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Calcitonin-Specific Transcription and Splicing Targets Gene-Directed Enzyme Prodrug Therapy to Medullary Thyroid Carcinoma Cells

Department of Cancer Genetics (M.M., D.M.T.Y., B.G.R.), Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, New South Wales, Australia 2065; Department of Medicine (B.G.R.), University of Sydney, Sydney, Australia; and Commonwealth Scientific and Industrial Research Organization Molecular Science (G.W.B., P.L.M.), Sydney, New South Wales, Australia 1670

Address all correspondence and requests for reprints to: Prof. Bruce G. Robinson, Cancer Gentics Unit, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales, Australia 2065. E-mail: bgr{at}med.usyd.edu.au.

Recurrent and metastatic medullary thyroid carcinoma (MTC) remains difficult to treat due to its limited responsiveness to chemotherapy, radiotherapy, and imaging. To investigate an alternative therapeutic approach, we examined the feasibility of targeting gene-directed enzyme/prodrug therapy delivered by adenoviral vectors to MTC. We previously described a modified human calcitonin (CT)/CT gene-related peptide promoter that produced increased expression while maintaining specificity for MTC cells. In this study, we introduced an additional level of specificity by using cell-specific splicing and examined whether the selectivity of the gene-directed enzyme/prodrug therapy for MTC was enhanced when both the promoter and splicing features were combined in a single transcription unit. Two replication-defective adenoviruses were constructed that expressed the Escherichia coli purine nucleoside phosphorylase (PNP) gene under the transcriptional control of a modified T2 promoter (Ad.T2-PNP) or the T2 promoter in combination with a CT minigene cassette in which the PNP gene was imbedded within the CT gene exon 4 (Ad.T2-CT/PNP). The specificity of PNP expression by Ad.T2-PNP, Ad.T2-CT/PNP, and control viruses in the MTC cell line, TT, and in a panel of non-MTC cell lines was evaluated. The highest level of PNP gene expression and the most effective cell killing in the presence of prodrug occurred in TT cells infected with Ad.T2-PNP, followed by Ad.T2-CT/PNP. Infection of most non-MTC cell lines, even with high multiplicities of Ad.T2-PNP, produced only low-level PNP expression that resulted in minimal cell killing in the presence of prodrug. High-level expression of PNP and effective cell killing was observed with both adenoviral gene constructs. The highest level of cell specificity was achieved with the combined use of promoter and splicing regulation in the Ad.T2-CT/PNP virus.

This study was supported by grants from the University of Sydney Cancer Research Fund and The Elizabeth Rosenthal Trust.

Abbreviations: CGRP, CT gene-related peptide; CT, calcitonin; CT/PNP, CT minigene with PNP gene; GDEPT, gene-directed enzyme prodrug therapy; 6-MeP, 6-methylpurine; MOI, multiplicity of infection; 6-MPDR, 6-methylpurine-2-deoxyriboside; MTC, medullary thyroid carcinoma; pA, polyadenylation; PNP, purine nucleoside phosphorylase; RSV.LTR, rous sarcoma virus long terminal repeat; T2, modified CT/CGRP promoter containing tandem dual TSE; TCID₅₀, 50% tissue culture infectious dose; TSE, tissue-specific enhancer.

Plasmids: pCT3.KS, pBluescript vector containing CT exon 3 sequence; pCT/mg, modified pGEM4Z vector containing CT minigene; pRSV.CAT, pBluescript vector containing rous sarcoma virus promoter driving the chloramphenicol acetyl transferase gene; pXCX3, shuttle plasmid containing the left end of adenoviral genome; pJM17, plasmid containing the adenoviral genome; TSE2.CP1.GL3, Luciferase vector containing the modified CT/CGRP promoter, adenoviral constructs; Ad.R-PNP, adenovirus containing PNP gene driven by rous sarcoma virus promoter; Ad.R-CT/PNP, adenovirus containing CT/PNP minigene driven by rous sarcoma virus promoter; Ad.T2-PNP, adenovirus containing PNP gene driven by CT/CGRP modified promoter (T2); and Ad.T2-CT/PNP, adenovirus containing CT/PNP minigene CT/CGRP modified promoter (T2).

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