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Vidyullatha Vasireddy, Matthew Sochor, Theodore Drivas, Adam Wojno, Ivan Shpylchak, Jeannette Bennicelli, Daniel C Chung, Jean Bennett, Mitch Lewis; Development of an Autogenous Transgene Regulation System for Ocular and Systemic Applications. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3638.
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© ARVO (1962-2015); The Authors (2016-present)
The ability to introduce transgenes into a cell and restore normal physiology holds the future for ameliorating a variety of inherited and acquired diseases. While gene augmentation therapy using constitutive promoters appears promising in conditions such as Leber’s congenital amaurosis (LCA2), optimization of therapeutic effect may require that transgene expression be titrated according to disease progression. The goal of this study is to develop an inducible transgene regulatory system for somatic gene therapy.
We created an inducible autogenously regulated switch using the regulatory components of the lac operon. This regulatory switch can be induced in both prokaryotes and eukaryotes by a small galactoside. Inducible gene expression was determined by measuring production of reporter protein as a function of inducer in HEK 293T, and HTRT-RPE cells. Bioavailability of the inducer was tested by oral administration to CD1 mice for 3 days. Lysates from liver, kidney, skeletal muscle and neural retina were used to measure the steady state concentration of inducer in those tissues. Reporter gene luciferase was packaged into AAV particles using an AAV8 packaging plasmid. AAV was delivered by tail vein (TV) or subretinal injection in CD1 mice and inducible expression of a reporter (GFP or luciferase) was evaluated using an IVIS imaging system.
Here we demonstrate of the utility of autogenously inducible switch in bacteria, eukaryotic cells, and both retina and liver of living mice. Expression of the reporter gene increased proportionally with the concentration of the inducer. Mice injected with AAV-luciferase showed a robust induction and returned to baseline through two on/off cycles. The removal of inducer led to cessation of reporter gene expression. There was no evidence of cellular infiltrate after histological evaluation of the retina and liver of these animals.
To evaluate the utility of this regulatory system as a tool for gene therapy, we built an inducible autogenous switch and tested it in both in vitro and in vivo models. The ability to induce transgene expression repeatedly via an oral inducer in two different organ systems holds a great deal of promise for applications to human gene therapy.
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