Evaluation of polyethylene glycol modified adenovirus for innate response reduction and ligand specific cell targeting
Mok, Hoyin Sunny
Barry, Michael A.
Doctor of Philosophy
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.