Development of a rapid method for the PEGylation of adenoviruses with enhanced transduction and improved stability under harsh storage conditions.

PEGylation is the covalent attachment of activated monomethoxy poly(ethylene) glycols (MPEGs) to free lysine groups of therapeutic proteins. This technology has enhanced the physical stability of proteins and ablated humoral immune responses generated against them. In this study, adenoviral vectors were modified with MPEGs activated by cyanuric chloride, succinimidyl succinate, and tresyl chloride. Under proper buffering conditions, reactions were complete within 2 hr. Transduction efficiency of PEGylated adenoviruses was not compromised by neutralizing antibodies to native adenovirus in vitro. These preparations retained titers that were significantly greater than those of the unconjugated virus after storage at 42, 25, 4, and -20 degrees C. Stability profiles of PEGylated preparations at -20 degrees C suggest that glycerol could be eliminated from formulations without significant loss of viral titer. PEGylated adenoviruses produced a two- to threefold increase in transduction in the lung when administered by intratracheal injection and a fivefold increase in transduction in the liver when administered intravenously.