April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Dual AAV vector development for CEP290-LCA
Author Affiliations & Notes
  • Renee C Ryals
    Ophthalmology, University of Florida, Gainesville, FL
  • Frank M Dyka
    Ophthalmology, University of Florida, Gainesville, FL
  • Jingfen Sun
    Ophthalmology, University of Florida, Gainesville, FL
  • Oliver Sroka
    Ophthalmology, University of Florida, Gainesville, FL
  • William W Hauswirth
    Ophthalmology, University of Florida, Gainesville, FL
  • Footnotes
    Commercial Relationships Renee Ryals, None; Frank Dyka, None; Jingfen Sun, None; Oliver Sroka, None; William Hauswirth, AGTC (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3329. doi:
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      Renee C Ryals, Frank M Dyka, Jingfen Sun, Oliver Sroka, William W Hauswirth; Dual AAV vector development for CEP290-LCA. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3329.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: Approximately 20% of LCA cases are due to mutations in CEP290, a gene that plays an important role in protein trafficking in photoreceptors. Although RPE65-LCA patients have been successfully treated with AAV gene therapy, treatments for patients with CEP290-LCA have yet to be developed. CEP290 cDNA is approximately 8kb, which is beyond the carrying capacity of one AAV capsid. We hypothesize that creating dual AAV vector systems for CEP290 can overcome this packaging limitation and allow for AAV mediated CEP290 expression. The purpose of this study is to investigate the ability of our simple overlap and hybrid dual AAV vector systems to mediate full-length CEP290 expression in vitro.

Methods: First, simple overlap and hybrid dual AAV vector systems were constructed. Secondly, a control full-length CEP290myc plasmid was transfected in mouse inner medullar collecting duct (mIMCD) cells. Three days post transfection cells were stained with antibodies specific to myc and acetylated alpha tubulin. Finally, we infected our simple overlap and hybrid vectors in human embryonic kidney (HEK293) cells. Three days post infection protein was harvested and Western blots were run to assess myc labeled CEP290 protein expression.

Results: Simple overlap and hybrid dual vector systems were successfully constructed, sequence confirmed and packaged into AAV2. Exogenously expressed CEP290 localized to the cytoplasm, nucleus, centrioles and the base of the primary cilia in mIMCD cells. Full-length CEP290 expression, which was confirmed by simultaneous myc expression, was evident after transfection in HEK293 cells with full-length control plasmid.

Conclusions: These results indicate that exogenously expressed CEP290 can be expressed in biologically relevant intracellular locations. Our dual AAV vector systems appear to be competent for therapeutic testing of CEP290 expression in animal models of CEP290-LCA.

Keywords: 538 gene transfer/gene therapy • 695 retinal degenerations: cell biology • 554 immunohistochemistry  

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