The hypothesis is that the scarring which occurs following trabeculectomy is primarily controlled by transforming growth factor beta (TGF-b) that is produced by cells in the area of the trabeculectomy. Thus, if the amount of TGF-b protein released in the trabeculectomy wound area can be limited, the amount of scarring would be reduced while causing minimal side effects. To accomplish this goal, plasmids will be synthesized that transcribe ribozymes which will selectively destroy TGF-b mRNA. The plasmid will be transiently transfected into cells of the trabeculectomy wound at the time of surgery using either liposome or adenovirus vectors. Long-term inhibition of TGF-b action would not be required to prevent bleb-scarring since bleb-failure is initiated during the first two-week post-operative period.
We have begun development of ribozymes specific for TGF-b1, TGF-b2 and the type II TGF-b receptor, have synthesized plasmids which contain autocatalytic processing hairpin ribozymes and are currently evaluating their ability to cleave TGF-b and receptor mRNAs in vitro. We will continue to develop and refine the ribozymes for TGF-b1, TGF-b2 and type II TGF-b receptor. This will involve construction of plasmids which contain hairpin and hammerhead ribozymes. The ribozymes will be tested for catalytic activity both in vitro and in vivo. After the ribozymes have been optimized for maximum effectiveness, we would test them in patients at high risk for trabeculectomy failure. Initially we expect to utilize an expression plasmid with a strong promoter for human cells such as SV40 or CMV. Production of GMP grade cationic liposome or adenovirus victors for humans would require the use of the special gene therapy facilities of the HAL. Administration of the ribozymes and monitoring of the patients would require the use of the OCI and GCRC outpatient facilities.
- PT Khaw, NL Occleston, G Schultz, I Grierson, MB Sherwood, G. Larkin. Activation and suppression of fibroblast function. Eye 8:188-195, 1994.
- PT Khaw, NL Occleston, K Sethl, RA Alexander, GS Schultz, I Grierson. The effects of growth factors (EGF, bFGF, TGF-b1, and IGF-1) on cellular and molecular aspects of ocular fibroblast wound healing behavior In Vitro. European Tissue Repair Meeting, August 1994.
- R Tarnuzzer, R Stiff, GS Schultz. Development of hairpin ribozymes for TGF-b1, TGF-b2, and type II receptor with defined 3’termini by autocatalytic processing. J Cellular Biochem 19A:A6-319, 1995.