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Astra Dinculescu, Frank M Dyka, Seok-Hong Min, Rachel Michelle Stupay, William W Hauswirth; Co-expression and interaction of wild-type and mutant C1QTNF5 in vivo: implications for gene-therapy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1173.
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© 2017 Association for Research in Vision and Ophthalmology.
The S163R mutation in complement C1q tumor necrosis factor-related protein-5 (C1QTNF5) causes an autosomal dominant disorder known as late-onset retinal degeneration (L-ORD). Here, our goal was to test the effects of co-expressing wild-type and mutant S163R C1QTNF5 protein in vivo in RPE cells following AAV-mediated delivery to wild-type mouse eyes.
We generated scAAV vectors with the AAV2 quad YF capsid containing either human wild-type C1QTNF5-myc tagged cDNA or mutant S163R C1QTNF5-HA tagged cDNA, driven by an RPE-specific BEST1 promoter (1012 vector genomes/mL), and delivered 1 μL subretinally into one eye of adult C57BL/6 mice, while the contralateral eyes remained untreated and served as controls. The vectors were either delivered individually, or as a mixture containing equal amounts of wild-type and mutant S163R vectors. Retinal function was assessed monthly by full-field electroretinography (ERG) under scotopic (dark-adapted) and photopic conditions. The eyes were also examined by digital fundus imaging and spectral-domain optical coherence tomography (SD-OCT). Transgene expression was detected by immunohistochemistry, and retinal morphology was examined by hematoxylin and eosin (H&E) staining of paraffin sections.
Consistent with our previous studies, RPE targeted overexpression of S163R C1QTNF5 mutant led to accumulation of mutant protein as large, spherical aggregates in the RPE, while wild-type C1QTNF5 was secreted apically towards the OLM. In contrast, when both wild-type and mutant S163R C1QTNF5 are simultaneously delivered, the mutant no longer forms the characteristic spherical aggregates, and is found dispersed towards the RPE lateral membrane, where it co-localizes with the AAV-expressed wild-type protein. There was a small, but not significant decrease in the scotopic ERG amplitudes in the eyes injected with the vector mixture.
These results suggest that: 1) wild-type and mutant C1QTNF5 interact in-vivo, 2) the wild-type protein prevents the S163R mutant from forming aggregates, 3) the wild-type protein continues its normal secretion apically from RPE cells towards the OLM. This suggests that a combination therapy of the appropriate dose of wild-type C1QTNF5 vector and an siRNA to inhibit mutant protein expression may represent a therapeutic option for L-ORD patients.
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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