentration, in vitro, using both rAAV2 and rAAV8 luciferase reporter vectors. These results have been able to replicated using other reporter vectors: rAAV2-LacZ reporter, rAAV2-GFP, rAAV2-hAAT, and a rAAV8-GFP in vitro. Results have shown a dose-dependent increase in rAAV genomes with CDV pretreatment in HelaS3 cells via Southern Blot. Also southern blot analysis of cells pretreated with CDV then infected with rAAV revealed no difference in the amount of vector present between 0 and 2 hours post infection, suggesting CDV does not enhance viral entry but that CDV may enhance at steps downstream of viral entry. Using RNA sequencing and Ingenuity software analysis of HelaS3 cells pretreated with CDV, an increase in several genes of interest including those involved in the mechanism of viral exit were observed. These genes include Actin and vacuolar proteins, these molecules are involved in and associated with endosomal sorting complexes as well as required for transport inside the cell. This finding along with southern blot data supports the theory that CDV may enhance rAAV trafficking since we observed that CDV pretreatment enhances viral accumulation of rAAV vectors in both the cytoplasm and nucleus 24 hours post-infection. Also utilizing the dual luciferase reporter system an increase in transgene expression present with CDV pretreatment compared to PBS in both the cytoplasm and nucleus was observed suggesting that CDV may enhance with rAAV trafficking. These results taken together demonstrate that this dual reporter system is a powerful tool for understanding and improving rAAV trafficking. Also drugs like CDV can greatly contribute to the understanding of rAAV trafficking, and eventually lead to the development of novel strategies to increase overall efficiency of AAV transduction. / Microbiology and Immunology