Gene Augmentation Therapy for a Missense Substitution in the cGMP-Binding Domain of Ovine CNGA3 Gene Restores Vision in Day-Blind Sheep.
Gene Augmentation Therapy for a Missense Substitution in the cGMP-Binding Domain of Ovine CNGA3 Gene Restores Vision in Day-Blind Sheep.
复制标题
DOI：
10.1167/iovs.16-20986
复制
发表时间：
2017-03-01
影响因子：
4.4
通讯作者：
Seroussi E
中科院分区：
文献类型：
--
作者：
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
应用CNGA3基因增强疗法治疗绵羊色盲（ACHM）的一种新型致病突变。新生羔羊自发出现的视力受损通过行为学、视网膜电图（ERG）和组织学技术进行了表征。研究人员比较了一只受影响的羔羊和一只未受影响的羔羊的深度测序结果，这些数据位于与类似视力障碍相关的人类基因同源的保守基因组区域内。观察到的非同义氨基酸取代按其有害程度评分进行分类。通过产生复合CNGA3杂合子和使用同源人cDNA进行基因增强治疗来评估推定的致病突变。行为评估显示日盲，随后的ERG检查显示光反应减弱。病变羊眼的组织学和免疫组化检查未见变性，存在表达CNGA3的视锥光感受器。生物信息学和测序分析表明，CNGA3的gmp结合域的c.1618G> a, p.Gly540Ser突变是致病突变。遗传一致性检验和遗传互补实验证实了这一点：5个复合CNGA3杂合子均为日盲，同时携带CNGA3的p.a g236*和p.g el540ser突变。此外，使用腺相关病毒载体（AAV）将完整的人CNGA3基因在视网膜下传递，使两只受影响的p.Gly540Ser纯合公羊恢复了光性视力。CNGA3中的c.1618G>A, p.Gly540Ser突变被确定为Awassi羊一种新型ACHM的致病突变。基因增强疗法恢复了患病羊的视力。这种新的突变提供了一种适用于大多数人类CNGA3 ACHM患者的大型动物模型；它们中的大多数携带错义突变而不是过早终止突变。
Applying CNGA3 gene augmentation therapy to cure a novel causative mutation underlying achromatopsia (ACHM) in sheep. 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. 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. 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.