Preclinical evaluation of rAAV8.CNGA3 in the Cnga3 knockout mouse model of ACHM2

Abstract

Purpose : Mutations in the cyclic nucleotide-gated channel alpha 3 subunit (CNGA3) gene are known to cause achromatopsia type 2 (ACHM2) characterized by poor visual acuity, photophobia and lack of color discrimination. Here, a novel recombinant adeno-associated virus (AAV) vector for gene supplementation therapy of CNGA3-linked ACHM2 was developed and tested for efficacy in the preclinical Cnga3 knockout (KO) mouse model of ACHM2.

Methods : An AAV-based gene supplementation vector coding for full length human CNGA3 under control of the human, cone-specific cone arrestin promoter was generated and used for packaging of AAV8-pseudotyped vectors. The resulting vector (rAAV8.CNGA3) was tested for efficacy by subretinal injections in two-week-old Cnga3 KO mice with short-term and long-term analysis at 8 weeks and 12 months post-injection, respectively. AAV vector-mediated expression of human CNGA3 protein was assessed by immunohistochemistry using a CNGA3-specific antibody combined with confocal microscopy. Efficacy was assessed in vivo by electroretinography (ERG) with protocols designed to isolate cone-specific light responses.

Results : We designed and generated rAAV8.CNGA3, a novel AAV vector optimized for efficient CNGA3 gene expression in human cone photoreceptors. Transgene expression assay of rAAV8.CNGA3 in Cnga3 KO mice confirmed efficient and specific human CNGA3 protein expression that localized to cone photoreceptor outer segments. A biological activity assay for rAAV8.CNGA3 based on ERG analysis confirmed a beneficial effect of the treatment at both observation time points. Additionally, we found prolonged survival of cone photoreceptors up to 12 months after treatment.

Conclusions : The novel rAAV8.CNGA3 vector supports efficient and specific transgene expression and biological activity in the preclinical Cnga3 KO mouse model of ACHM2.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.