Browsing by Author "Barry, Michael A."

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    Engineering adenoviral gene delivery vectors for improved gene-based immunization
    (2006) Mercier, George Timothy, IV; Barry, Michael A.
    Gene-based vaccines have been shown to elicit protective immune responses against a number of pathogens; however, conventional gene delivery methods fail to elicit potent systemic and mucosal responses required to prevent infection by certain pathogens. With the aim of inducing more potent responses, two complementary targeting strategies were employed for the delivery of antigen genes to immunologically-relevant cells, namely mucosal cells and antigen-presenting cells (APC). This thesis explores the identification and application of ligands that target Ad vectors to APC and mucosal sites using genetic engineering and biotin-avidin coupling methods with the aim of increasing immune responses to a model transgene. For APC-targeting, biotinylated Ad (Ad-BAP) was used as a novel ligand screening platform to identify the antigen uptake receptors as the most efficient targets for increased transduction. Specifically, Ad-BAP complexed to a mannosylated ligand demonstrated efficient transduction of mouse dendritic cells and macrophages. Vaccination experiments in mice indicate this vector elicits decreased antibody responses and similar cellular responses compared to unmodified Ad. More work is required to determine the benefit of targeting APC for vaccination, because not all types of APC in mice harbor mannose receptors in situ. For targeting mucosal sites, a chimeric Ad vector (Ad-sigma1) was genetically engineered to display the mucosal-targeting sigma1 protein of reovirus. The striking structural homology between the Ad fiber protein and the reovirus sigma1 protein was exploited for the development of a functional chimeric fiber-sigma1 protein to allow virion encapsidation. Adal binds and infects cells through the reovirus receptors, junctional adhesion molecule 1 (JAM1) and cell-surface sialic acid, and not through the Ad receptor, coxsackievirus and adenovirus receptor (CAR). However, Ad-sigma1 transduction of mucosal cells in vitro shows markedly decreased efficiency as compared to unmodified Ad. Despite these defects, Ad-sigma1 elicited similar immune responses compared to unmodified Ad after mucosal vaccination of mice. These results suggest that re-engineering of the fiber-sigma1 chimera to better enable JAM1 interactions may offer promise for using Ad-sigma1 for mucosal vaccination. Further work needs to establish the relative importance of the barriers to Ad-based vaccination, namely low pH, digestive enzymes, glycocalyx, and apical receptor expression.
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    Enhancing transduction of breast and ovarian cancer using EGF and herceptin complexed adenoviral vectors
    (2006) Adams, Kristen E.; Barry, Michael A.
    Successful gene therapy for breast and ovarian cancer will likely require that anti-cancer genes be delivered specifically to primary and metastatic tumor sites while avoiding normal tissues. Adenoviral vectors are attractive for cancer gene therapy, since they can deliver transgenes to many different tumors. While adenovirus is quite potent at gene delivery, it is also non-specific and delivers genes into tumor and non-tumor cells in vivo. For effective gene therapy, the natural tropism of adenovirus must be removed and the virus re-targeted to tumor cells using cancer-specific ligands. To identify new cell binding ligand, peptide presenting phage libraries were selected against human breast cancer cell lines. Displayed on phage, these peptides bound specifically to their selection target, cross-reacted to varying degrees on other breast cancer cell lines, and did not bind to normal breast epithelial cells. The binding properties of these peptides were compared with those of commercially available antibodies such as Herceptin and binding proteins such as EGF to determine viable candidates for vector targeting. Viral targeting methods developed in our laboratory show promise in both ablating the natural tropism of adenovirus and retargeting the virus. The targeting ligands were complexed to biotinylated adenovirus through avidin bridges and chemically cross-linked to adenovirus using bifunctional PEG molecules. The viral complexes were tested in vitro before delivery was evaluated in the in vivo xenograft tumor models. Fluorescent and luminescent reporter genes were used to determine the location of vector delivery through gene expression in vivo. Targeted adenovirus had reduced background transduction and somewhat increased breast and ovarian cancer transduction. Finally to better evaluate ligand performance, real time, dynamic imaging was used to track ligand distribution and kinetics in vivo in tumor models. Fluorescent conditions were first evaluated in mouse models, demonstrating that imaging in the near infrared had superior signal to noise profiles over fluorescence in the visual range. Therefore ligands were labeled with the near infrared dye IR800 and their distribution was tracked in real time. To evaluate the feasibility of tracking virus (not transgene products), adenovirus was labeled with IR800, given to mice and virion trafficking was successfully imaged.
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    Evaluation of polyethylene glycol modified adenovirus for innate response reduction and ligand specific cell targeting
    (2005) Mok, Hoyin Sunny; Barry, Michael A.
    Clinical applications of adenoviruses as gene delivery vectors are limited by their propensity to invoke strong immune responses and toxicity. In addition, the inherent tropism of adenovirus prevents them from reaching the desired cell targets in vivo. This thesis evaluates the ability of chemically modified adenoviral vectors to evade innate immune responses, binding to blood cells, and to target specific cell types with cell-specific ligands. Previous studies have shown that polyethylene glycol (PEG) modification can protect vectors from pre-existing and adaptive immune responses by reducing protein-protein interactions. In this work, we have optimized PEGylation methods and have compared the induction of innate immune responses between modified and unmodified first generation and helper dependent adenoviruses in mouse models. The levels of interleukin-6, a cytokine induced during acute immune response, were found to be significantly lower in the most heavily PEGylated viruses in the murine models. We also observed that the uptake of PEG-modified vectors by macrophages and hepatic Kupffer cells were significantly reduced in vivo. Besides immunogenicity of the vectors, we also explored the binding affinity of chemically modified Ad to blood cells in vitro. PEG-modified Ad not only had reduced binding to erythrocytes and platelets, but also provoked reduced in vivo thrombocytopenia and prevented in vitro hemagglutination. To achieve clinically relevant gene transfer in cell types not susceptible to adenoviral transduction, we conjugated a wide array of cell-specific ligands onto adenoviruses via PEG crosslinkers to retarget the vectors to new receptors. Specifically, we conjugated epidermal growth factors (EGF) and anti-CD59 antibodies to Ad. Conjugation of these new ligands increased transduction on epidermal carcinoma and acute myeloid leukemia cell lines 5--10 fold over PEGylated vectors in vitro. However, the ability of targeted vectors to transduce cells varied greatly and is dependent on receptor densities, ligand functionality after conjugation and size of the conjugated vectors. Nonetheless, this strategy of incorporating cell-specific ligands to PEG modified adenovirus is valuable in creating a safer vector, thereby improving the overall safety and efficacy of adenoviral vectors for future cancer and metabolic disease gene therapy treatments.
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    Ligand discovery and applications for vector targeting
    (2006) Ghosh, Debadyuti; Barry, Michael A.
    Adenoviral (Ad) and adeno-associated viral (AAV) vectors have great promise as gene delivery vehicles for gene therapy and genetic immunization. However, these vectors can non-specifically target tissues and cell types in vivo. Redirected targeting of these vectors by the addition of cell-specific ligands would improve the therapeutic efficacy and safety of these vectors by reducing the effective dosage needed for gene therapy. Phage display technology has been exploited to discover novel cell-specific ligands for vector targeting. However, these ligands are selected in the context of phage and translation of the ligands back into the viral capsid can ablate viral assembly and function or inactivate the targeting function of the ligand itself. To circumvent this ligand-vector compatibility problem, a novel approach to identify cell-specific ligands is described. We have introduced structural "context" onto filamentous bacteriophage and generated random peptide libraries within these contexts for use in ligand selection. The HI loop of the adenoviral capsid was displayed on phage and a random peptide library was generated within this scaffold and used to identify cell-specific ligands against mouse skeletal muscle in vitro. A cell-specific peptide ligand, designated 12.51, was incorporated back into Ad capsid and the redirected Ad vector improved targeting in vitro, suggesting the viability of this approach for ligand discovery. This "context"-based approach was extended towards generating random peptide libraries within streptavidin protein for ligand selection. In addition, a system for conjugation of targeting ligands to the AAV capsid based on the streptavidin-biotin interaction, has been developed. A biotin acceptor peptide was engineered into the AAV capsid and resulted in the development of vectors that are metabolically biotinylated during production in cell lines. This avidin-biotin technology was previously utilized for construction of metabolically biotinylated Ad vectors. However, Ad vectors are extremely immunogenic compared to AAV and may not be suitable for in vivo applications. We constructed metabolically biotinylated AAV vectors and demonstrated proof-of-principle targeting in vitro using various biotinylated ligands. Eventually, streptavidin-context ligands can be conjugated to biotinylated vectors for targeted delivery.
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    Metabolic biotinylation of the adenoviral capsid: Avidin-based applications and studies of ligand-targeted gene delivery
    (2005) Campos, Samuel Knox; Barry, Michael A.
    Adenoviral vectors have great potential for use in gene therapy and genetic immunization. The targeting of Ad vectors to the relevant tissue and cell types in vivo could greatly improve their safety and performance by lowering the effective dosage required for therapeutic levels of gene expression. Redirection of Ad vector tropism will require physical modifications of the adenoviral capsid but direct genetic modification of the Ad capsid has so far been limited to small peptides. A novel system for the attachment of targeting ligands to the Ad capsid, based on the extremely strong avidin-biotin interaction, is described herein. The genetic insertion of a biotin acceptor peptide (BAP) into the fiber, protein IX, or hexon components of the Ad capsid has resulted in vectors that are metabolically biotinylated upon production in host cells. Avidin-dependent redirection of transduction through a variety of biotinylated ligands is greatly dependent on the nature of the biotinylated capsid protein. While targeted transduction via the fiber was efficient through a broad array of ligand-receptor interactions, redirection of binding and uptake through the more abundant protein IX and hexon resulted in poor transduction. Although the basis of these differences has not been determined, it most likely reflects functional differences between the capsomeres during the process of vector uptake and trafficking. This study represents the first direct comparison of transduction through the various capsomeres and strongly suggests that future targeting efforts should be focused on fiber modification. In addition to the functional studies on Ad-IX-BAP, structural analysis by cryoelectron microscopy and particle reconstruction is presented. The C-terminal BAP fusion was used as a structural tag to visualize the position of IX within the capsid. Results contradict all previous reports on the location of IX and suggest the surface accessible density currently assigned as IIIa is actually attributable to protein IX. These studies highlight the need for a more thorough analysis of adenoviral structure and the complex interactions between its components.