871. Surface Modification of Adenovectors with TAT- and Antp-Containing Polymers Protects Them from Neutralizing Antibodies and Increases Expression of Reporter Genes In Vitro and In Vivo

Abstract

The relative inefficiency of adenovirus (Ad)-based transduction vectors in cardiovascular tissues is related to the low expression of the CAR receptor on the surface of endothelial and smooth muscle cells as well as to the rapid neutralization of Ad by anti-Ad antibodies. Polymer modification of Ad surface potentially addresses these problems by masking Ad particles from neutralizing antibodies and providing a convenient platform for the conjugation of ligands possessing transduction-facilitating properties, such as TAT and Antp peptides. Our present studies investigated the hypothesis that by surface modifying Ad using photoreactive polymers to attach either TAT or Antp increased transduction could be achieved while reducing susceptibility to neutralizing antibodies. This was studied with reporter gene transduction with surface-modified Ad vectors in cell culture and in vivo, in rat carotid arteries. A series of fluorescently labeled polyallylamine (PAA)-based photochemically activatable (either via benzophenone, or azide chemistry) thiol reactive linear polymers were custom synthesized and conjugated with TAT-SH or Antp-SH. Ad-GFP or Ad-Luc were suspended in PBS x5 and admixed with either TAT-, Antp-derivatized or unmodified polymers to obtain the final polymer/Ad ratios ranging from 1:60 to 1:10 (w/w). Cellular uptake of vectors and ensuing transgene expression in rat aortic smooth muscle cell line (A10), primary bovine aortic endothelial cells (BAEC) and human alveolar epithelial cell line (A549) were examined by the fluorescence microscopy and fluorometry. Additionally, naïve and surface- modified Ad vectors were exposed to escalating concentrations of neutralizing anti-knob antibodies (50 to 500 nM) prior to the cell infection. 6x108 pfu of either naïve or polymeric TAT-conjugated Ad-Luc were locally delivered to the balloon-injured common carotid arteries of SD rats (n=6). The transgene expression was assessed 2 days after gene delivery by bioluminescence imaging. Model experiments have shown faster kinetics of photochemical polymer attachment to the capsid proteins with azide-compared to benzophenone-containing polymers. The surface modification of Ad-GFP with TAT- and Antp-derivatized polymers resulted in 3–10-fold increase of GFP expression in the studied cell types. 15 min incubation of naïve Ad with neutralizing antibodies resulted in a 94% decrease of GFP expression in A10 cells, while the surface modification of Ad with TAT- and Antp-containing polymers bestowed protection against neutralizing antibodies in proportion with the extent of surface modification (59%, 14% and 6% inhibition for the polymer/Ad ratios of 1:60, 1:30 and 1:10, respectively). In the initial in vivo experiments local delivery of polymeric TAT- conjugated Ad resulted in 4.4-fold enhancement of luciferase activity in comparison with unmodified Ad. In conclusion, light-induced covalent conjugation of TAT- and Antp-modified PAA-based polymers to Ad capsid proteins protects adenovectors from preformed neutralizing antibodies and significantly augments gene transduction both in vitro and in vivo.