Hammerhead Ribozyme-Mediated Knockdown of mRNA for Fibrotic Growth Factors: Transforming Growth Factor-Beta 1 and Connective Tissue Growth Factor
Excessive scarring (fibrosis) is a major cause of pathologies in multiple tissues, including lung, liver, kidney, heart, cornea, and skin. The transforming growth factor-β (TGF-β) system has been shown to play a key role in regulating the formation of scar tissue throughout the body. Furthermore, connective tissue growth factor (CTGF) has been shown to mediate most of the fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. Currently, no approved drugs selectively and specifically regulate scar formation. Thus, there is a need for a drug that selectively targets the TGF-β cascade at the molecular level and has minimal off-target side effects. This chapter focuses on the design of hammerhead ribozymes, measurement of kinetic activity, and assessment of knockdown mRNAs of TGF-β and CTGF in cell cultures.
Key wordsRibozymes TGF-β CTGF Scar formation Transduction Oligonucleotides
- 5.Lee, P.A., Blatt, L.M., Blanchard, K.S., Bouhana, K.S., Pavco, P.A., Bellon, L., Sandberg, J.A. (2000) Pharmacokinetics and tissue distribution of a ribozyme directed against hepatitis C virus RNA following subcutaneous or intravenous administration in mice. Hepatology 32: 640–646.PubMedCrossRefGoogle Scholar
- 9.Heid, C.A., Stevens, J., Livak, K.J., Williams, D.M. (1996) Real time quantitative PCR. Genome Res. 10: 984–986.Google Scholar
- 10.Blalock, T.D., Duncan, M.R., Varela, J.C., Goldstein, M.H., Tuli, S.S., Grotendorst, G.R., Schultz, G.S. (2003) Connective tissue growth factor expression and action in human corneal fibroblast cultures and rat corneas after photorefractive keratectomy. Invest. Ophthalmol. Vis. Sci. 44: 1879–1887.PubMedCrossRefGoogle Scholar
- 11.Tamatani, T., Kobayashi, H., Tezuka, K., Sakamoto, S., Suzuki, K., Nakanishi, T., Takigawa, M., Miyano, T. (1998) Establishment of the enzyme-linked immunosorbent assay for connective tissue growth factor (CTGF) and its detection in the sera of biliary atresia. Biochem. Biophys. Res. Commun. 251: 748–752.PubMedCrossRefGoogle Scholar