Abstract
Purpose: :
To compare transduction onset time and cellular tropism of GFP-expressing AAV vectors with Try-to-Phe capsid mutations compared with self-complimentary AAV vectors following either intravitreal or subretinal injection in canine eyes.
Methods: :
12 normal beagle dogs (24 eyes) were used. All AAV genomes included eGFP driven by the chicken beta actin promoter. Vectors were tittered and diluted to 5x1011 vg/ml. 4 right eyes (OD) received intravitreal AAV serotype 2/2 with 4 Tyr-to-Phe capsid mutations (mutAAV2); 4 contralateral left eyes (OS) received intravitreal AAV serotype 2/2 with a self-complimentary genome (scAAV2). Following 3-port pars plana vitrectomy, 4 OD received subretinal mutAAV2; 4 OS received self-complimentary AAV serotype 2/5 (scAAV5). Following vitrectomy, the remaining 4 OD received subretinal AAV serotype 2/8 with 1 Tyr-to-Phe capsid mutation (mutAAV8); 4 OS received scAAV5. Ophthalmic examination and color and fluorescent fundus photography were performed regularly. Fundic images were identically computer enhanced to detect low-level fluorescence. Eyes were fixed following euthanasia at 28 days post-injection. Retinal cryosections were evaluated for GFP. Contralateral eyes were compared statistically via paired t-test.
Results: :
Onset of GFP expression was detected in all dogs with intravitreal injection of mutAAV2 by 9 days and scAAV2 by 8 days; fluorescence was similar for both vectors throughout the study. GFP expression was detected in all dogs with subretinal injection of mutAAV2 and mutAAV8 by 3 days and scAAV5 by 5 days. However, fluroescence was greater for mutAAV8 compared to both mutAAV2 and scAAV5. Histology showed no difference in transduction of inner retinal cells between intravitreal mutAAV2 and scAAV2; however, mutAAV2 more efficiently transduced the outer retina and RPE. All subretinal injected vectors efficiently transduced outer retina and RPE.
Conclusions: :
AAV 2/2 vectors with engineered capsid mutations are efficient in transduction of outer retina following intravitreal delivery. AAV 2/2 and 2/8 vectors with engineered capsid mutations produce more rapid and robust gene expression following subretinal delivery. AAV vectors with engineered capsid mutations are likely to be a critical resource in gene replacement therapy for rapidly progressive heritable retinal degenerations.
Keywords: retina • gene transfer/gene therapy • retinal degenerations: hereditary