IN-VIVO GENERATION OF HIGHLY ABUNDANT SEQUENCE-SPECIFIC OLIGONUCLEOTIDES FOR ANTISENSE AND TRIPLER GENE-REGULATION

Antisense and tripler oligonucleotides continue to demonstrate potenti al as mediators of gene-specific repression of protein synthesis. Howe ver, inefficient and heterogeneous cellular uptake, intracellular sequ estration, and rapid intracellular and extracellular degradation repre sent obstacles to their eventual clinical utility. Efficient cellular delivery of targeted ribozymes can present similar problems. In this r eport we describe a system for circumventing these obstacles and produ cing large quantities of short, sequence-specific RNA oligonucleotides for use in these gene regulation strategies. The oligonucleotides are generated from a vector containing promoter, capping, and termination sequences from the human small nuclear U6 gene, surrounding a synthet ic sequence incorporating the oligonucleotide of interest. In vivo, th ese oligonucleotides are produced constitutively and without cell type specificity in levels up to 5 x 10(6) copies per cell, reach steady-s tate levels of expression within 9 hours post-transfection, and are st ill readily detectable 7 days post-transfection. In addition, these ol igonucleotides are retained in the nucleus, obtain a 5' gamma-monometh yl phosphate cap, and have an intracellular half-life of approximately one hour. This expression vector provides a novel and efficient metho d of intracellular delivery of antisense or tripler RNA oligonucleotid es (and/or ribozymes) for gene regulation, as well as a cost-effective means of comparing the biological activity arising from a variety of different potential oligonucleotide sequences.