In VivoAdenovirus-Mediated Gene Transfer of theEscherichia coliCytosine Deaminase Gene to Human Colon Carcinoma-Derived Tumors Induces Chemosensitivity to 5-Fluorocytosine

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Abstract
To evaluate the concept that in vivo transfer of the Escherichia coli cytosine deaminase gene will confer sensitivity of a solid tumor to the prodrug 5-fluorocytosine (5FC), we constructed an adenovirus vector (AdCMV.CD) carrying the cytosine deaminase gene driven by the cytomegalovirus (CMV) promoter, infected HT29 colon carcinoma cells in vitro and in vivo, and evaluated cell growth over time. AdCMV.CD produced a functional cytosine deaminase protein in HT29 cells in vitro as evidenced by the ability of lysates from the infected cells to convert [3H]5FC to its active metabolite 5-fluorouracil (5FU). The AdCMV.CD vector effectively suppressed HT29 cell growth in vitro in the presence of 5FC in a dose-dependent manner. Infection with AdCMV.CD, when as few as 10% of cells expressed the cytosine deaminase gene, was associated with a bystander effect when combined with 5FC in cell mixing studies. Further, this bystander effect was not dependent on cell-to-cell contact as demonstrated by suppression of [3H]thymidine incorporation in HT29 cells when supernatant from AdCMV.CD-infected cells treated with 5FC was transferred to uninfected cells. Consistent with these in vitro observations, when AdCMV.CD was directly injected into established subcutaneous HT29 tumors in nude mice receiving 5FC, there was a four-fold reduction in tumor size at day 15 compared to controls, and a five-fold reduction at day 28. These observations suggest that adenovirus-mediated gene transfer of the E. coli cytosine deaminase gene and concomitant administration of 5FC may have potential as a strategy for local control of the growth of tumor cells susceptible to 5FU. A replication deficient adenovirus vector was constructed to transfer the E. coli cytosine deaminase gene to human colon carcinoma cells in vitro and in vivo. Expression of this gene within target cells leads to a functional enzyme that confers sensitivity to the nontoxic antifungal compound 5-fluorocytosine (5FC) by converting it to the active chemotherapeutic agent 5-fluorouracil (5FU). Furthermore, cells expressing the gene, exposed to 5FC, liberated enough of the active product to suppress growth of surrounding, uninfected cells. This observed “bystander effect” is useful in vivo in circumstances where adenovirus-mediated gene transfer to an established tumor mass is not able to transduce 100% of cells comprising the tumor. This study shows adenovirus-mediated transfer of the cytosine deaminase gene to subcutaneous tumors in vivo, combined with systemically administered 5FC, is able to suppress tumor growth, a model that serves as a paradigm for use of this strategy in local control of cancers sensitive to 5FU.