Antisense RNA is a valuable, though often unpredictable, strategy for experimental gene inhibition as well as a potential rational approach to genetic therapeutics. We propose to investigate sophisticated chimeric antisense genes constructed by inserting antisense DNA fragments into structural RNA genes including snRNA and tRNA genes. The novel design of these antisense RNAs exploits the biological activity of both the snRNP and tRNA parent genes and has shown significant antisense inhibition activity in cultured cells and in transgenic mice. We will test enhanced designs of the chimeric genes including improvements in secondary structure, enhancer-like flanking sequences, enzymatic RNA motifs, Ul, and U7 snRNP genes. The details of the molecular activities of the chimeric antisense RNAs will initially be studied in Xenopus oocytes and cultured mouse cells. Transgenic mice containing chimeric antisense genes will be constructed as a model for anti-sense gene therapy because no culture system can adequately mimic the complexity of cell function within intact mammals. The anti-sense inserts will be designed to be complementary to both a readily assayed CAT gene and to oncogenic murine retroviruses. Transgenic mouse lines will be screened for the production of chimeric RNA and then challenged with a target CAT gene (by crossing with a CAT transgenic mouse). We will quantify and characterize the degree of inhibition using the ectopic CAT system and then test the capacity of these constructs to suppress retroviral proliferation and pathogenesis in cultured cells and in whole mice. These studies will provide a critical test of the prospects of antisense RNA gene therapy for the treatment of mammalian retroviruses such as MSV and HIV.
