Yongblah Kevin, Alford Spencer C, Ryan Bridget C, Chow Robert L, Howard Perry L
Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 1700 STN CSC, Victoria, BC V8W2Y2, Canada.
Department of Biology, University of Victoria, P.O. Box 1700 STN CSC, Victoria, BC V8W 2Y2, Canada.
Mol Ther Nucleic Acids. 2018 Dec 7;13:144-153. doi: 10.1016/j.omtn.2018.08.018. Epub 2018 Sep 1.
Aniridia is a rare congenital syndrome that is associated with reduced visual acuity and progressive loss of vision. Aniridia patients may also develop systemic health issues associated with defects in the pancreas, digestive, and central nervous systems. The spectrum of symptoms associated with aniridia is due to haploinsufficiency of the paired box 6 gene (PAX6) and its role in the development and maintenance of the affected tissues. Here, we isolated pancreatic islets from mice heterozygous for Pax6 to test whether a Pax6-specific miRNA suppression (target protector) strategy can restore PAX6 protein levels. We show that miR-7 and miR-375 target specific sites within the Pax6 3' UTR in a mouse pancreatic β-insulinoma cell line. Tough decoys (Tuds) against miR-7 and miR-375 increase expression of a mouse Pax6 3' UTR luciferase reporter and increase PAX6 protein levels in these cells. Finally, we demonstrate that the shielding of the miR-7 binding site with a target protector restores PAX6 protein levels in the Pax6 heterozygous islets. The data presented here represent a proof of concept for RNA-based therapy for the progressive defects associated with aniridia and suggest the target protector approach may be a useful therapeutic strategy for other haploinsufficiency diseases.
无虹膜症是一种罕见的先天性综合征，与视力下降和视力逐渐丧失有关。无虹膜症患者还可能出现与胰腺、消化系统和中枢神经系统缺陷相关的全身性健康问题。与无虹膜症相关的一系列症状是由于配对盒6基因（PAX6）的单倍剂量不足及其在受影响组织的发育和维持中的作用。在这里，我们从Pax6杂合小鼠中分离出胰岛，以测试Pax6特异性miRNA抑制（靶标保护）策略是否可以恢复PAX6蛋白水平。我们表明，miR-7和miR-375在小鼠胰腺β胰岛素瘤细胞系中靶向Pax6 3'UTR内的特定位点。针对miR-7和miR-375的刚性诱饵（Tuds）增加了小鼠Pax6 3'UTR荧光素酶报告基因的表达，并增加了这些细胞中的PAX6蛋白水平。最后，我们证明用靶标保护剂屏蔽miR-7结合位点可恢复Pax6杂合胰岛中的PAX6蛋白水平。此处呈现的数据代表了针对与无虹膜症相关的进行性缺陷的基于RNA的治疗的概念验证，并表明靶标保护剂方法可能是治疗其他单倍剂量不足疾病的有用治疗策略。
