Abstract
Dog models with spontaneously occurring mutations in retinal dystrophy genes are an invaluable resource for preclinical development of retinal gene therapy. Adeno-associated virus (AAV) vectors have been most successful; to target the outer retina and RPE they are delivered by subretinal injection, causing a temporary retinal detachment with some potential for retinal morbidity. A recent reporter gene study using an AAV2/8 vector in dogs reported transgene expression beyond the boundary of the subretinal bleb. This could be a desirable feature which increases the area of retina treated while minimizing the retinal detachment and any associated morbidity. We performed a detailed study of the lateral spread of transgene expression beyond the subretinal injection site following subretinally delivered AAV vectors in normal dogs. Vectors expressed green fluorescent protein (GFP) using a small chicken beta-actin promoter. AAV2/2 (quadruple tyrosine to phenylalanine (Y-F) capsid mutant), self-complementary (sc) AAV2/8 (single Y-F capsid mutant) and a scAAV2/5 were used. We found that in all eyes GFP expression involved retina beyond the initial post-injection subretinal bleb boundary. In all eyes there was post-injection spread of the retinal detachment within the first 3 days post procedure and prior to retinal reattachment. In 11/16 eyes this accounted for the entire “lateral spread” of GFP expression while in 5/16 eyes a very slight extension of GFP expression beyond the final boundary of the subretinal bleb could be detected. All 3 AAV constructs induced GFP expression in the nerve fiber layer with spread to the optic nerve. Patients treated by subretinal injection should be monitored for possible expansion of the subretinal injection bleb prior to reattachment. Injections in the para-foveal region may expand to lead to a foveal detachment that may be undesirable. Cell-specific promoters may be required to limit spread of expressed transgene to the brain with these AAV serotypes.
Highlights
Retinal dystrophies such as the genetically heterogenous conditions Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP) are important causes of human vision loss
Expressing green fluorescent protein driven by smCBA (subsequently referred to as AAV2(quadY-F); and a self-complementary expressing AAV2/8 construct with a single capsid tyrosine to phenylalanine mutation (Y733F) expressing humanized green fluorescent protein (hGFP) driven by smCBA (subsequently referred to as scAAV8(Y733F)
We show in this study that retinal GFP expression extends beyond the boundary of the subretinal bleb immediately postinjection
Summary
Retinal dystrophies such as the genetically heterogenous conditions Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP) are important causes of human vision loss (see reviews [1,2]). Similar retinal degenerative conditions occur spontaneously in animal species including dogs and cats [3,4]. The dog in particular has been important in preclinical gene therapy trials. For example dogs with a spontaneous mutation in the Rpe gene were pivotal in the development of phase I/II clinical trial programs for treatment of patients with mutations in the RPE65 gene, an important cause of LCA [5]. Several other spontaneously occurring dog models have been identified and include those with mutations in BEST1, CNGB3, rhodopsin, RPGR, PDE6A and PDE6B [6,7,8,9,10,11]. Preclinical gene therapy trials using some of these additional dog models have been reported [12,13,14,15]
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