Purchase this article with an account.
P. M. Kuznia, W. Hauswirth, A. Lewin, G. Schultz; Use of Gene Silencing Technologies, Ribozymes and Small Interfering RNAs, to Mediate Profibrotic Growth Factors. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3139.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Profibrotic growth factors, transforming growth factor- β1 (TGF-β1) and connective tissue growth factor (CTGF) stimulate the production of haze in the eye after infection, surgery and trauma. Currently, there is no clinically available agent to selectively reduce the activity of TGF- β1 or CTGF. In this study, we investigate the use of gene silencing technologies, ribozymes and siRNAs that target these profibrotic transcripts to reduce both mRNA levels and protein levels of these growth factors.
The hammerhead ribozymes were designed based on optimal nucleotide cleavage sites and predicted folding of mRNA target sequences and kinetic properties determined using in vitro cleavage of synthetic targets. Both hammerhead ribozymes were cloned into self- complementary adeno-associated viral (scAAV) expression plasmids. The TGF-β1 hammerhead ribozyme expression plasmid was transfected into HEK293 cells, and protein levels of TGFβ-1 in conditioned media were assessed using ELISA. The expression plasmid containing the CTGF ribozyme was packaged into AAV. The sc-AAV-CTGF-Rz was applied to rat corneas after ablation and mRNA levels were assessed at 0, 1, 2, 4, 7, and 14 days. At each time point one eye of the rat was given the scAAV ribozyme and the other eye received no treatment. Several siRNAs targeting TGF-β1 or CTGF were bought from Applied Biosystems. Targets for the siRNAs were ligated into a secreted alkaline phosphatase plasmid to test siRNA knockdown efficiency.
Transfection of the TGF- β1 ribozyme into HEK293 cells showed a significant (p<0.05) reduction of ~55% of relative TGF- β1 protein in the media as compared to control, GFP, and inactive cell media. Analysis of CTGF mRNA levels in control rat corneas compared to treated eyes showed a decrease in CTGF mRNA levels by 20% in treated eyes at 7 days post treatment. Target sequences for TGF β1 and CTGF have been cloned into the secreted alkaline phophatase plasmids.
These data indicate that topically applied ribozymes may be therapeutically useful in reducing levels of profibrotic growth factors. The development of the target secreted alkaline phosphatase plasmid will allow for in vitro analysis of the siRNAs without the need to upregulate or induce the endogenous genes. Simultaneous expression of a ribozyme and a siRNA to TGF- β1 and CTGF may work synergistically to reduce scarring.
This PDF is available to Subscribers Only