Purchase this article with an account.
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.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
This PDF is available to Subscribers Only