TITLE:
Reduction of corneal scarring in rabbits by targeting the TGFB1 pathway with a triple siRNA combination
AUTHORS:
Sriniwas Sriram, Daniel Gibson, Paulette Robinson, Sonal Tuli, Alfred S. Lewin, Gregory Schultz
KEYWORDS:
RNA Interference; siRNA Combination; Corneal Scarring; TGFB1; CTGF
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.4 No.10C,
October
18,
2013
ABSTRACT:
Purpose:
The transforming growth factor beta1 (TGFB1) pathway has been linked to
fibrosis in several tissues including skin, liver, kidney and the cornea. In this study, a RNA interference-based approach using siRNAs targeting three critical scarring genes, TGFB1, TGFB receptor 2
(TGFBR2) and connective tissue growth factor (CTGF), was tested for effects on
reducing alpha smooth muscle actin (SMA) and corneal scarring (haze) in
excimer laser ablated rabbit corneas. Methods:
Levels of TGFB1 and CTGF mRNAs were measured using qRT-PCR in the
epithelial and endothelial cell layers of normal and excimer ablated rabbit
corneas at 30 minutes, 1 day and 2 days after ablation. Two different scarring
models were utilized to assess the effects of the triple siRNA combination on
corneal scarring. In the first model, rabbit corneas were unevenly ablated
creating a mesh pattern then treated immediately with the triple siRNA combination. After 1 day the ablated areas of corneas were collected and levels of
mRNAs for TGFB1, TGFBR2 and CTGF were measured. After 14 days, levels of mRNA for SMA were measured and SMA protein immunolocalized
in frozen sections. In the second model, rabbit corneas were uniformly ablated
to a depth of 155 microns followed by three daily doses of the triple combination
of siRNA. After 14 days, corneas were photographed and images were analyzed
using Image J software to assess corneal
scarring. Corneas were also analyzed for levels of SMA mRNA. Results: In
both unwounded and wounded corneas, levels of TGFB1 and CTGF mRNA were always
significantly higher in endothelial cells than in epithelial cells (10 to 30
fold). Thirty minutes after injury, levels of both TGFB1 and CTGF mRNAs
increased approximately 20-fold in both epithelial and endothelial cells, and
further increased approximately 60-fold in 2 days. In the first therapeutic
experiment with a single siRNA dose, two of three rabbits showed substantial
reductions of all three target genes after 1 day with a maximum knock down of
80% of TGFb 1, 50% reduction of TGFBR2 and
40% reduction of CTGF mRNA levels and reduced SMA mRNA at day 14. In
the second therapeutic experiment with multiple doses of siRNA treatment, both
rabbits showed a ~22% reduction in scar formation at day 14 as calculated by
image analysis. There was also a corresponding 70% and 60% reduction of SMA RNA
expression. Conclusion: These results demonstrate that both TGFB1 and CTGF
dramatically increase in rabbit corneal epithelial and endothelial cells after
injury. Treatment of excimer ablated rabbit corneas with a triple combination
of siRNAs effectively reduced levels of
the target genes and SMA, leading to reduced corneal scarring at 14
days, suggesting that this triple siRNA
combination may be an effective new approach to reducing scarring in
cornea and other tissues.