REGULATION OF OUTFLOW FACILITY BY GENE TRANSFER

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DESCRIPTION: One effective means of judging the contribution of a protein to cellular function is to modulate its expression by introducing into the cell nucleic acid, sequences that are identical (sense) or complementary (antisense) to the mRNA sequence that encodes the target protein. The PI hypothesis is that expression of genes involved in cytoskeleton dynamics in cells of the outflow pathway can influence aqueous humor outflow facility; that manipulation of the concentration of the proteins coded by these genes will alter outflow facility; that replication deficient adenovirus vectors will successfully deliver genes to the outflow pathway cells; and that the rigorous evaluation of the efficacy and toxicity of this delivery system will advance the potential development of ocular gene therapy for glaucoma. Because of the increase in outflow facility observed both in vivo and in vitro after treatment with microtubule depolymerization drugs, we wish to initially investigate B-tubulins, microtubule-associated proteins and the transcription factor NFkB. The PI will characterize which isoforms from these gene families are expressed in the trabecular meshwork. She will employ adenovirus transfer technology to overexpress and underexpress their specific regions in organ culture perfused porcine and human eyes. She will measure outflow facility. Preliminary results indicate that replication-deficient adenoviruses are likely ideal vectors to investigate our hypotheses. They enter the cells of the trabecular meshwork very efficiently and, by themselves, do not interfere with the cytoskeleton network nor with the rate of outflow. Even if these initial manipulations do not alter aqueous outflow, this investigation should still contribute valuable insight about cellular mechanisms involved in aqueous humor outflow. Furthermore, the complete development of this system, coupling molecular biology with physiological measurements, will allow testing of future multiple hypotheses involving proteins/enzymes (receptors, growth factors, ion channels, kinases...) that have been conceptualized as being important in aqueous humor physiology.