Selective delivery of virus vector systems to Fc-receptor positive cells : targeting, uptake, and vector-mediated gene delivery

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2007.The central goal of the studies presented in this Thesis was to explore Fc receptor targeting as an approach to enhancing virally-vectored gene transfer in mammalian cells. To approach this question, two sets of experiments were performed. First, studies were conducted to test the hypothesis that the efficiency of gene transfer to dendritic cells (DC) by recombinant adenoviral (Ad) vectors could be improved by targeting the vector to the high affinity Fc-gamma receptor I (FcγRI). The long-term goal of these experiments was to improve Ad-based vaccine delivery. To ‘retarget’ Ad vectors to FcγRI, a targeting complex consisting of a trimeric adenovirus fiber-binding moiety fused to a single-chain antibody specific for FcγRI was generated. Transduction studies revealed that FcγRI-targeted Ad transduced primary monocyte-derived DC with greater efficiency than unmodified Ad vectors. In addition, FcγRI-targeted Ad elicited more efficient activation of antigen-specific autologous memory CD8+ T-cells than unmodified Ad vectors. Thus, FcγRI-targeted Ad demonstrated enhanced antigen delivery to DC. A second experimental model system was also used to study effects of Fc receptor targeting on virally-vectors gene transfer. In this case, a bacterial virus vector (recombinant bacteriophage λ) was decorated with intact antibody molecules, and the resulting immune complexes were then used to study the molecular mechanisms involved in FcγRI internalization and intracellular routing following receptor ligation. Bacteriophage λ immune complexes were found to transduce FcγRI expressing mammalian cells with approximately 20-50 fold greater efficiency than unmodified vii bacteriophage λ particles. The ability of other Fc receptor subtypes to support gene transfer by bacteriophage λ immune complexes was also assessed, and FcγRI was found to confer the greatest permissivity to phage-mediated gene expression. Pharmacologic studies revealed that actin microfilaments, which are important for clathrin-mediated endocytosis, are essential for efficient gene expression, following addition of bacteriophage λ immune complexes to FcγRI positive cells. In contrast, endosomotropic and microtubule-disrupting agents increased bacteriophage λ- mediated gene expression. Overall, these studies suggest that bacteriophage λ immune complexes bind to FcγRI and then enter mammalian cells through clathrincoated pits; most of the incoming bacteriophage particles then appear to enter endosomes, with the majority of bacteriophage λ particles presumably becoming degraded in lysosomes. Overall, these studies show that Fc receptor targeting can enhance gene transfer efficiency in a wide range of mammalian cell types, by both bacterial and eukaryotic virus vectors. This suggests that Fc receptor targeting may be a broadly useful approach to improve virally-vectored gene transfer to FcR-positive host cells such as dendritic cells