The successful restoration of visual function with recombinant adeno-associated virus (rAAV)-mediated gene replacement therapy in animals and humans with an inherited disease of the retinal pigment epithelium has ushered in a new era of retinal therapeutics. For many retinal disorders, however, targeting of therapeutic vectors to mutant rods and/or cones will be required. In this study, the primary cone photoreceptor disorder achromatopsia served as the ideal translational model to develop gene therapy directed to cone photoreceptors. We demonstrate that rAAV-mediated gene replacement therapy with different forms of the human red cone opsin promoter led to the restoration of cone function and day vision in two canine models of CNGB3 achromatopsia, a neuronal channelopathy that is the most common form of achromatopsia in man. The robustness and stability of the observed treatment effect was mutation independent, but promoter and age dependent. Subretinal administration of rAAV5-hCNGB3 with a long version of the red cone opsin promoter in younger animals led to a stable therapeutic effect for at least 33 months. Our results hold promise for future clinical trials of cone-directed gene therapy in achromatopsia and other cone-specific disorders.

Achromatopsia is a genetic disorder of cones, and one of the most common forms is a channelopathy caused by mutations in the β-subunit, CNGB3, of the cone cyclic nucleotide-gated (CNG) channel. Recombinant adeno-associated virus of serotype 5 (rAAV5)-mediated gene transfer of human CNGB3 cDNA to mutant dog cones results in functional and structural rescue in dogs <0.5 years of age, but treatment is minimally effective in dogs >1 year. We now test a new therapeutic concept by combining gene therapy with the administration of ciliary neurotrophic factor (CNTF). Intravitreal CNTF causes transient dedifferentiation of photoreceptors, a process called deconstruction, whereby visual cells become immature with short outer segments, and decreased retinal function and gene expression that subsequently return to normal. Cone function was successfully rescued in all mutant dogs treated between 14 and 42 months of age with this strategy. CNTF-mediated deconstruction and regeneration of the photoreceptor outer segments prepares the mutant cones optimally for gene augmentation therapy.

PurposeHuman long (L) and middle (M) wavelength cone opsin genes are highly variable due to intermixing. Two L/M cone opsin interchange mutants, designated LIAVA and LVAVA, are associated with clinical diagnoses, including red-green color vision deficiency, blue cone monochromacy, cone degeneration, myopia, and Bornholm Eye Disease. Because the protein and splicing codes are carried by the same nucleotides, intermixing L and M genes can cause disease by affecting protein structure and splicing.MethodsGenetically engineered mice were created to allow investigation of the consequences of altered protein structure alone, and the effects on cone morphology were examined using immunohistochemistry. In humans and mice, cone function was evaluated using the electroretinogram (ERG) under L/M- or short (S) wavelength cone isolating conditions. Effects of LIAVA and LVAVA genes on splicing were evaluated using a minigene assay.ResultsERGs and histology in mice revealed protein toxicity for the LVAVA but not for the LIAVA opsin. Minigene assays showed that the dominant messenger RNA (mRNA) was aberrantly spliced for both variants; however, the LVAVA gene produced a small but significant amount of full-length mRNA and LVAVA subjects had correspondingly reduced ERG amplitudes. In contrast, the LIAVA subject had no L/M cone ERG.ConclusionsDramatic differences in phenotype can result from seemingly minor differences in genotype through divergent effects on the dual amino acid and splicing codes.Translational RelevanceThe mechanism by which individual mutations contribute to clinical phenotypes provides valuable information for diagnosis and prognosis of vision disorders associated with L/M interchange mutations, and it informs strategies for developing therapies.

Carotid atherosclerosis is the primary cause of ischemic stroke. To identify genetic factors contributing to carotid atherosclerosis, we performed quantitative trait locus (QTL) analysis using female mice derived from an intercross between C57BL/6J (B6) and BALB/cJ (BALB) apolipoprotein E (Apoe−/−) mice. We started 266 F2 mice on a Western diet at 6 wk of age and fed them the diet for 12 wk. Atherosclerotic lesions in the left carotid bifurcation and plasma lipid levels were measured. We genotyped 130 microsatellite markers across the entire genome. Three significant QTLs, Cath1 on chromosome (Chr) 12, Cath2 on Chr5, and Cath3 on Chr13, and four suggestive QTLs on Chr6, Chr9, Chr17, and Chr18 were identified for carotid lesions. The Chr6 locus replicated a suggestive QTL and was named Cath4. Six QTLs for HDL, three QTLs for non-HDL cholesterol, and three QTLs for triglyceride were found. Of these, a significant QTL for non-HDL on Chr1 at 60.3 cM, named Nhdl13, and a suggestive QTL for HDL on ChrX were new. A significant locus for HDL (Hdlq5) was overlapping with a suggestive locus for carotid lesions on Chr9. A significant correlation between carotid lesion sizes and HDL cholesterol levels was observed in the F2 population (R = −0.153, P = 0.0133). Thus, we have identified several new QTLs for carotid atherosclerosis and the locus on Chr9 may exert effect through interactions with HDL.

Background Diabetic patients have an increased risk of developing atherosclerosis and related complications compared to non-diabetic individuals. The increased cardiovascular risk associated with diabetes is due in part to genetic variations that influence both glucose homeostasis and atherosclerotic lesion growth. Mouse strains C57BL/6J (B6) and BALB/cJ (BALB) exhibit distinct differences in fasting plasma glucose and atherosclerotic lesion size when deficient in apolipoprotein E (Apoe−/− . Quantitative trait locus (QTL) analysis was performed to determine genetic factors influencing the two phenotypes. Methods and Results 266 female F2 mice were generated from an intercross between B6.Apoe−/− and BALB.Apoe−/− mice and fed a Western diet for 12 weeks. Atherosclerotic lesions in the aortic root, fasting plasma glucose, and body weight were measured. 130 microsatellite markers across the entire genome were genotyped. Four significant QTLs, Ath1 on chromosome (Chr) 1, Ath41 on Chr2, Ath42 on Chr5, and Ath29 on Chr9, and one suggestive QTL on Chr4, were identified for atherosclerotic lesion size. Four significant QTLs, Bglu3 and Bglu12 on Chr1, Bglu13 on Chr5, Bglu15 on Chr12, and two suggestive QTLs on Chr9 and Chr15 were identified for fasting glucose levels on the chow diet. Two significant QTLs, Bglu3 and Bglu13, and one suggestive locus on Chr8 were identified for fasting glucose on the Western diet. One significant locus on Chr1 and two suggestive loci on Chr9 and Chr19 were identified for body weight. Ath1 and Ath42 coincided with Bglu3 and Bglu13, respectively, in the confidence interval. Conclusions We have identified novel QTLs that have major influences on atherosclerotic lesion size and glucose homeostasis. The colocalization of QTLs for atherosclerosis and diabetes suggests possible genetic connections between the two diseases.