title: In vivo generation of highly abundant sequence-specific oligonucleotides for antisense and triplex gene regulation.
abstract: Antisense and triplex oligonucleotides continue to demonstrate potential as mediators of gene-specific repression of protein synthesis. However, inefficient and heterogeneous cellular uptake, intracellular sequestration, and rapid intracellular and extracellular degradation represent obstacles to their eventual clinical utility. Efficient cellular delivery of targeted ribozymes can present similar problems. In this report we describe a system for circumventing these obstacles and producing 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 synthetic sequence incorporating the oligonucleotide of interest. In vivo, these oligonucleotides are produced constitutively and without cell type specificity in levels up to 5 x 10(6) copies per cell, reach steady-state levels of expression within 9 hours post-transfection, and are still readily detectable 7 days post-transfection. In addition, these oligonucleotides are retained in the nucleus, obtain a 5' gamma-monomethyl phosphate cap, and have an intracellular half-life of approximately one hour. This expression vector provides a novel and efficient method of intracellular delivery of antisense or triplex RNA oligonucleotides (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.
authors.affiliation: Bioengineering Graduate Group, University of California, San Francisco 94143.
authors.forename: S B
authors.initials: SB
authors.lastname: Noonberg
authors.forename: G K
authors.initials: GK
authors.lastname: Scott
authors.forename: M R
authors.initials: MR
authors.lastname: Garovoy
authors.forename: C C
authors.initials: CC
authors.lastname: Benz
authors.forename: C A
authors.initials: CA
authors.lastname: Hunt
mesh_terms: D001483:Base Sequence; D001943:Breast Neoplasms; D002460:Cell Line; D002678:Chimera; D003001:Cloning, Molecular; D003609:Dactinomycin / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000494:pharmacology*; D012342:RNA, Small Nuclear / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D012342:RNA, Small Nuclear; D003609:Dactinomycin