The discovery and characterization of oncogenes and tumor suppressor genes have provided excellent targets for the development of novel gene-directed antiproliferative agents. Antisense and anti-gene approaches have the goal of specifically inhibiting gene expression. During the previous funding period, the principal investigator of this application has demonstrated that the selective inhibition of expression of the c-myc gene by the GC-specific DNA binding drug mithramycin is caused by its ability to prevent the binding of specific regulatory proteins to the c-myc promoter. However, currently available DNA binding drugs are inadequately sequence specific to inhibit expression of single genes in a therapeutically useful manner. Triplex DNA provides such a sequence- specific approach. During the previous funding period, the applicant has identified triplex forming sequences in the promoters of the c-myc, c-Ha- Ras, c-Ki-Ras, and cyclin D genes. Triplex based DNA binding compounds have been developed which interact with the c-myc, N-Ras, and cyclin D1 promoters in a sequence-specific manner and which inhibit c- myc transcription in vitro and in vivo. In addition, a cDNA partially complementary to a putative triplex forming transcript targeted to the c-myc promoter has been cloned and partially sequenced. Thus, the overall goal of the application is to fully characterize the antiproliferative effects of a series of c-myc targeted agents and to develop them into therapeutically useful anti-leukemic compounds.
The specific aims i nclude: 1. To characterize the ability of oligonucleotide-drug conjugates targeted to the c-myc P1 and P2 promoters to form triplex and inhibit c-myc transcription by leukemic cells in vivo. 2. To characterize the potential antiproliferative synergy of simultaneous inhibition of c-myc and cyclin D1. 3. To determine the ability of c-myc, cyclin D1, and N-Ras targeted phosphorothioate triplex forming oligonucleotides (TFO) and oligonucleotide-drug conjugates to inhibit proliferation of human and murine leukemic cells in vivo and to investigate the potential synergistic effects of DNA binding drugs. 4. To develop adenovirus/polylysine and liposome systems to enhance nuclear delivery of c-myc targeted oligonucleotide in animal systems. 5. To characterize the biological significance and therapeutic potential of naturally occurring triplex forming RNA molecules which are complementary to the c-myc promoter.
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