Therapeutic Potential of Rescued FMR1 Mis-Splicing in Fragile X Syndrome

脆性X综合征（FXS）是一种神经发育障碍，是最常见的遗传形式， 智力障碍和孤独症最普遍的单基因原因。FXS是由膨胀200引起的 在FMR 1基因的5'非翻译区（UTR）中存在一个或多个CGG三联体，这导致FMR 1甲基化 和转录沉默。FMR 1基因产物FMRP的缺失导致过量的蛋白质合成 这可能导致了这种疾病的几种表现。 我们还对FXS患者的白色血细胞（WBC）的RNA进行了深度测序 作为年龄匹配的典型发育（TD）个体。我们发现有数百种RNA 与TD相比，FXS WBC中的调节。我们还检测到FXS中数百种错误剪接的RNA， 相比TD。这些错误调节的RNA事件具有统计学显著性，并且可能构成了一个强大的干扰。 FXS个体的生物标志物。令我们惊讶的是，我们还发现，在50%的FXS WBC样本中，FMR 1 RNA是 合成了在这些情况下，FMR 1 RNA被错误剪接，使得外显子被剪接成“假外显子”。 在FMR 1内含子中。这种错误剪接的RNA是多聚腺苷酸化的，可以编码一种小多肽， 功能（如果有的话）未知。我们产生了2 '-O-甲氧基乙基（2'-莫伊）反义寡核苷酸（ASO）， 平铺在内含子、内含子-外显子连接处并进入假外显子。转染入FXS WBC时 我们发现，两个ASO阻断了FMR 1的不正确剪接，挽救了正确的FMR 1剪接， 重要的是，将FMRP恢复到TD水平。我们还检测到FXS死后大脑中的FMR 1错误剪接， 表明FMR 1错误剪接在FXS个体中的广泛性。这些和其他数据表明， 阿索对FMR 1错误剪接的纠正和FMRP的恢复可能为治疗FXS提供一种创新的疗法。 为了评估阿索治疗FXS的治疗潜力，我们将研究CGG-1的机制。 依赖性FMR 1错误剪接，确定是否存在具有不同连接、长度或序列的其他ASO 更有效地抑制FMR 1错误剪接和恢复FMRP，检查阿索拯救是否发生在iPSC中， 衍生的FXS神经元，并评估阿索毒性、药代动力学和脑靶向， 脑室内（ICV）注射至小鼠。

Fragile X Syndrome (FXS) is a neurodevelopmental disorder that is the most common inherited form of intellectual impairment and most prevalent single gene cause of autism. FXS is caused by an expansion of 200 or more CGG triplets in the 5’ untranslated region (UTR) of the FMR1 gene, which leads to FMR1 methylation and transcriptional silencing. The loss of the FMR1 gene product FMRP results in excessive protein synthesis in the brain, which likely contributes to several manifestations of the disorder. We performed deep sequencing of RNA from white bloods cells (WBCs) of individuals with FXS as well as age-matched typically developing (TD) individuals. We found that hundreds of RNAs were up or down regulated in FXS WBCs compared to TD. We also detected hundreds of RNAs that were mis-spliced in FXS compared to TD. These mis-regulated RNA events were statistically significant and may constitute a robust biomarker for FXS individuals. To our surprise, we also found that in 50% FXS WBC samples, FMR1 RNA was synthesized. In these cases, FMR1 RNA was mis-spliced such that an exon was spliced to a “pseudo-exon” within an FMR1 intron. This mis-spliced RNA is polyadenylated and could encode a small polypeptide whose function, if any, is unknown. We generated 2‘-O-methoxyethyl (2’-MOE) antisense oligonucleotides (ASOs) that tiled across the intron, the intron-exon junction, and into the pseudo-exon. When transfected into FXS WBC lines, we found that two ASOs blocked improper FMR1 splicing, rescued proper FMR1 splicing, and most importantly, restored FMRP to TD levels. We also detected FMR1 mis-splicing in FXS postmortem brain, indicating the widespread nature of FMR1 mis-splicing in FXS individuals. These and other data suggest that ASO correction of FMR1 mis-splicing and restoration of FMRP may provide an innovative therapy to treat FXS. To assess the therapeutic potential of ASO treatment of FXS, we will investigate the mechanism of CGG- dependent FMR1 mis-splicing, determine whether additional ASOs with different linkages, lengths, or sequences more efficiently inhibit FMR1 mis-splicing and restore FMRP, examine whether ASO rescue occurs in iPSC- derived FXS neurons, and assess ASO toxicity, pharmacokinetics, and brain targeting following intracerebroventricular (ICV) injection into mice.