Optimization of Cone-Directed AAV-Mediated Gene Augmentation Therapy for CNGB3-Achromatopsia by Use of the IRBP/GNAT2-Promoter and Intravitreal CNTF Administration

Abstract

Purpose: AAV5-mediated cone-directed gene therapy results in rescue of cone function and day vision in canine models of CNGB3-achromatopsia. However, the treatment success rate is <80%, and the human red-cone opsin promoter does not target S-cones. The objective of this study was to optimize therapeutic outcome (1) by use of an enhanced cone transducin promoter and (2) by intravitreal CNTF administration in non-responders.

Methods: The IRBP/GNAT2 promoter consists of the 277-bp 5'-flanking sequence of the human GNAT2 promoter coupled with the 214-bp IRBP enhancer (Ying et al. 1998). The ability of this promoter to target cone subclasses was evaluated by subretinal injection of AAV5-IRBP/GNAT2-GFP in normal dog eyes. GFP reporter gene expression was evaluated by observation of in vivo fluorescence and by immunohistochemistry. Subsequently, 14 young CNGB3-mutant dogs between 9-11 weeks of age were treated by bilateral subretinal injection of either AAV5-IRBP/GNAT2-hCNGB3 or AAV8-IRBP/GNAT2-hCNGB3. The animals were followed for 5-15 months post injection by standard full-field electroretinography and visual behavior testing in an obstacle avoidance course. Six months post therapy, 3 “non-responder” dogs were intravitreally injected with 12 μg of CNTF (n = 3 eyes) or PBS (n = 3 eyes) and followed for 2 months.

Results: The IRBP/GNAT2 promoter allowed robust and specific targeting of GFP-reporter gene expression in both cone subclasses. Rescue of cone function and day vision was achieved with both AAV serotypes; best results were obtained by use of ~10¹² vector genomes/mL (2/6 and 4/6 injected eyes responding with AAV5 and AAV8 respectively). A therapeutic effect was not obtained in all AAV-treated eyes. However, all 3 eyes treated with intravitreal CNTF 6 months following unsuccessful gene augmentation therapy showed robust rescue of cone function; this was not the case with intravitreal PBS injection.

Conclusions: Gene expression can be targeted to both canine cone subclasses and their function restored in CNGB3-achromatopsia with the IRBP/GNAT2 promoter in either AAV5 or AAV8. It remains unclear why some animals do not respond to AAV-mediated gene replacement therapy alone; however, treatment outcome can be enhanced by combination with intravitreal CNTF injection in eyes that do not respond.

Keywords: 648 photoreceptors • 490 cytokines/chemokines • 503 drug toxicity/drug effects