Regulation of gene expression for therapy of age related macular degeneration

Drug inducible gene modulation technology allows for an ideal therapy for treating neovascular ocular diseases. The dose-dependent and temporal properties inherent in these drug inducible systems substantiate their suitability for use in clinical applications. A major limitation of this system is "leaky" gene expression during non-induced states. Recent advances have established better inducible systems that improve upon this concern. My goal is to assess neovascular abatement in a mouse laser CNV model while utilizing the inducible hammerhead ribozyme system (riboswitch) in order to regulate the expression of an antineovascularity gene.;HEK 293 cells were transfected with either a construct containing the double riboswitch, which included two inducible ribozymes in tandem-orientation, or a single riboswitch construct regulating GFP and sFlt-01 transgene expression. Cells transfected with the ribozyme constructs were induced with a cocktail of Toyocamycin plus 5-fluorouridine. The single riboswitch construct exhibited a 2.8 fold higher level of GFP expression than the double riboswitch construct. However, leaky expression of the sFlt-01 transgene by the single riboswitch plasmid was 7.3 fold higher than that of the double riboswitch plasmid. Adult C57BL6 mice were intravitreally injected within the right eye with AAV2 virus vectors containing the double riboswitch system regulating the antineovascularity gene sFlt-01 six weeks prior to implementation of the laser CNV model. One day before retinal laser burns, animals were implanted with time release pellets containing the inducer agents. Neovascular growth was determined by the product of the CNV area and the fluorescence intensity. For two of the riboswitch induction experiments, CNV was 2.6 (p = 0.003) and 5.2 (p = 0.03) fold higher in non-injected versus injected eyes. Placebo treated animals did not show a significant difference in CNV development for injected and non-injected eyes (p = 0.6). CNV growth was reduced by 93% in riboswitch injected eyes versus the 87% reduction seen in eyes injected with constructs expression sFlt-01driven by the chicken beta actin promoter. These results indicate that the inducible riboswitch was functional in the retinas of mice and may serve as a prototype of similar regulatory systems for use in human gene therapy.