September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Co-expression and interaction of wild-type and mutant C1QTNF5 in vivo: implications for gene-therapy
Author Affiliations & Notes
  • Astra Dinculescu
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Frank M Dyka
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Seok-Hong Min
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Rachel Michelle Stupay
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • William W Hauswirth
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Footnotes
    Commercial Relationships   Astra Dinculescu, None; Frank Dyka, None; Seok-Hong Min, None; Rachel Stupay, None; William Hauswirth, AGTC (P), AGTC (F), AGTC (C)
  • Footnotes
    Support  NIH grants EY021721 and EY018331, FFB, MVRF, and RPB, Inc. WWH: P, F, C, AGTC
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1173. doi:
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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.

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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