Comparison of Gene Replacement Therapy With AdV–Rb and AdV–Rb94 to Suicide Gene Therapy With AdV–TK Plus Ganciclovir in a Murine Model of Retinoblastoma

Abstract

Abstract: : Purpose: To compare the effectiveness of gene replacement therapy using adenoviral vectors to deliver the Rb gene or a truncated Rb94 gene (one that expresses an Rb protein lacking the 112 N–terminal amino acids) with AdV–TK/ganciclovir suicide gene therapy. Methods: AdV–TK, AdV–Rb, or AdV–Rb94 (10–10⁶ i.u./ml) were added to cultures of Y79Rb retinoblastoma cells. Ganciclovir (10 µg/ml) was added to AdV–TK–containing cultures 18 hours later. After 6 days in culture, cell viability was assessed using alamar blue and/or trypan blue exclusion. A xenograft model of retinoblastoma was used to assess in vivo efficacy. Adenoviral vector constructs (10⁷ or 10⁸ i.u. in a volume of 1 µl) were injected into Rb tumors in the vitreous space. Ganciclovir was injected into the vitreous of animals receiving AdV–TK 1 and 4 days later. The eyes were examined histologically for tumor response on day 7. Results: AdV–Rb can express the Rb protein in Y79Rb cells in vitro. One order of magnitude more of either AdV–Rb or AdV–Rb94 than AdV–TK followed by ganciclovir is needed to kill Y79Rb cells. Transduction of ∼20% of Y79Rb cells with AdV–TK followed by ganciclovir results in close to 100% cell death. In vivo, while the higher dose of AdV–Rb or AdV–Rb94 (10⁸ i.u.) results in a measurable tumor response, the lower dose of either viral vector (10⁷ i.u.) does not have a measurable affect on the tumor. This observation contrasts to the effect of AdV–TK followed by ganciclovir where the lower dose of viral vector (10⁷ i.u.) can be used to eradicate retinoblastoma tumors. Conclusions: The difference between gene replacement therapy and suicide gene therapy appears to be the bystander effect elicited when phosphorylated ganciclovir diffuses to adjacent tumor cells when the TK–ganciclovir method is used.

Keywords: gene transfer/gene therapy • retinoblastoma • adenovirus