Conventional cancer chemotherapy often lacks specificity and is consequently associated with significant normal tissue toxicities. Molecular chemotherapy offers the potential to target the activation of inert prodrugs by utilising tumour-specific catalytic enzymes to restrict cytotoxicity to neoplastic tissues. Appropriate expression of therapeutic enzymes can be achieved by exploiting the genetic distinctions that exist between tumour and normal tissues through the use of tissue- or disease-specific promoters. Alternatively, the unique physiological differences that arise in solid tumour masses as a consequence of the abnormal vascular architecture might also be exploited to achieve therapeutic selectivity. The most conspicuous of these differences is the presence of areas of low oxygen tension (hypoxia) arising through both diffusion and perfusion-limited oxygen availability. Hypoxia is an important cause of radioresistance and is a independent prognostic indicator for local recurrence, metastatic spread and overall survival. Evidence also implicates hypoxia in chemotherapeutic resistance and genetic instability, as well as the progression of and selection for an aggressive neoplastic phenotype. Attempts to eliminate tumour hypoxia have met with some success, but the opportunity to utilise thisphenomenonfor therapeutic gain, through the exploitation of the unique reductive tissue environment have lead to the development of hypoxic-specific cytotoxins. These bioreductive prodrugs rely. on the natural complement of tumour enzymes to catalyse their activation under low tissue oxygen tensions. Levels of these reductases are potentially heterogeneous and are often down-regulated in the neoplastic state. The artificial reintroduction of high levels of reductive enzyme expression may be of significant therapeutic value, particularly if expression is restricted to the hypoxic tissue enviroment in which the prodrugs will be activated. This might be achieved through the utilisation of the specific cisacting sequences that are responsive to hypoxia-regulated transcription factors. A diverse spectrum of genes are known to be induced as a consequence of oxygen deprivation, being involved in systemic oxygen supply, vascular tone, neovascularisation, iron homeostasis, glucose metabolism, drug detoxification and protein chaperoning. The details of the cis-acting sequences and transcription factors that mediate this oxygen-sensitive gene control are beginning to emerge. This provides the opportunity to exploit these defined sequences to regulate therapeutic genes in a hypoxia-responsive manner. This thesis describes the evaluation of three potential prodruglenzyme paradigms that may have application in this context. Further, the potential of hypoxia-response-elements to specifically regulate heterologous genes in response to low oxygen tension is described. The application of such an oxygen-regulated gene-directed enzyme/prodrug therapy to solid tumours may provide chemotherapeutic specificity aimed at a clinically important tumour subpopulation.

The aim of the work presented in this thesis was to develop vectors for gene transfer, that are based on herpes simplex virus type 1, for possible application in the field of surgery.

The kynurenine pathway plays a critical role in regulating immunological homeostasis in the brain. Evidence supporting the hypothesis that kynurenine pathway dysfunction may exacerbate progression of neurodegenerative diseases like Parkinson’s is growing. First, we investigate the effects of Interferon-γ, Lipopolysaccharide, and Interleukin-4 on several key kynurenine pathway metabolites using high performance liquid chromatography. We found that Interferon-γ had significant effects on the extracellular concentration of kynurenine metabolites in astrocytes, microglia, and macrophage. GCSF gene therapy is previously demonstrated to exert neuroprotective effects on models of Parkinson’s and Alzheimer’s disease. Seven days after receiving GCSF gene therapy, A53T Parkinson’s mice were found to have increased levels of GCSF and tyrosine hydroxylase positive neurons. A concurrent increase in expression of the kynurenine pathway enzyme kynurenine aminotransferase 2 was observed. GCSF gene therapy may exhibit neuroprotective effects in a Parkinson’s disease mouse model by restoring this key kynurenine pathway enzyme. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Parkinson’s Disease (PD) is a neurological disorder affecting the basal ganglia in which the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) manifests as a complex array of motor and non-motor symptoms. Due to the lack of treatment for preventing the neurodegenerative process of PD, the only available therapy options involve managing the clinical symptoms resulting from dopamine (DA) depletion in the basal ganglia. The most widely implemented treatment is the pharmacological agent L-DOPA which serves as the precursor to dopamine. Although L-DOPA administration is initially effective in improving motor function and patient life quality, its therapeutic effect diminishes as PD pathology progressively worsens over time and side effects such as L-DOPA induced dyskinesia become apparent. Researchers are now seeking to alleviate the symptoms of PD on a molecular basis with gene therapy in which the three therapeutic strategies target specific genes involved in either increasing dopamine production, regulating the pathways of the basal ganglia, or protecting dopaminergic neurons of the nigrostriatal pathway. Current research is focused on investigating the efficacy and overall safety of gene therapy through delivery of the genes responsible for aromatic L-acid decarboxylase (AADC), glutamic acid decarboxylase (GAD), glial cell derived neurotrophic factor (GDNF), and neurturin (NRTN). Although these methods of gene therapy are relatively new and still developing, they present a promising direction for PD treatment. In this review, the various gene therapy strategies designed for improving parkinsonism are evaluated for safety and efficacy.

<p> The effectiveness of vitamin C in treatment of cancer and heart disease is a matter of debate. While some studies show that vitamin C intake is correlated with improved clinical outcome in cancer patients and is associated with better cardiovascular health, others did not. In this thesis, we examine the biochemical and pharmacological properties of this vitamin in the hope that they will be conducive to resolving this controversy. </p> <p>In Chapter 1 of this thesis, we present a compilation of three publications reviewing the current knowledge about this nutrient, including its chemical and biological properties, with focus placed on its therapeutic potentials. From these literatures, we arrived at the hypothesis that vitamin C, at pharmacological concentrations in the serum, may have mitigative effect on cancer and cardiovascular disease. </p> <p> In Chapter 2 of this thesis, we examine the effectiveness of an alternative vitamin C delivery method using gene therapeutic vectors in a humanized transgenic mouse model. These mice have been rendered defective in endogenous vitamin C production by genetic knockout of gulonolactone oxidase ( GULO -/- encoding gene ( Gulo ), which is responsible for catalyzing ascorbic acid biosynthesis. In an earlier study, we constructed gene therapeutic helper-dependent adenoviral vectors (HDAd) carrying the coding sequence for Gulo under either human phosphoenolpyruvate carboxykinase (PEPCK) promoter (HDAd-PEPCK-Gulo) or munne cytomegalovirus(mCMV) immediate-early promoter (HDAd-mCMV-Gulo ). In this study, we sought to examine the ability of these vectors to mediate the expression of GULO and the production of ascorbic acid in human hepatocellular carcinoma (HEPG-2) and Gulo-knockout (Gulo -/-) mice. We found that HEPG-2 infected with HDAd-mCMV-Gulo expressed GULO, which can be readily detected in cells infected at a multiplicity of infection (MOl) of 10 viral particles per cell (vp/cell) using immuno-based blot. Immunoblot also showed that GULO expression occurred at 18 h post-infection in cells treated with HDAd-mCMV-Gulo at a MOl of 500 vp/cell. Vitamin C production was observed in HEPG-2 treated with HDAdmCMV- Gulo as measured by HPLC-electrochemical detection (HPLC-ECD). We showed that vitamin C production is dependent on the substrate, gulonolactone, concentrations. Gu/a-knockout mice treated with 2X1011 vp expressed GULO in the liver. Using HPLCECD, we showed that the serum vitamin C concentrations of these mice were elevated to levels comparable to those of the wild type mice (60 J.LM) after 4 days of infection and were maintained at 30 J.LM for the duration of the experiment (23 days and ongoing). Similar elevation was observed in urine and tissue vitamin C concentrations in vectortreated animals. In conclusion, we demonstrated here that gene therapeutic HDAdmCMV- Gulo vectors are able to mediate the expression of GULO and endogenous production of vitamin C in human cells and in Gulo -/- transgenic mice. Taken together, these findings support the feasibility of gene therapy as a novel vitamin C delivery method to achieve supra-physiological concentrations of vitamin C in the blood. </p> / Thesis / Master of Science (MSc)

Hemophilia A is an X-linked recessive bleeding disorder caused by the deficiency of coagulation factor VIII [1]. Current treatment for hemophilia A consists of prophylactic or on demand replacement therapy of either plasma-derived or recombinant FVIII concentrates [2]. Albeit effective, there are several limitations associated with factor concentrates, including high cost that limits its availability for close to 80% of hemophilia patients in developing countries [3-5]. An alternative treatment would thus be desirable. Gene therapy for hemophilia has seen many successes in animal models and represents a more cost-effective alternative to the current treatment modalities [6]. In the current work, I present a gene therapy system for hemophilia that uses mouse fetal myoblasts engineered to secrete FVIII, enclosed in immuno-protective alginate-poly-L-lysine-alginate (APA) microcapsules, as a sustained source of FVIII. In this study, a thorough examination of the encapsulated myoblasts using a novel flow cytometry assay was performed. This method yielded an accurate and precise method for encapsulated cell viability calculation, and also allowed for analysis of several other parameters such as health (cell morphology), cell size and distribution. Flow cytometry was also used to monitor the time-course proliferation profile of encapsulated myoblasts secreting cFVIII, using the division tracking dye CFSE. We found that encapsulated cells display a decreased proliferation rate as well as lower viability than non-encapsulated cells. Implantation of encapsulated G8 myoblasts secreting cFVIII into hemophilia A mice resulted in maximum plasma levels of protein on day 1 ( ~18% of normal canine FVIII levels). Delivery of cFVIII in hemophilic mice also offered protection against blood loss after the mice were subjected to injury (as measured by hematocrit levels); indicating that biologically functional cFVIII continued for at least 7 days post-capsule implantation. Low levels of FVIII antigen returned on day 28 after a transient disappearance on day 14. However, the presence of antigen must be reconciled with appearance of anti-cFVIII antibodies that were detected in the plasma of treated mice at the end of five weeks. The neutralizing nature of these antibodies still needs to be characterized by Bethesda assay Overall, our study demonstrates the feasibility of delivering therapeutic levels of FVIII using encapsulated G8 fetal myoblasts. The presence of functional FVIII protein on day 7, suggests that this treatment was not met by transcriptional repression in vivo, thereby overcoming one of the major obstacles faced by using the transformed C2C12 cell line secreting hFVIII [7] If such levels of FVIII were achieved in humans, it would be sufficient to convert a severe hemophiliac into a mild phenotype. Thus, this gene therapy strategy may be a suitable therapeutic alternative for hemophilia patients. Further work ought to focus on the long-term persistence of FVIII in hemophilia A mice, and also determining the protection following trauma over time to determine if the FVIII remains functional. Other cell lines should be explored for higher expression, reduced immunogenicity and improved viability Still, there is a need to develop human cells expressing high levels of biologically active hFVIII with similar properties to the fetal cells described in this study [8, 9] / Thesis / Master of Applied Science (MASc)

Hamm et al., 2021 reported that voluntary wheel running (R) was complementary to micro-dystrophin gene therapy (GT) in mdx mice, a model of Duchenne muscular dystrophy (DMD). After 21 weeks of running, time to fatigue on a treadmill for the mdxRGT mice was increased 1.8-fold compared to mdxGT mice (no run) and ~5-fold compared to mdx mice (no micro-dystrophin, no run). Fatigue times for mdxRGT were similar to wild type runners (WTR), while mdxGT and WT (no run) were also similar. The diaphragm is an important muscle for endurance exercise. Remarkably, diaphragm power in mdxRGT was depressed compared to mdxGT, suggesting a negative impact of running on GT. To explore mechanisms to explain this decrease, transcriptome profiles for each of the study groups were assessed. RNASeq data revealed differentially expressed genes (DEGs) from groupwise comparisons. Transcripts identified using the Jackson Labs' Gene Expression Database and extensive literature review were organized into a master signaling pathway composed of two sub-pathways: muscle regeneration and fast-slow fiber type shift. Both sub-pathways were hypothesized to explain the improved treadmill performance despite decreased diaphragm power in mdxRGT as potential adaptive mechanisms. Analysis revealed that GT alone (mdxGT) rescued transcriptome expression to WT values in the mdx phenotype more than GT and running combined (mdxRGT). This outcome indicates that, at the 26-week timepoint of sacrifice, the signaling of the transcripts in the muscle regeneration and fast-slow fiber type shift sub-pathways was likely not responsible for the observed improved running performance of mdxRGT compared to mdx. / Master of Science / Muscular dystrophy is a group of diseases characterized by progressive muscle wasting and loss of function. Duchenne muscular dystrophy (DMD) is the most common of these conditions, with an occurrence of 18 per 100,000 live births. The muscles of people with DMD lack a protein called dystrophin, which provides structural integrity for muscle fibers during contraction. This lack of dystrophin leads to muscle deterioration over time, leading to people with DMD typically being wheelchair-bound by ten years of age. Animal models of DMD have been created over time to help study this condition. One such model, the mdx mouse, was used in the study that led to this thesis project. In this study, Hamm et al., 2021, some of these mdx mice were given a micro-dystrophin gene therapy (GT). This GT aimed to deliver a smaller, but still functional version of the missing dystrophin protein to the mice, which has been shown to be beneficial in other studies. This study aimed to measure the effect of this GT when combined with voluntary wheel running. As people with DMD cannot exercise under current clinical guidelines, measuring the response of mdx mice to GT and running combined is an important step in determining the safety of such a treatment in human patients. In the study conducted by Hamm et al., 2021, the mice that received the micro-dystrophin GT and access to running wheels (mdxRGT) performed almost twice as well on a treadmill running test than the mice that received GT alone (mdxGT). Despite this positive result, the mdxRGT mice showed decreased diaphragm power generation compared to mdxGT mice. As the diaphragm is the most important breathing muscle, it is also very important for running performance; therefore, the decreased diaphragm power generation seen in mdxRGT mice is apparently contrary to their improved running performance. To explain this discrepancy, this thesis project examined the diaphragm transcriptome of the different groups in Hamm et al., 2021. The transcriptome is the sum total of messenger ribonucleic acid (mRNA) expressed in a given tissue. Deoxyribonucleic acid, or DNA, is transcribed into mRNA, which is then translated into protein. As such, this thesis project looked at the mRNA expressed in the diaphragm of the mice in the various groups of Hamm et al., 2021, specifically comparing the mdxGT and mdxRGT groups. Important mRNA transcripts, or genes, were identified and assembled into signaling pathways, cascades that highlight how transcripts affect each other and ultimately lead to function once they are translated into their corresponding proteins. Two such signaling pathways were generated based on mechanisms that were thought to contribute to the improved running performance-decreased diaphragm power discrepancy in mdxRGT mice -slow fiber type shifting, and muscle fiber regeneration. The expression of many of the mRNA transcripts in these resulting pathways was closer to the control group in mdxGT compared to mdxRGT. The control group was made up of healthy mice, and as such, their transcript expression level is seen as normal. This outcome of mdxGT having more similar expression to the control group than mdxRGT suggests that the expression of the transcripts included in the two signaling pathways (fast-slow fiber type shift and muscle fiber regeneration) likely did not explain the improved running performance despite decreased diaphragm power in mdxRGT mice. As such, future studies are warranted.

Introduction: Gene therapy is currently being developed for people with cystic fibrosis (CF), a life-threatening condition for which there is no cure. The UK CF Gene Therapy Consortium are preparing for a multi-dose gene therapy trial of sufficient duration that clinical benefit may be seen. Aims: The current study aimed to explore the expectations and beliefs of cystic fibrosis (CF) patients involved in the preparatory phase of the gene therapy trial (the Run-in study), from which participants will be selected for the multi-dose actual gene therapy trial. Method: Twelve participants (six with mild and six with moderate CF) were interviewed using a semi-structured interview. Interviews were recorded, transcribed verbatim and then analysed using a Constructivist Grounded Theory approach. Results: Since entering the Run-in study, half of the patients had increased their expectations of gene therapy being an effective future treatment. Most of the participants hoped to derive clinical benefit from the trial itself though half were unsure of what to expect. Whilst half of the participants expressed the hope of a future cure for CF, the remainder saw gene therapy only in terms of an improved treatment. Participants used several strategies to manage their expectations including not thinking too far ahead and trusting the research team. Discussion: The findings indicate that participants in the Run-in trial are generally eager to be involved in the gene therapy trial and have developed a strong sense of trust in the research team conducting the trials. The levels of optimism expressed for personal benefit from trial were higher than those from earlier studies. Some of the positive expectations were unlikely to be met by the gene therapy trial and participants risk disappointment. However other patients participated with apparently realistic expectations and it seems likely that some patients would have participated even without prospect for personal benefit. Possible areas of psychological support are discussed e.g. a standard clinical interview for all those not accepted for the gene therapy trial; screening for anxiety pre-, during and post-participation.

Despite expressing antigens that can induce immune surveillance and immune eradication, tumours demonstrate the capacity to evade anti-tumour immunity. Recently, this has been attributed to the ability of tumours to induce a local immunosuppressed micro-environment, which is a major obstacle to successful natural and vaccine induced anti-tumour immunity. Soluble factors such as transforming growth factor beta (TGFβ), and interleukin 10 (IL-10), released by cancer and stromal cells, are thought to play a significant role in this local immunosuppression. In order to assess the influence of antagonising these soluble factors locally on tumour biology and tumour immunity, a murine CT26 colorectal carcinoma model that can express cytokine antagonists under Doxycycline (Dox) control was engineered. Two stable CT26 cell lines expressing Dox-inducible soluble extracellular domain of TGFβ receptor II (STGFβRII) or soluble extracellular domain of IL-10 receptor (SIL-10R), were established. Expression of STGFβRII in vitro and in vivo was only evident after Dox treatment. When Dox was administered directly following subcutaneous (s.c.) inoculation of STGFβRII-expressing CT26 cells into Balb/c mice, tumour growth was significantly suppressed. Interestingly, inducing STGFβRII in well-established tumours showed less suppression of tumour growth. To assess the effect of expressing STGFβRII on tumour immunity the RNA expression of 22 common cytokines was measured in the tumours of mice receiving Dox and control mice. The levels of some of these cytokines were modulated by STGFβRII expression (e.g TGFβ,Tnfsf9, IL-2). We also tested for any additive effect between expressing STGFβRII, and the administration of anti-CTLA-4 antibody on tumour growth, and tumour immunity. The model described here could help address various limitations seen previously in studies of TGFβ blockade. It provides means of effective local antagonism, and it addresses immunological endpoints which have been limited in previous studies. Because of its tight regulation, the model also allows identification of the best timing of TGFβ blockade alone or in combination with other immunotherapeutics.

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy