Targeting of Gene Expression to the wildtype and ADAMTS10-Mutant Canine Trabecular Meshwork by Non-Self-Complementary AAV2

Abstract

Purpose: The purpose of this study was to target green fluorescent protein (GFP) reporter gene expression to the trabecular meshwork (TM) of wt and ADAMTS10-mutant dogs. Our ultimate goal is to perform gene augmentation therapy in dogs with primary open-angle glaucoma (POAG) due to a G661R missense mutation in ADAMTS10. Even though the use of self-complementary (sc) AAV provides the most robust gene expression to the TM, the ADAMTS10 cDNA (~3.3 kb) is too long for inclusion into scAAV. In this study we evaluated the ability of non-sc AAV2-based capsid mutants to target the canine TM.

Methods: Young adult beagle dogs (mean age: 10.9 ± 4.8 mos), both wt (n=6) and ADAMTS10-mutants (n=4), received a 50-μL intracameral injection of capsid mutant rAAV2 vector at concentrations between 2 x 10¹⁰ - 2 x 10¹³ vg/mL; a total of 13 eyes were injected. All AAVs were expressing GFP under the constitutive small CMV-chicken beta-actin (smCBA) promoter. The following capsid mutants were used: Y444F, triple Y-F, and triple Y-F + T-V. The animals were monitored for 3-6 mos by routine ophthalmic examination and weekly diurnal intraocular pressure (IOP) measurement. GFP expression was evaluated histologically in all four quadrants of the iridocorneal angle.

Results: Of the three capsid mutants evaluated, only AAV2(Y444F) provided GFP expression in the canine iridocorneal angle. The level of GFP expression was dose-dependent and clearly detectable in all quadrants, with all vector concentrations, and in both wt and mutant eyes. Green fluorescence was strongest along the conventional aqueous humor outflow pathway, including the TM, but weak GFP expression was also found on the anterior iris surface. Despite the use of the non-specific smCBA promoter, no other cell types were transfected. No severe adverse reactions were observed, and GFP expression did not affect IOP.

Conclusions: Conclusions: The use of AAV2(Y444F) provides robust targeting of the canine TM despite being non-sc, and it will be considered for future gene therapy trials, such as for canine ADAMTS10-POAG.

Keywords: 472 comparative anatomy • 538 gene transfer/gene therapy • 735 trabecular meshwork