OLIGODEOXYNUCLEOTIDE INHIBITION OF CELL PROLIFERATION

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The goal of this work is to test novel, gene-specific DNA therapeutics for treatment of human solid tumors and leukemias induced by activated c-myc oncogene in two animal models. The central hypothesis is that some combination of backbone and terminal modification will yield maximum potency and specificity. Antisense or antigene DNA efficacy against oncogenes and viral genes has been observed in cell culture in this laboratory and others, in a dose dependent and sequence specific manner. Preliminary results indicate that antisense DNA methylphosphonates inhibit c-myc expression in B-cells of c-myc transgenic mice, and that normal antisense DNA inhibits tumorigenesis by pretreated c-Ha-ras-transformed NIH3T3 cells in athymic nude mice. In this proposal, normal, phosphorothioate, and methylphosphonate DNAs, with and without 5' dimethoxytrityl and/or 3' cholesteryl derivatization to accelerate cellular uptake and slow excretion, will be synthesized. Three sensitive c-myc mRNA targets are known: the 5' Pl transcription start site [5'-dGCA CAG CTC GGG GGT], the first intron/second exon junction [5'-dGGC TGC TGG AGC GGG], and the initiation codon loop [5'-dAAC GTT GAG GGG CAT]; two sensitive DNA targets are known upstream of Pl: the putative antiparallel triplex site [5'-dTGG GTG GGG TGG GTG], and the overlapping putative H-DNA site [5'-dACC CTC CCC ACC CTC]. Scrambled, sense, and wild type controls will also be synthesized, as well as shorter oligomers. The different derivatives will then be tested for their relative ability to inhibit tumor growth in c-myc-transgenic mice, an animal model for B-cell lymphoma, and in C57BL/Balb/c athymic nude mice implanted subcutaneously with human promyelocytic HL-60 cells. Transformed cells from treated and control animals will be analyzed for c-myc p65 expression, proliferation, and differentiation, and rate of tumor growth will be measured. Sequence specificity, length dependence, dose dependence, and time course of inhibition will be determined for each derivative. Three regimens of administration will be studied: daily via intraperitoneal bolus, continuous infusion via subcutaneous Alza pump, and daily via intravenous liposomes. Pharmacokinetics of the oligomers will be measured in serum, urine, and feces. In the event that tumors are significantly reduced in size and growth rate by antisense or antigene treatment, cells from treated tumors will be tested for tumorigenicity in order to determine whether the treated tumor cells underwent terminal differentiation as a result of DNA therapy. The interrelationship of c-myc expression with MHC class I antigens will be studied relative to immune surveillance and cytotoxic T lymphocyte sensitivity. These results should allow convergence of the candidate gene-specific DNAs to an optimum combination of derivatization, length, and sequence which will be suitable for preclinical trials.