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Raghavi Sudharsan, Simone Iwabe, Tatyana Appelbaum, Alfred S Lewin, William W Hauswirth, Gustavo D Aguirre, William A Beltran; Evaluation of the efficacy of mouse opsin promoter for rhodopsin gene augmentation in dogs. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5408.
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© ARVO (1962-2015); The Authors (2016-present)
An AAV2/5 carrying human Rhodopsin (hRHO) cDNA under the control of a mouse opsin promoter (mOP) did not prevent light-induced retinal degeneration in the light-sensitive canine model of autosomal dominant Retinitis Pigmentosa (ADRP) (Iwabe et al, ARVO 2014). We now have evaluated in canine retinas the levels of rhodopsin expression following treatment with this same viral vector construct to determine the lack of rescue effect.
Wild-type dogs were used; an AAV2/5-mOP-hRHO construct was subretinally injected (150 ul at 5x1012 vg/ml) in 5 eyes, and one eye received equal volume of BSS. Dogs were euthanatized 8-9 weeks post injection. Punches of neuroretina (3 mm diameter) were collected from the bleb (treated) and non-injected areas, and used to analyze levels of expression of hRHO transgene by western blot and qRT-PCR. RHO protein levels were evaluated using an antibody that recognizes both canine and human proteins. Transcription of hRHO was evaluated by RT-PCR using a set of primers specific to hRHO. In addition, absolute quantitation was performed using the qRT-PCR Standard Curve method to determine the absolute amounts of human versus endogenous canine RHO mRNA transcribed.
No significant differences in total RHO protein amounts between treated and non-treated regions were found by western blotting. Transcription of hRHO in bleb regions retina was confirmed based on Ct values obtained with hRHO specific primers. However, using absolute quantitation, we determined that the hRHO transgene was being transcribed at only 1-4% of the mRNA levels of the endogenous canine RHO.
A similar AAV construct as used here has been previously shown to efficiently augment RHO expression in the transgenic P23H mouse model of ADRP, yielding a two-fold increase in total RHO mRNA, and a 1.5 times increase in protein (Mao et. al., Hum. Gene Ther., 2011). In the canine retina, our current results show that hRHO transgene expression is low, thus explaining the lack of rescue effect in the canine T4R RHO model of ADRP that we had previously reported. This may be due either to limited efficiency of the mouse opsin promoter, or to instability of hRHO transcripts in the canine retina. Ongoing studies now focus on the human opsin promoter instead, and adding enhancer elements to the viral construct to improve transgene expression.
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