Abstract
Abstract: :
Purpose:The control over time and space of the levels of therapeutic proteins is crucial for successful retinal gene therapy. We tested the ability of viral vectors to deliver a model secreted protein (erythropoietin; Epo) intraocularly, either constitutively or in a pharmacologically-regulated manner using the rapamycin-inducible transcriptional regulatory system. Methods:We cloned Epo into a single adeno-associated viral vector (AAV) under the control of the constitutive CMV promoter, or into the two-vector rapamycin-inducible system. Recombinant AAV vectors were produced and delivered to the intravitreal or subretinal space of nude Sprague Dawley rats. Following systemic administration of rapamycin (for the regulated vectors), the levels of Epo in the anterior chamber, vitreous fluid and serum was determined by ELISA, and blood hematocrits were also measured. Results:Administration of AAV2-CMV-rhEpo led to expression of Epo in the anterior chamber or vitreous fluid, and intravitreal or subretinal administration gave similar levels of expression. Following subretinal delivery of the dual-vector rapamycin-inducible system, no Epo protein was detected in the anterior chamber fluid in the absence of rapamycin. Three days after rapamycin adminstration, Epo levels peaked in the ocular fluid in a dose-dependent manner and returned to basal levels 14-21 days later (a ≷100-fold induction). Drug inductions were repeated several times over a 3 months period. No increases in circulating Epo levels or hematocrit were observed following rapamycin administration. Conclusion:We demonstrate that Epo secretion after intraocular administration of vector is measurable in the ocular fluids of rats. In addition, the use of the rapamycin system allows inducible secretion of Epo and reversal to baseline levels upon withdrawal of rapamycin. Epo does not accumulate to significant levels in serum and thus does not lead to changes in hematocrit. The rapamycin inducible system promises to be useful for developing gene therapy for inherited retinal degeneration and ocular neovascularization.
Keywords: 419 gene transfer/gene therapy