Hammerhead Ribozyme-Mediated Reduction of Connective Tissue Growth Factor Expression and Action

Abstract

Abstract: : Purpose: To develop a hammerhead ribozyme to target and specifically cleave connective tissue growth factor (CTGF) mRNA to reduce expression in human embryonic fibroblasts. Methods: In vitro cleavage time course and multiturnover kinetic experiments were performed on synthetic ribozyme and ³²P-labeled target oligos specific for two regions of CTGF mRNA. Products were separated on a 19% PAGE gel followed by quantitation using phosphoimaging. The more efficient ribozyme was chosen to clone into an expression plasmid (pTR-UF21) for use in cell culture experiments and its effects were compared to a kinetically inactive ribozyme. Human embryonic fibroblasts were transfected with the appropriate plasmid using lipofection and selected with neomycin. The effects of transfection with kinetically active and inactive ribozyme on CTGF expression were measured. CTGF protein was measured using an ELISA and CTGF mRNA was measured using TaqMan quantitative RT-PCR. The effect of CTGF ribozyme on transforming growth factor beta- (TGF-ß) induced cell proliferation was also measured using a non-radioactive cell proliferation assay. Results: In vitro kinetics experiments for ribozyme #1 showed a Km of 1.56 µM and a Kcat of 2.97/min while ribozyme #2 had a Km of 7.80 µM and a Kcat of 5.7/min. The turnover numbers (Kcat/Km) of ribozyme #1 and #2 were 1.9 x10⁶ M/min and 7.4 x10⁵ M/min, respectively, thus ribozyme #1 is 2.6 times more efficient. Human embryonic fibroblasts transfected with the CTGF ribozyme #1 plasmid showed that CTGF protein was significantly reduced (by 72% in conditioned medium and 71% in the cytoplasm, p < 0.01, n = 6) compared with control groups. CTGF mRNA expression was also decreased by 55% in CTGF ribozyme #1-transfected cells (p < 0.01, n = 6) compared with control groups. Also, cells transfected with the CTGF ribozyme #1 showed a marked decrease (92%, p < 0.01, n = 6) in TGF-ß-induced cell proliferation. Conclusion: These results show that the CTGF ribozyme is efficient in reducing CTGF expression in cell culture. Also, the CTGF ribozyme has a physiological effect on cells in addition to decreasing CTGF mRNA and protein expression. This suggests that the CTGF ribozyme is suitable for treatment in animal models of scarring, such as photorefractive keratectomy (PRK) on rat corneas, where CTGF expression has been shown previously to be significantly elevated.

Keywords: gene transfer/gene therapy • growth factors/growth factor receptors • cornea: basic science