OLIGONUCLEOTIDE INHIBITION OF CELL PROLIFERATION

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The goal of this work is to develop novel, gene-specific DNA therapeutics for treatment of solid tumors induced by activated C-myc oncogene in E(mu)-myc transgenic mice, an animal model for B-cell lymphoma. The central hypothesis is that some combination of DNA backbone modifications will yield maximum potency and specificity. Antisense or antigene DNA efficacy against oncogenes and viral genes has been observed in cell culture and in animals in this laboratory and others, in a dose dependent and sequence specific manner. Progress to date indicates that antisense DNA inhibits c-myc expression in B-cells of E(mu)-myc transgenic mice, and prophylactic antisense DNA therapy inhibits congenital tumorigenesis in this line. Three sensitive c-myc mRNA targets are known: the 5' P1 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'-dCAC GTT GAG GGG CAT]; two sensitive DNA targets are known upstream of Pi: the putative antiparallel triplex site [5'-dTGG GTG GGG TGG GTG], and the overlapping putative H-DNA site [5'-dACC CTC CCC ACC CTC]. In this proposal, stereoregular all-R methylphosphonate and all-S phosphorothioate DNAs will be synthesized. The stereoregular oligomers will be more potent because they basepair much more tightly than the racemic oligomers. Scrambled, sense, and wild type controls will also be synthesized, as well as shorter oligomers. Later in the project, chimeras with 2-3 central phosphodiester residues and 10-13 outlying stereoregular methylphosphonate or 2'-O-methyl methylphosphonate residues will be synthesized. The different derivatives will be administered by micro- osmotic pumps, and tested for their relative ability to inhibit tumor growth in E(mu)-myc transgenic mice, both prophylactically before tumor onset, and therapeutically after tumor onset. The rates of tumor onset and progression will be measured, and transformed cells from treated and control animals will be analyzed for c-myc mRNA and MYC p65 protein expression, proliferation, and differentiation characteristics. Dose dependence, sequence specificity, length dependence, and time course of inhibition will be determined for each backbone derivative. Pharmacokinetics of the oligomers will be measured in serum, urine, and feces. Where anti-c-myc therapy fails to ablate tumors, biopsy samples will be screened to identify the subsequent activated oncogenes, which will then be attacked with new antisense DNAs. Cells from antisense- regressed tumors will be tested for tumorigenicity in order to determine whether the treated tumor cells underwent terminal differentiation as a result of DNA therapy, or constitute residual disease. These results should allow convergence of antisense DNAs to an optimum combination of derivatization, length, and sequence which will be suitable for translation to clinical trials.