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Timothy P Day, Cecile Fortuny, Yebon Oh, David Schaffer, John Gerard Flannery; Evaluation of CRB1 gene delivery in the rd8 mouse. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1183.
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© ARVO (1962-2015); The Authors (2016-present)
Recent gene therapy studies have shown that expression of crb1 delivered by a viral vector leads to an immune response in mice null for crb1. We hypothesized that this immune response would not be observed in the naturally occurring rd8 mouse model of crb1 because a truncated version of the protein is expressed. Retinal degeneration in the rd8 mouse is found only in the inferior portion of the retina with relative sparing in the superior retina. Taking advantage of this observed phenotype, we compared the expression of genes associated with crb1 in the superior versus the inferior retina in the rd8 mouse to assess if any of the associated proteins play a compensatory role. In addition, we continued to evaluate small promoters to increase the expression levels of the relatively large crb1 gene to enhance its therapeutic potential.
Intravitreal injections of crb1 via the Müller glia-specific adeno-associated virus, ShH10, was performed in rd8 mice. Immunohistochemistry, optical coherence tomography (OCT), and electroretinograms (ERGs) were used to determine the immune response and degeneration in the mouse model after gene delivery. Comparative quantitative PCR was used to determine the expression levels of crb1-associated proteins in the superior versus the inferior retina. Several versions of a minimal CMV promoter were packaged into ShH10 and injected into the rd8 mouse to determine if expression levels could be increased.
Immunohistochemistry, ERG, and OCT data did not show a difference between crb1 treated and untreated eyes in the rd8 mouse 6 months post injection. Quantitative PCR was able to determine the mRNA expression levels of relevant genes in both the superior and inferior retina. Additional small promoters showed increased expression.
Mutations in crb1 are associated with multiple clinically relevant retinal degenerations including retinitis pigmentosa. Our study found that an immune response was not observed in the rd8 mouse model after crb1 gene delivery opening up the potential for therapy in patients who express a truncated form of crb1. Also, by evaluating the expression profile of different crb1-associated genes, we can begin to examine the potential compensatory roles proteins play in the absence of functional crb1. Finally, by evaluating small promoters for greater expression we increased the therapeutic potential of crb1 gene therapy.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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