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Hepatitis C virus (HCV) is a major global pathogen estimated to infect over 170 million people worldwide. A recent study has shown that vaccination with adenoviral vectors, based on rare human and simian serotypes encoding the non-structural (NS) proteins of HCV, induces highly potent, multi-specific and durable T cell responses in healthy human volunteers. In this thesis I assess the safety and immunogenicity of these vaccines (ChAd3–NSmut and Ad6-NSmut), for the first time in HCV infected patients. This work also explores whether vaccine-induced T cell responses target in vivo circulating HCV antigens and common naturally occurring epitope variants. Patients with treatment naive chronic genotype 1 HCV infection were vaccinated (i.m.) with ChAd3-NSmut and Ad6-NSmut in a heterologous prime boost schedule, either with or without current IFN and ribavirin (IFN/RBV). Epitope-specific T cell responses were defined by fine mapping using HCV peptides. Circulating viral genomic sequence was determined in vaccinated patients at baseline and at any point of viral relapse. Cross-reactivity of vaccine-induced T cell responses was determined in T cell assays, using peptides corresponding to both circulating host virus and common population HCV epitope variants. An in vitro dendritic cell /T cell priming model was used to identify possible candidates for a cross-reactive vaccine immunogen at the most immunodominant epitope, NS31406. 33 patients were vaccinated. Vaccination was well tolerated. At the highest vaccine dose (2.5 x 1010vp) vaccine-induced T cell responses were detectable in 11/20 patients receiving concurrent IFN/RBV and 2/4 patients receiving vaccination alone. In total 14 antigenic targets were identified, 2 of which have not previously been described. However, T cell responses were of lower magnitude and more narrowly focused than those observed in healthy volunteers vaccinated with the same regimen. Analysis of viral sequence showed that in many cases vaccine-induced T cells did not target the circulating virus. At the most immunodominant epitope (NS31406), T cells induced by vaccination failed to target common circulating genotype 1 HCV variants. An in vitro model suggested that in order to target all genotype 1 sequences at this epitope, it would be necessary to insert both a genotype 1a and 1b version of this epitope into a vaccine immunogen. Vaccination with adenoviral vectors induces T cell responses in patients with chronic HCV infection, however immune responses are attenuated compared with healthy volunteers. Ultimately a successful therapeutic or prophylactic vaccine strategy will rely on inducing responses that target conserved or cross-reactive epitopes.

The advanced stage of prostate cancer is the second leading cause of cancer-related death for American men. Novel, effective treatment options and more cancer-specific diagnostic tools are urgently needed to facilitate patient management. Here, we explored the construction and application of an array of gene-based molecular imaging and therapeutic vectors in a variety of clinically relevant settings. These vectors exploit prostate cancer-specific promoters to control the transcription of imaging reporter genes or therapeutic genes, and thus can achieve stringent cancer selectivity. We showed that gene-based imaging vectors can detect prostate cancer metastasis earlier than conventional imaging modalities; we also developed a dual-reporter imaging system that can specifically interrogate the activation/inhibition status of androgen receptor, and thus monitor the efficacy of androgen deprivation and androgen receptor blockade therapies. Further, we explored the applicability of gene-based vectors in detecting circulating tumor cells and have obtained promising results. Moreover, we employed two strategies to improve the clinical usability of viral vectors in immunocompetent hosts (such as humans). In the first strategy, we used a broad-acting immunosuppressant, rapamycin, to reduce the scale of adenovirus-induced inflammatory responses and also to decrease the eliminating effects of host adaptive immune system on viral vectors and viral-infected, transgene-expressing cells. Rapamycin significantly augmented the magnitude and prolonged the duration of virus-delivered transgene expression, and thus enhanced the diagnostic capability of these imaging vectors. The second approach took advantage of the versatile surface modifying ability of non-viral reagents. We designed a polypeptide coating that can non-covalently bind to adenoviral particles and confer protections against blood components such as neutralizing antibodies and coagulation factors. The coating significantly decreased the inhibitory effects of neutralizing antibodies on adenoviral transduction and markedly abrogated coagulation factors-mediated hepatocyte infection. Furthermore, this polypeptide coating reduced the antigenicity of adenoviral vectors in vivo, greatly diminished anti-adenovirus antibody production, and therefore could facilitate repeated administration of viral vectors in immunocompetent hosts. Overall, we herein present a substantial amount of evidence supporting the pre-clinical and translational use of gene-based imaging and therapeutic vectors to assist the management of patients with advanced, treatment-refractory prostate cancer.

Ex vivo gene transfer can improve the outcome of islet transplantation for treating Type I diabetes. Earlier we have shown co‑expression of human vascular endothelial growth factor (hVEGF) and human interleukin‑1 receptor antagonist (hI‑1Ra) after transfection of plasmid DNA encoding these two genes. Due to poor transfection efficiency of plasmid DNA and the better known islet transduction efficiency of adenoviral (Adv) vectors, in this study, we constructed Adv‑hVEGF‑hIL‑1Ra by cloning hVEGF and hIL‑1Ra coding sequences and polyA signal under separate cytomegalovirus (CMV) promoters in Adenoquick plasmid (Ad 13.1). There was dose and time dependent expression of these genes after transduction of Adv‑hVEGF‑hIL‑1Ra into human islets. The mRNA expression of hVEGF and hIL‑1Ra was more than 100 times higher than that of the non‑transduced and bicistronic plasmid transfected control islets. Transduced islets were viable as evidenced by insulin release upon glucose challenge. Co‑expression of hVEGF and hIL‑1Ra by islets showed decrease in caspase‑3 activity and apoptosis induced by a cocktail of inflammatory cytokines such as TNF‑α IL‑1βand IFN‑γ. Compared to non‑treated or Adv‑LacZ transduced islets, transduction of islets with Adv‑hVEGF‑hIL‑1Ra prior to transplantation under the kidney capsules of diabetic NOD‑SCID mice reduced the blood glucose levels, and increased serum insulin and c‑peptide levels. Immunohistochemical staining of the islet bearing kidney sections at day 30 after transplantation was positive for human insulin, hVEGF and von Willebrand factor. Further, we decided to replace VEGF with hepatocyte growth factor (HGF) as it not only can promote revascularization of islets but also increases β ‑cell proliferation. It can also protect the islets from apoptosis. Adv‑hHGF‑hIL‑1Ra showed dose and time dependent expression of these genes after transduction into human islets and further showed significant decrease in caspase‑3 induced by the cytokines. Compared to un‑transduced islets, transduction of islets with Adv‑hHGF‑hIL‑1Ra at 1000 MOI prior to transplantation under the kidney capsules of streptozotocin‑induced‑diabetic NOD‑SCID mice reduced blood glucose levels, and increased serum insulin and c‑peptide levels. These results indicate that the bicistronic Adv vector efficiently expresses both growth factor and antiapoptotic genes, decreases apoptosis and improves the outcome of islet transplantation. Liver fibrosis is a consequence of chronic liver disorders which lead to accumulation of extra cellular matrix (ECM). Particularly, there is an increased accumulation of collagen in the fibrotic liver. We have therefore used a triplex forming oligonucleotide (TFO) against the type α1 (I) collagen and evaluated, whether it can attenuate liver fibrosis induced by common bile duct ligation (CBDL). There was a significant decrease in hydroxyproline levels in TFO‑treated groups compared to non‑treated CBDL group. Masson’s trichrome staining showed weak staining for collagen in TFO‑treated liver tissues and there was a predominantly high staining in non‑treated CBDL group. There was over expression of α ‑smooth muscle actin (α ‑SMA), marker for myofibroblasts following CBDL. Western blotting analysis also revealed an increase in TGF‑β1 expression, which is a potential marker for fibrosis in the CBDL group compared to TFO‑treated group. These results suggest that TFO can be used to downregulate Type I collagen gene expression and can alleviate liver fibrosis induced by common bile duct ligation.

Current therapies for inflammatory bowel disease (IBD) fail to address disease pathogenesis, providing only symptomatic alleviation, and lack organ specificity, with most of the drug reaching the systemic circulation rather than being targeted locally to the diseased intestine. The objective of this research project was to study two novel approaches to IBD therapy; adenoviral gene delivery and local liposomal drug delivery after intralumenal administration. Caco-2 cells in in vitro models of mucosal wound healing, ex vivo intestinal biopsy cultures and an in vitro M-cell model were employed to study interactions of adenoviral vectors and intestinal epithelium. Transgene expression was quantified by biophotonic imaging. The rat was used as an animal model for investigation of intestinal absorption and metabolism of the model compound, 6-mercaptopurine, administered in solution or as a liposomal formulation, coupled with HPLC and liquid scintillation counting bioanalytical methods. It was determined that restituting intestinal epithelial cells are transfected much more efficiently by adenoviral vectors than their healthy, confluent counterparts. The effect appears to be related to integrin and CAR expression by the restituting cells, associated with the migration process. Significant differences in adenoviral transduction of Caco-2 cells and M-cells compared to other epithelia cell types were observed. Liposomal formulation of 6-mercaptopurine, despite mucosal adherence, did not prove advantageous in terms of local delivery compared to solution. This is in contrast to previous studies with a 5-aminosalicylic acid liposomal formulation. The differences can be explained by the different physicochemical properties and absorption characteristics of the two drugs. This study suggests that gastrointestinal epithelium, in disorders such as IBD where epithelial restitution takes place, represents a valid target for gene transfer. As gastrointestinal gene delivery is still in its infancy, liposomal formulations that offer an increased drug residence time at the inflamed intestinal tissue due to mucosal adherence are an attractive alternative to traditionally used controlled/delayed release formulations of small drug molecules. However, drug physicochemical properties and intestinal absorption characteristics will dictate the potential for these formulations.

First-generation adenovirus (Ad) vectors are unsuitable for gene replacement therapy, due to immune responses and low cloning capacity. These problems may be exacerbated in the inflammatory milieu of dystrophic muscle. It is hoped that gutted Ad vectors, which do not carry viral genes, may provide effective gene transfer without eliciting acute toxicity or a longer-term, antigen-specific anti-adenovirus response. Unfortunately, gutted Ad vectors share some drawbacks with conventional Ad vectors and present new challenges. First, gutted vectors share the tropism of conventional adenovirus and could elicit an anti-transgene response in patients with null alleles. Second, clinical grade production of gutted Ad vectors has not been achieved. The goal of this thesis is to determine whether these two problems with gutted adenovirus, low virus yield and immune response to transgene products, can be overcome. Two hypotheses were tested: (1) poor growth of gutted adenovirus vectors in tissue culture is caused primarily by failure of sub-optimal replication origins to compete for replication factors; and (2) in healthy muscle tissue, but not necessarily in dystrophic tissue, the immune response to transgene products delivered by a vector with promiscuous tropism can be eliminated by strict tissue-specific gene expression. We explored whether gutted virus production would be facilitated by transfection into cells expressing replication factors or by use of synthetic terminal protein-DNA complex. Both methods were effective, together yielding a 1000-fold increase in gutted virus titer. Muscle tissue from the mdx mouse, a model for Duchenne muscular dystrophy (DMD), is characterized by infiltrating antigen-presenting cells (APCs). We found that mdx mouse muscles mounted a stronger immune response to antigens that can be directly presented by those APCs. However, we did not detect an immune response to beta-galactosidase expressed specifically in muscle by an Ad vector, even at high expression levels. This suggests that cross-presentation is not dramatically more effective in mdx mouse muscle, and that targeted vectors and tissue-specific promoters may be useful tools for evasion of the immune response in dystrophic muscle. The results of our studies indicate that gutted vectors carrying muscle-specific transgenes have great potential for gene therapy of DMD.