A major clinical problem that occurs frequently after excimer PRK is the development of subepithelial haze or stromal scar. Histologically, the scar appears to be composed primarily of collagen types I and III and proteoglycans that presumably are synthesized by stromal fibroblasts s and epithelial cells. No treatment has been proven to be clinically effective for reducing subepithelial haze although long term topical corticosteroid treatment is frequently used in spite of the risks of glaucoma and secondary cataracts. Thus, there is a need for treatments that will selectively and safely reduce scar formation following PRK. Based on the PI initial experimental results with excimer PRK ablated rat corneas, he hypothesize that stromal scarring following PRK is stimulated predominately by the TGF-B system. This suggest that the TGF-B system is a prime target for treatments to reduce corneal scarring. To test this hypothesis, Aim number 1 will measure the levels of mRNAs and proteins for the TGF-B isoforms and TGF-B IIR along with important components of the ECM in rat corneas at key times following PRK using competition-based RT-PCR and ELISAs. These data will determine the time frame in which the TGF-B system is most active in rat corneas after PRK and thus will help in the design of recombinant adeno-associated virus (rAAV)-antisense vectors that will be developed in Aim number 3 to selective reduce TGF-B II receptor synthesis.
Aim number 2 will use in situ hybridization and immunohistochemistry to identify corneal cells synthesizing ECM scar proteins in response to activation by the TGF-B system. These data will indicated which corneal cells are targets for transduction by the rAAV-antisense vectors as described in the in vivo experiments in Aim number 4.
Aim number 3 will synthesized different rAAV-vectors expressing antisense RNA directed against TGF-B IIR mRNA and will evaluate the effectiveness of the vectors in reducing levels of TGF-B IIR mRNA and protein in cultures of transfected corneal fibroblasts.
Specific Aim number 4 will perform in vivo eperiments on excimer PRK ablated rat corneas. rAAV-antisense vectors containing optimal promoter and antisense sequences developed in specific Aim number 3 will be used to transduce rat corneal cells following PRK. The effects of the rAAV-antisense transfection on expression of TGF-B type II receptor mRNA and protein will be assessed together with the effects on transfection on expression of TGF-B type II receptor mRNA and protein will be assessed together with effects on formation of corneal haze in the rats. Ribozymes have the potential to be more effective than antisense RNA. Following the development of the rAAV-antisense plasmid, we will synthesize rAAV-ribozyme vectors that are targeted to cut sequences for TGF-B type II receptor mRNA and evaluate their effectiveness in reducing expression of TGF-B system in regulating stromal scarring and will develop new approaches for prevention of corneal haze following PRK ablations.
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