Figure 6.

Poly(maleic anhydrides): amphiphilic compounds that improve adenovirus transduction efficiency with minimal toxicity. A series of zwitterionic polymers of varying size were screened for their ability to improve the transduction efficiency of recombinant adenoviruses in lung epithelial cells. Initial screening of formulations in vitro and in vivo was performed with AdlacZ containing the beta-galactosidase transgene (panels A and D). Use of an E1/E3 deleted recombinant adenovirus expressing green fluorescent protein (AdGFP) and quantitation of infected cells by flow cytometry enhanced sensitivity of the screening assay so that subtle differences in transduction efficiency in the presence of anti-adenovirus neutralizing antibodies could be detected (panel C). (A) Transduction efficiency of formulated AdlacZ in the presence of neutralizing antibody. Formulations containing 1 × 10⁸ infectious particles of AdlacZ were incubated with aliquots of a highly characterized neutralizing antibody stock for 1 h prior to infection of Calu-3 cells. Forty-eight hours later, beta-galactosidase positive cells were identified by histochemical staining. The number of infectious virus particles was tallied and calculated as described previously.¹⁸ (B) Toxicity profile of F16. Formulations were placed on differentiated Calu-3 cell monolayers for a period of 2 h. Culture medium was then assessed for LDH activity. Lysis buffer served as a positive control (100% lysis) and KPBS as a negative control. (C) Quantitative assessment of transduction efficiency of formulated AdGFP over a range of neutralizing antibody concentrations. In this experiment, 1 × 10⁸ infectious particles of AdGFP were incubated in solution containing concentrations of anti-adenovirus antibody reflective of that found in the global population^28,41 as described in panel A. Twenty-four hours after infection, infected cells, positive for GFP, were counted by flow cytometery. (D) Histological evaluation of transgene expression in the lung 4 days after intranasal administration of formulated virus. A single dose of 5 × 10¹⁰ infectious particles of AdlacZ was given to naive mice or mice with PEI to adenovirus induced by the intranasal route. Four days later, mice were sacrificed, and tissue was harvested and stained for transgene expression. Sections illustrate representative transgene expression patterns found in tissue collected from 6 animals per treatment. Magnification for unformulated panels: 200×. Magnification for F16 panels: 400×. Results in panels A–C are reported as the mean ± standard error of the mean of data generated from triplicate samples collected from four separate experiments.