No description available.

No description available.

Multiple DNA transactions are at the center of almost all processes regulating the AAV life cycle. A common feature shared by all transactions is the binding of the large AAV Rep proteins Rep78/Rep68 onto DNA sites harboring multiple GCTC repeats. AAV mediated site-specific integration is contingent upon the formation of a productive complex between Rep78/Rep68 and the AAVS1 site located at chromosome 19. In order to understand the mechanistic details of the initial assembly process we carried out equilibrium binding experiments of Rep68 and its individual domains with a 42-mer AAVS1 site. Results show that although Rep68 binds AAVS1 with high affinity (69 nM), both the OBD and helicase individual domains bind DNA weakly with affinities of >>60μM and 22μM respectively under our experimental conditions. Mutant Rep68 proteins that have a defective oligomerization interface bind DNA poorly suggesting that productive binding requires both the concerted interaction of the individual domains with DNA and oligomerization. Moreover, we show that a minimal number of two repeats is required to form a stable complex. In addition, initial studies were done to characterize the interaction between Rep68 and the viral ITR DNA sequences using AUC and electron microscopy.

No description available.

While the last century of medical discoveries has made a significant impact on improving the lives of human populations across the globe, a perfect solution to the yearly infection cycle from the influenza virus has yet to be discovered. Although vaccines stand the best chance at targeting yearly epidemics, new treatment options must be created to combat the arrival of rapidly mutating and antiviral-resistant strains of the virus that could lead to another pandemic such as the 1918 Spanish flu that killed millions worldwide. We describe a method to create functionally enhanced monoclonal antibodies targeting influenza via genetic engineering of fragment crystallizable glycan structures. Muscle and liver cell lines were lentivirally-transduced to produce the broadly neutralizing antibody, Fi6v3, while also overexpressing a critical glycosylation enzyme, B-1,4-N-acetyl-glucosaminyltransferase III. Secreted antibodies were tested for effector functionality using a Natural Killer cell degranulation assay and an antibody-dependent cellular phagocytosis assay. Results conclude that modified antibodies from both muscle and liver cells lines exhibit enhanced function in comparison to their unmodified counterparts, providing support to the future creation of an influenza prophylactic or treatment option using antibodies with the ability to more effectively activate innate immune killing mechanisms.

En thérapie génique ex vivo, les vecteurs lentiviraux peuvent être utilisés pour transduire les progéniteurs hématopoïétiques. Mais l’utilisation de vecteurs intégratifs non site-spécifique peut conduire à une mutagénèse insertionnelle. J’ai évalué le niveau de transduction des progéniteurs hématopoïétiques en mesurant le nombre de copies de vecteur intégrées dans des colonies cellulaires isolées. Il a été montré que la fréquence des cellules transduites et la distribution du nombre de copies de vecteur intégrées peut dépendre des conditions expérimentales. L’utilisation de vecteurs ciblant l’insertion du transgène à un site précis du génome permettrait de surmonter les problèmes de génotoxicité. Les introns de groupe II sont des éléments génétiques auto-épissable mobiles pouvant d’intégrer à un site précis du génome. Ils sont utilisés en ciblage génomique chez les procaryotes, mais pas chez les eucaryotes, probablement dû à une activité catalytique limitée dans ces cellules. J’ai étudié l’intron de groupe II Pl.LSU/2 de Pylaiella littoralis, qui est le seul capable de s’auto-épisser à des concentrations très faibles de magnésium. La protéine codée par l’intron Pl.LSU/2 (IEP) exprimée chez Escherichia coli a été purifiée et a montré une activité de transcriptase inverse soit seule, soit associée à l’intron ARN. Il a été montré que l’intron Pl.LSU/2 peut s’épisser chez Saccharomyces cerevisiae et que l’efficacité de l’épissage est augmentée par l’activité maturase de l’IEP. Cependant, les transcrits épissés ne sont pas traduits, et l’épissage de l’intron n’a pas été démontré dans les cellules humaines, tout comme le homing de l’intron chez E. coli et S. cerevisiae. / In ex vivo hematopoietic gene therapy, lentiviral vectors can be used to transduce hematopoietic progenitors. However, the use of non site-specific integrating vectors may lead to insertional mutagenesis. I evaluated the level of transduction of hematopoietic progenitor cells at the single-cell level by measuring vector copy number in individual colony-forming cell units. It was shown that the frequency of transduced progenitor cells and the distribution of VCN in hematopoietic colonies may depend upon experimental conditions. Nevertheless, the use of vectors that can target the integration of the transgene into a specific-site of the host genome would overcome genotoxicity issues. I evaluated the use of a group II intron for genomic targeting. Group II introns are self-splicing mobile elements that can integrate into precise genomic locations by homing. Engineered group II introns are commonly used for targeted genomic modifications in prokaryotes but not in eukaryotes, probably due limited catalytic activation in these cells. I studied the brown algae Pylaiella littoralis Pl.LSU/2 group II intron which is uniquely capable of in vitro ribozyme activity at unusually low level of magnesium. Recombinant Pl.LSU/2 IEP expressed in Escherichia coli was purified and showed a reverse transcriptase activity either alone or associated with intronic RNA. The Pl.LSU/2 intron was showed to splice accurately in Saccharomyces cerevisiae and splicing efficiency was improved by the maturase activity of the intron-encoded protein. However, spliced transcripts were not expressed, and intron splicing was not detected in human cells, as well as homing of Pl.LSU/2 in E. coli and S. cerevisiae.

No description available.

Gene therapy is an attractive treatment for many genetic diseases because rather than treat the symptoms of the disease, it has the potential to correct the underlying defect. Cystic fibrosis and hemophilia A are two monogenic disorders that are particularly well-suited to treatment with gene therapy as a relatively small increase in the function is needed to see improvement. Gene therapy has provided some correction in both diseases using a variety of vector systems but sustained expression and long term correction have yet to be demonstrated in the clinic. It is unclear in which cell type(s) correction of the underlying defect in cystic fibrosis will be most effective. Studies indicate that the majority of CFTR expression is in the submucosal glands and ciliated epithelia – a terminally differentiated cell type (Engelhardt, J.F. et al, 2004, Journal of Clinical Investigation). Therapeutic gene transfer would thus be most effective if achieved in a progenitor cell type. Additionally, the native regulation of CFTR has not been definitively elucidated. To this end, one goal of our studies is to develop a lentiviral vector system with heterologous promoters of varying strengths and cell specificity to aid in our selection of optimal reagents for appropriate CFTR expression. We show that use of novel internal promoters from the human PLUNC and WDR65 genes direct persistent expression in the airway. Additionally, disruption of the nasal epithelium with the detergent polidocanol eliminated reporter expression in mouse airway. Two weeks post-treatment, expression returned indicating targeting of a progenitor cell population with our novel vectors. Integrating vector systems can treat chronic diseases such as cystic fibrosis because expression can persist long term from these vectors if cells with progenitor capacity are targeted (Sinn, P.L. et al, 2005, Journal of Virology). However, the potential for genotoxicity from vector-related dysregulation is a concern. Thus, a second aim of these studies was to develop a lentiviral vector that can target a specific locus in the genome. We developed a FIV vector in which the integrase was modified with a protein-binding domain that when co-delivered with a fusion consisting of the cognate protein and a DNA binding domain would tether the vector to the appropriate locus. Unfortunately, integrase modification rendered the vector catalytically inactive. Lastly, we hoped to develop a non-viral transposon vector system (piggyBac) for gene transfer applications to the liver for treatment of hemophilia A. The recent demonstration that piggyBac transposase is highly active in mammalian cells warrants further development of this vector as an alternative to other non-viral integrating vector systems currently under investigation. We showed persistent reporter and therapeutic transgene expression in the livers of mice treated with the piggyBac vector. Furthermore, we show for the first time in vivo persistence and increased expression from the recently developed hyperactive transposase. The development of integrating vectors targeted to specific tissues or genomic loci is important in for treatment of the monogenic diseases cystic fibrosis and hemophilia A.

Novel approaches to cancer treatment in the context of Gene Therapy have been gaining popularity as an alternative to conventional therapies which have proven to lack specificity, resulting in tumour cell resistance, tumour progression and mortality. As a consequence the use of adenoviruses has been widely developed not only as a replication deficient vector for gene therapy but also as a replication competent oncolytic agent designed to selectively target and kill tumour cells. Unfortunately their success in clinical application has been limited, and it has been suggested that a lack of the primary viral attachment receptor 'CAR' could be a barrier to infection by limiting access to target cells. If Ad/CAR binding is the rate limiting step for successful Ad therapy, it is essential to establish a CAR expression profile in normal and tumour tissue, and in tumour progression, to enable more effective targeted therapy. Furthermore, in the context of using adenovirus as an anticancer strategy by exploiting its replicative lysis, it is important to explore whether Ad success is affected by CAR expression and to identify factors downstream of CAR that may be influential in this process. In the first experimental chapter, an in vivo immunohistochemical analysis of tissue array slides determined CAR expression in a range of normal and tumour tissue. CAR was differentially expressed dependent on cell of origin, with normal stem cells and basal cells displaying very high CAR, signifying its importance in early development and differentiation. Epithelial cells were also high in CAR but its expression was negligible in mesenchymal, lymphoid and neural cells. This trend was also reflected in most tumour tissue albeit with a general decrease in CAR compared to corresponding normal tissue of the same organ. An exception was the blastic tumours which displayed high CAR reflecting their embryonic state of derivation. CAR expression also decreased in high grade, poorly differentiated tumours of the prostate, stomach and breast compared to their well differentiated counterparts. In the second experimental chapter, a more comprehensive study of breast cancer biopsy specimens was undertaken, to determine both the expression of CAR and the tumour suppressor gene p53 in relation to tumour grade. The rationale being that both loss of CAR expression and p53 mutation (resulting in loss of function), have been associated with tumour progression. It is possible that CAR and p53 interact directly or indirectly and may be modulated by each other. This study revealed a decrease in both CAR and hormone receptor expression and an increase in p53 'mutational' status with increasing tumour grade. These three factors when compared independently to tumour grade are statistically significant associations, implying that CAR expression and hormone responsiveness decrease with tumour progression and p53 function is compromised or lost via mutation. There was also a significant association between CAR expression and hormone receptor status, however a significant association between CAR expression and p53 status within the tumour grades was not found. Treatment outcome with Ads will also depend on defining factors downstream of CAR attachment that affect adenovirus 'permissivity', which is ultimately measured by viral replication and cell death, relying on the bystander effect to eradicate all tumour cells. The in vitro analysis revealed statistically significant associations between CAR receptor expression, 'infectivity' (virus infection) and permissivity. Cell lines that were more susceptible to Ad5 were generally of epithelial origin, had high CAR, and were easily infected. However there were exceptions and CAR was not the sole determinant in adenovirus cell entry nor in its ability to replicate and kill the cell. Permissivity was also related to p53 status. Thus, although CAR expression may indeed be a limiting factor, it is apparent that a combination of other events contributes to a deficient infection, especially in the deregulated tumour environment. The results presented in this thesis clearly demonstrate that there is more to the story of 'CAR' which hints that its role in viro-oncotherapy is not limited solely to its function as an attachment receptor for adenovirus but may also involve its function as a cell adhesion molecule and signal transducer. The further elucidation of these aspects of CAR�s potential role in the scheme of tumour biology may alter the course and strategy of cancer therapy in the future.

Endogenously, VEGF initiates angiogenesis, then later Angiopoietin (Ang)-1 matures vessels. We hypothesized that multigene therapy of VEGF before Ang1 to ischemic hindlimb tissue would result in persistent angiogenesis. At 2, 4 and 8 wks after inducing ischemia, blood flow was assessed by contrast-enhanced ultrasound. Animals were treated with VEGF at 2 wks, VEGF/Ang1 at 2 wks, or VEGF at 2 wks and Ang1 at 4 wks. In untreated controls, blood flow remained reduced. After VEGF delivery, resting flow and vessel density increased; however, flow reserve remained reduced, and vasculature was capillary-rich and eventually regressed. After VEGF/Ang1 co-delivery, flow increased marginally, flow reserve improved and vascular architecture remained normal. After separated VEGF and Ang1 delivery, flow, vessel density and flow reserve increased and were sustained, while vascular architecture remained normal. In conclusion, temporally separated VEGF and Ang1 delivery promotes sustained angiogenesis and improved vessel functionality.