EVALUATION OF GENE TRANSFER EFFICIENCY BY VIRAL VECTORS TO MURINE BLADDER EPITHELIUM

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Abstract
Purpose: In pre-clinical gene therapy studies of bladder cancer there is tremendous variation in the ability of viral vectors to deliver genetic material to bladder epithelium. Possible explanations for this variability may involve the physical parameters of delivering vectors in these experimental models. We examined the effects of intravesical volume and pressure during instillation as well as chemical modification of the bladder epithelium on subsequent gene expression in the bladder in mice. Materials and Methods: Female C57B1/6 mice underwent intravesical instillation of the replication restricted canarypox virus (ALVAC) recombinant for the reporter genes luciferase or β-galactosidase. Similar viral titers were instilled at different volumes and a pressure transducer measured intravesical pressure when the vector was instilled. Also, various agents, including 0.6 N hydrochloric acid, 0.4% oxychlorosene, poly-L-lysine and 0.25 M. ammonium chloride, were used to modify the bladder surface before vector instillation and then assayed for transgene expression. Results: As expected, maximum intravesical pressure measured during instillation was significantly greater in mice instilled with a higher volume (33.1 versus 9.8 mm. Hg). Significantly more gene expression was detected in bladders instilled with a higher volume of viral vectors (p <0.05). Likewise, higher instillation pressures resulted in higher transgene expression in distant organs. Modification of the bladder epithelium with agents such as oxychlorosene and poly-L-lysine resulted in elevated gene expression with only minimal increases in systemic activity. Conclusions: Significant differences in gene expression are achieved by varying physical parameters during intravesical instillation. Increased gene expression associated with larger volume instillation may be responsible for some reported variability of gene transfer to the bladder. Alternate manipulations, such as modifying the bladder surface, may be done to enhance gene transfer to the urothelium without increasing systemic distribution.