We have developed a novel antisense technology based on catalytic RNA which can potentially be applied to human therapeutics. These catalytic RNA molecules, called ATR (antisense/triplex/ribozyme), hybridize with specific target mRNAs in mammalian cells by base-pairing interactions. Alteration in the structure of the ATR RNA upon hybridization then induces it to circularize around the target mRNA, forming a topologically linked strong complex. Strong complexes between ATR and target mRNAs cannot be displaced by scanning or translocating ribosomes in cell free systems. ATR RNA can be delivered to mammalian cells complexed with cationic lipids. ATR molecules which were designed to bind specifically to mouse TNFalpha mRNA inhibited TNFalpha protein production by 90% in the mouse macrophage-like cell line RAW264.7, with an IC-50 of 41 nM. Following intraperitoneal administration to mice, specific anti-TNFalpha ATR-lipid complexes inhibited TNFalpha secretion by peritoneal macrophages by 50% relative to an ATR molecule targeted to a heterologous mRNA. ATR molecules require only native ribonucleosides for activity, and therefore may potentially be produced inexpensively by fermentation in E. coli or isolation from tobacco. They may also be expressed in situ from gene therapy vectors. We now intend to apply ATR technology and more sophisticated nucleic acid drug delivery techniques to the development of a new therapeutic for the treatment of rheumatoid arthritis. This therapy will be based on delivery of anti-TNFalpha ATR RNA or gene therapy vectors to primary macrophages and type A synoviocytes, which will inhibit the production of this key mediator of chronic inflammation at the site of disease in patients with rheumatoid arthritis.
Inappropriate expression of TNFalpha has been implicated in the pathogenesis of several important human diseases, including septic shock, insulin dependent diabetes, and rheumatoid arthritis. The combined incidence of these three diseases represents approximately 4.3 million people in the United States afflicted with diseases for which there is no cure and for which current treatments are inadequate. The incidence of rheumatoid arthritis alone is 1.44%. The combined expenditures for drugs to treat were approximately $9.7 billion (b)in 1996, with non- steroidal anti-inflammatory drugs for arthritis treatment contributing $7b.
| Dallas, Anne; Balatskaya, Svetlana V; Kuo, Tai-Chih et al. (2008) Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos. Nucleic Acids Res 36:6752-66 |
