In Vivo Gene Augmentation in the ADAMTS10-Mutant Canine Trabecular Meshwork (TM) with Y444F-Capsid Mutant, Single-Stranded AAV2

Abstract

Purpose: We have previously reported robust targeting of GFP reporter gene expression to the rodent and canine TM with capsid-mutant AAV2(Y444F), both self-complementary and single-stranded. The purpose of our study was to use this vector construct for in vivo TM-targeted gene augmentation in order to prevent/reverse intraocular pressure (IOP) elevation in dogs with primary open-angle glaucoma (POAG) due to a G661R missense mutation in ADAMTS10.

Methods: Eleven adult, ADAMTS10-mutant dogs (mean age: 10.9 ± 4.8 months) were treated unilaterally by intracameral injection of single-stranded AAV2(Y444F)-smCBA-hADAMTS10 vector with hADAMTS10 expression under the constitutive small CMV-chicken beta-actin (smCBA) promoter. Ten dogs received 50-μL and 1 dog received 150 µL of vector solution at a concentration of 2 x 10¹² vg/mL. The fellow eyes served as non-injected controls. The dogs were closely followed over 6 months by routine ophthalmic examination and weekly diurnal IOP measurements with a rebound tonometer (Icare TONOVET). IOPs were compared between treated and non-treated eyes by use of a generalized linear model and estimating equation. hADAMTS10 mRNA expression in the treated TM was evaluated by qPCR and compared to untreated control eyes.

Results: The AAV2 vector was well tolerated with no detectable adverse effects. Consistent with our previous findings using single stranded AAV2(Y444F) vector expressing GFP reporter gene, hADAMTS10 transgene expression was detected in the TM of AAV-treated but not in untreated eyes. Unfortunately, no therapeutic IOP effect was observed. Characteristic for the ADAMTS10-POAG disease phenotype, the gradual increase in IOP continued in both treated and untreated eyes during the 6-month follow-up. Among 7 of the study dogs at a comparable POAG disease stage no significant difference in average IOP was observed between treated (25.0 mmHg ± 1.0 mmHg) and untreated control eyes (24.4 mmHg ± 1.2 mmHg) over the 6-month post-treatment study period (p=0.56).

Conclusions: The intracameral administration of AAV2(Y444F) at the quantity administered was safe and resulted in TM-transgene expression but did not achieve a rescue effect. Possible explanations for this treatment failure are insufficient transfection efficiency and/or irreversibility of TM fibrosis/plaque formation in the POAG-affected eyes.