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Nasreen Jacobson, Chang-Xin Shi, Frank Graham, Keith Stewart, Hua Xin, J. C. Millar, Allan R. Shepard; Development of a Second-Generation, Helper-Dependent Adenovirus-Based Model of Glaucoma. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2480.
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© ARVO (1962-2015); The Authors (2016-present)
We previously developed a mouse model of glaucoma using adenoviral (Ad) transfer of a mutated human TGFβ2 gene (Ad.TGFβ2mut; IOVS 2010, 51:2067). The model is limited by the transient nature of transgene expression and adverse reaction to the Ad, likely due to host reaction to Ad viral proteins. In an effort to minimize these adverse cellular effects and prolong gene expression we have evaluated second-generation, helper-dependent Ad (HDAd) deleted in all viral protein-coding sequences.
For pilot studies, HDAd.EGFP-TGFβ1 (2.78E11 particles/mL) and HDAd.EGFP-LacZ (2.76E12 particles/mL) were readily available. Viruses were purified by double ultracentrifugation and dialyzed into 10 mM Tris-HCl, pH 8.0 with 10% glycerol. Viral competency was assessed in vitro using a transformed trabecular meshwork cell line (GTM3). In vivo efficacy was determined in mice by intravitreal injection (2 uL) at varying titers into BALB/cJ mice. IOP measurements were performed using TonoLab rebound tonometer and histology was performed by H&E, beta-galactosidase, and anti-CD45 staining.
GTM3 cells were efficiently transduced by HDAd.EGFP-LacZ and HDAd.EGFP-TGFβ1 as determined by beta-galactosidase staining and TGFβ1 ELISA, respectively. Mouse eyes were shown to be efficiently transduced with HDAd.LacZ as assessed by histological staining for beta-galactosidase expression. Eyes that were injected with HDAd.TGFβ1 responded with increased IOP similar to that seen previously with Ad.TGFβ2mut. Adverse events such as inflammation and hyperemia were observed only with higher (>1E8 particles) viral titers. CD45+ staining was only prevalent in HDAd.TGFβ1 transduced eyes. Anterior segment morphology of HDAd transduced eyes were significantly improved compared to Ad transduced eyes.
HDAd transduced eyes showed good transduction efficiency and gene expression in vitro and in vivo and, compared to Ad, showed fewer adverse events in vivo when used at an optimally determined titer (~1E7 particles). HDAd virus shows promise for use as a gene transfer vehicle for development of a second-generation mouse TGFβ2-based glaucoma model. Ongoing studies will determine whether HDAd results in longer duration of expression over Ad in the mouse eyes and whether a larger eye animal model (i.e. rabbit) is feasible.
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