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

Abstract：

Purpose:Applying CNGA3 gene augmentation therapy to cure a novel causative mutation underlying achromatopsia (ACHM) in sheep. Methods:Impaired 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. Results:Behavioral 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. Conclusions:The 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.

journal_name

Invest Ophthalmol Vis Sci

journal_title

Investigative ophthalmology & visual science

authors

Gootwine E,Abu-Siam M,Obolensky A,Rosov A,Honig H,Nitzan T,Shirak A,Ezra-Elia R,Yamin E,Banin E,Averbukh E,Hauswirth WW,Ofri R,Seroussi E

doi

10.1167/iovs.16-20986

subject

Has Abstract

pub_date

2017-03-01 00:00:00

pages

1577-1584

issue

eissn

0146-0404

issn

1552-5783

pii

2611261

journal_volume

pub_type

杂志文章

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