Abstract
Purpose: :
Corneal scarring remains a serious complication that can ultimately lead to functional vision loss. It is regulated by transforming growth factor ß1 (TGF-ß1), which binds to the TGFß receptor (TGF-ßR2) and induces their downstream mediator, connective tissue growth factor (CTGF). The purpose of this study was to determine the efficacy of a combination siRNA treatment in targeting these three key regulators of corneal scarring.
Methods: :
Short interfering RNA (siRNA) sequences were transfected into cultured rabbit corneal fibroblasts using Mirus transfection reagent. Levels of mRNA and protein for the three target genes, collagen, and α- smooth muscle actin were measured using ELISAs and qRT-PCR. Due to the interdependent action of the growth factors, it was hypothesized that a combination of the most potent mRNAs for the 3 targets was the most effective treatment to reduce the fibrotic response. Accordingly, single, double and triple siRNA combinations were tested.
Results: :
Three to five different siRNAs for each target gene were tested and two with ≥80% reduction in protein and mRNA levels were identified for each gene. Combinations of the most effective siRNAs for two genes were then tested for gene knockdown. For example, TGF-ß1 siRNA-#1 and CTGF siRNA-#2 at a combined concentration of 30nM (15 nM each) gave a TGF-ß1 knockdown of ~62% and a CTGF knockdown of ~79%, compared with knockdown of ~30% and 39%, respectively, by the individual siRNAs. The degree of synergism among the different siRNA combinations was quantified using a numerical parameter called the Combined Index (CI) value. The best triple combination of siRNAs at a combined concentration of 30 nM (10 nM each) each gave 83.1% collagen knockdown compared to a scrambled control, compared with 62.8%, 70.4% and 42% knockdown by 30nM of each individual TGF-ß1, CTGF and TGF-ßR2 siRNAs, respectively. At 60nM total concentration, the triple combination gave 97% knockdown of collagen mRNA
Conclusions: :
The results demonstrate that a triple combination of siRNAs targeting TGF-ß1, TGF-ßR2, and CTGF knocked down expression of collagen type 1 mRNA more effectively than single or double combination of siRNAs.