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L. R. Ferguson, S. Boye, T. Doyle, V. Chiodo, A. S. Lewin, W. W. Hauswirth; In-vitro and Retinal Regulation of Anti-neovascular Genes via Drug-Inducible Expression Systems. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3779.
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Diseases involving retinal and choroidal neovascularization are examples of situations where long-term devastating outcomes might be prevented if anti-neovascular treatments were provided periodically during the pre-neovascular stages of disease progression via a drug inducible expression system. Our goal was to compare the recently developed inducible ribozyme system with the Tet-on system both in-vitro and in-vivo in the retina.
In-vitro: HEK 293 cells were transfected with constructs containing either the inducible hammerhead ribozyme (riboswitch) or the Tet-on system. For the riboswitch, two plasmids were created, one containing a single ribozyme positioned in the 5’ untranslated region of the transgene and a second containing two inducible ribozymes, in tandem-orientation, in the same region. The Tet-on system involved co-transfection of a response plasmid and a regulator plasmid. These systems were used to compare expression of PEDF and sFLT-01 transgenes. In-vivo: Post-natal day 3 (P3) C57BL/6 mice right eyes were injected with a rAAV2 vector containing the riboswitch regulating an sFLT-01 transgene, while left eyes remained un-injected (N=6). At P12, mouse pups were implanted with a single 7-day time-release pellet containing 3.22µg of Toyocamycin (0.46µg/day). At P17 eyes were analyzed for expression of sFLT-01 using a VEGFRI sandwich ELISA.
In-vitro: The Tet-on system demonstrated 32 fold greater expression of the anti-neovascular transgenes than the construct containing two tandem-oriented inducible hammerhead ribozymes and 21 fold more expression than the single ribozyme construct. However, the Tet-on system had significantly more transgene expression in the non-induced state than either riboswitch system. In-vivo: In P17 mouse eyes the rAAV delivered riboswitch regulating sFLT-01 yielded 1.037 (SD = 2.323) ng/ml of transgene protein, while eyes not injected showed 0 (SD = 0.927) ng/ml.
When compared to the Tet-on system in-vitro, the inducible riboswitch exhibited tighter control of basal, uninduced expression of anti-neovascular transgenes and did not display an overproduction of the transfected transgenes. The riboswitch system also demonstrated retinal inducibility in-vivo.
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