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Maria Ines Patricio, Alun R Barnard, Harry Otway Orlans, Michelle McClements, Robert Maclaren; Transgene expression in the retina following AAV transduction: the WPRE effect. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1152.
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© 2017 Association for Research in Vision and Ophthalmology.
The use of adeno-associated virus (AAV) in retinal gene therapy for retinal diseases is increasing and an AAV1-based vector has now been approved for clinical use in the EU. The woodchuck hepatitis virus posttranslational regulatory element (WPRE) is widely used in a variety of gene therapy trials; however, it is still not clear the degree by which WPRE increases the transgene expression in retinal cells. This study investigates the effect of WPRE in driving AAV2-delivered green fluorescent protein (GFP) expression in vitro and in the mouse eye in vivo.
Two recombinant AAV2 vectors containing a hybrid human cytomegalovirus upstream enhancer with the chicken β-actin promoter (CAG) driving GFP were made with and without WPRE (rAAV2.CAG.GFP±WPRE). For in vitro analysis Human Embryonic Kidney (HEK) 293 cells were transduced with different multiplicities of infection (MOI) with both vectors, and GFP levels assessed by Western Blot. In vivo experiments involved bilateral subretinal injections of high and low dose of vectors (1E+09 and 1E+08 genome particles, respectively) in 7-week old C57BL/6J mice (N=6). Fundus autofluorescence was monitored over 5 weeks using a scanning laser ophthalmoscope. Eyes were processed after 5 weeks for histological analysis by confocal microscopy.
The levels of GFP expression were evaluated at 1, 3 and 5 days post-transduction (dpt), at MOI of 250, 1000, 5000, 10000 and 20000. The ratio GFP/actin was higher in rAAV2.CAG.GFP.WPRE-transduced cells at 3 and 5 dpt. Moreover, the WPRE effect was greater at MOI 5000 and 10000, compared to rAAV2.CAG.GFP-transduced cells. Analysis of in vivo measurement of retinal fluorescence intensity showed a significant increase of the mean grey value over time for both doses [high F(4, 20)=86, p<0.001; low F(4,20)=40, p<0.001]. For the low dose, rAAV2.CAG.GFP.WPRE-injected eyes expressed significantly more GFP compared to the contralateral eye [F(1,5)=8.082, p=0.017]. Confocal microscopy analysis revealed retinal pigment epithelium cells to be the main target of GFP expression in all treated eyes; the presence of the WPRE boosted the GFP signal in the outer nuclear layer.
Our results confirm that WPRE significantly increases the expression of transgenes in retinal layers following delivery by an AAV2 vector. This study fully supports the use of WPRE in ongoing and future AAV gene therapy trials for eye diseases.
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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