Gene augmentation therapy restores cone function in mice of PDE6c-acciocated achromatopsia

Abstract

Purpose : Achromatopsia is a rare autosomal recessive inherited retinal disease characterized by low visual acuity, photophobia, nystagmus, and color blindness. Our pervious study has reported a nonhuman primate (NHP) pedigree carrying a homozygous missense mutation in PDE6c, impairing the cone photoreceptor response. In this study we evaluated the rescue effect of PDE6c augmentation therapy in a spontaneous mouse model with PDE6c gene mutation, as the preliminary study of PDE6c gene therapy in NHP.

Methods : We used AAV5 vector containing cone-specific promoter (PR1.7) to delivery full length PDE6c cDNA. Cone photoreceptor function loss 1 (cpfl1) mice strain from Jackson lab with PDE6c gene mutation was used as animal model. AAV vectors were administered by subretinal injection on postnatal 14 days of cpfl1 mutant mice, one eye with AAV5-PR1.7-PDE6c, the other eye with AAV5-PR1.7-EGFP as control (1x10¹³gc/ml, 1ul per eye). In vitro validation of AAV5-PR1.7-PDE6c transfection efficiency on 661w cells was comfirmed by quantitative real-time PCR. Fundus photograph and SD-OCT was performed by MICRON IV (Phoenix MICRON™) one month after injection. Rescue was assessed by the electroretinogram (ERG, LKC technology). The protein expression in retina was detected by immunofluorescence.

Results : Real-time PCR validated the expression of PDE6c was increased by more than 10,000 fold change in 661w cells after AAV5-PR1.7-PDE6c transfection. GFP was posotive in the fundus 1 month after AAV5-PR1.7-EGFP SR injection, and the immunofluorescence staining showed GFP signal was specifically expressed by photoreceptor cell layer. ERG recording showed no light-adapted response in the untreated cpfl1 mice eyes. Eyes treated with AAV5-PR1.7-PDE6c exhibited significant improved photopic b-wave amplitudes 3 months after SR injection (10 dB flash: 22.86±5.78 uV in PDE6c injected eyes; 3.96 in±7.11 uV in GFP control eyes, P<0.01; 25 dB flash: 23.74±3.72 uV in PDE6c injected eyes; 6.51±4.80 uV in GFP control eyes). Immunofluorescence showed the PDE6c gene expression was increased in the treated eyes.

Conclusions : AAV-mediated PDE6c gene delivery effectively restores the cone function in cpfl1 mice. Our results provided the foundation for AAV5-based gene therapy for NHP of PDE6c-acciocated achromatopsia.

This is a 2020 ARVO Annual Meeting abstract.