Gene Augmentation Therapy for a Missense Substitution in the cGMP-Binding Domain of Ovine CNGA3 Gene Restores Vision in Day-Blind Sheep

Abstract

PurposeApplying CNGA3 gene augmentation therapy to cure a novel causative mutation underlying achromatopsia (ACHM) in sheep.MethodsImpaired vision that spontaneously appeared in newborn lambs was characterized by behavioral, electroretinographic (ERG), and histologic techniques. Deep-sequencing reads of an affected lamb and an unaffected lamb were compared within conserved genomic regions orthologous to human genes involved in similar visual impairment. Observed nonsynonymous amino acid substitutions were classified by their deleteriousness score. The putative causative mutation was assessed by producing compound CNGA3 heterozygotes and applying gene augmentation therapy using the orthologous human cDNA.ResultsBehavioral assessment revealed day blindness, and subsequent ERG examination showed attenuated photopic responses. Histologic and immunohistochemical examination of affected sheep eyes did not reveal degeneration, and cone photoreceptors expressing CNGA3 were present. Bioinformatics and sequencing analyses suggested a c.1618G>A, p.Gly540Ser substitution in the GMP-binding domain of CNGA3 as the causative mutation. This was confirmed by genetic concordance test and by genetic complementation experiment: All five compound CNGA3 heterozygotes, carrying both p.Arg236* and p.Gly540Ser mutations in CNGA3, were day-blind. Furthermore, subretinal delivery of the intact human CNGA3 gene using an adeno-associated viral vector (AAV) restored photopic vision in two affected p.Gly540Ser homozygous rams.ConclusionsThe c.1618G>A, p.Gly540Ser substitution in CNGA3 was identified as the causative mutation for a novel form of ACHM in Awassi sheep. Gene augmentation therapy restored vision in the affected sheep. This novel mutation provides a large-animal model that is valid for most human CNGA3 ACHM patients; the majority of them carry missense rather than premature-termination mutations.

Achromatopsia: clinical features, molecular genetics, animal models and therapeutic options

“…Promising results have been replicated in the Awassi sheep model of CNGA3-ACHM, where gene therapy led to an improvement in the cone ERG and daylight vision, with effects maintained for up to three years post-treatment [70]. A more recent study has similarly shown rescue with gene therapy in the Awassi model [71]. The success of gene therapy in this case is of particular significance, since it occurred in a large animal model with a genotype in keeping with majority of CNGA3-ACHM genotypes (missense variants) rather than knock-out/premature termination models.…”

Large Animal Models of Inherited Retinal Degenerations: A Review

“…Two spontaneous sheep models of Cnga3 achromatopsia have been identified [83]. The first identified has a nonsense mutation (p.Arg236Ter) and the second a missense mutation (p.Gly540Ser) [84]. Adeno-associated virus gene therapy was able to restore cone function in both models [84,85].…”

Naturally-occurring myopia and loss of cone function in a sheep model of achromatopsia

“…This autosomal recessive hereditary disease was determined to be caused by a C → T substitution leading to a premature stop codon at residue 236 of the ovine CNGA3 gene (c.706C > T, p.R236*), thus making affected sheep a naturally-occurring large animal model for human CNGA3 ACHM 32 . Later, a second (missense) mutation resulting in day blindness in sheep was identified in the same gene (c.1618G > A, p.Gly540Ser) 33 . Like most non-primate mammals, the retina of a sheep contains a specialized region called an “area centralis”, rather than a fovea.…”

“…Subsequently, we used these CNGA3 mutation models for successful gene therapy trials, based on subretinal injections of an adeno-associated virus carrying the CNGA3 transgene under the control of a cone-specific promoter 33 , 36 , 37 . Treatment restored cone function and photopic (daytime) vision within a few days in all treated sheep, as demonstrated both behaviorally and electroretinographically (ERG), with the oldest treated sheep still visual after more than 9 years 38 .…”

“…Though very successful, studies using large animal models of ACHM, such as sheep and dogs, are very laborious. Animals have to be anesthetized for ERG recordings 44,45 , and maze testing is time consuming and requires a special setup 33,46 . Therefore, our first aim was to develop rapid, non-invasive, and easily conducted tests for screening research animals for cone dysfunction.…”