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

挽救 FMR1 错误剪接对脆性 X 综合征的治疗潜力

• 批准号: 10713600
• 负责人: Joel D Richter
• 金额: $ 41.88万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713600
• 关键词: 5' Untranslated Regions; Age; Amino Acids; Antisense Oligonucleotides; Autopsy; Binding Sites; Biological Markers; Brain; CGG repeat; Cell Line; Cells; CpG Islands; Cytosine; Data; Developmental Delay Disorders; Disease; Drug Kinetics; Enhancers; Event; Exons; FDA approved; FMR1; FMRP; Fragile X Syndrome; Gene Silencing; Genes; Genetic Transcription; Glutamates; Hippocampus; Human; Impairment; Individual; Induced pluripotent stem cell derived neurons; Inherited; Injections; Innovative Therapy; Intellectual impairment; Introns; Length; Leukocytes; Methylation; Mitochondria; Mosaicism; Mus; Mutation; Nature; Neurodevelopmental Disorder; Neurons; Peptides; Poly A; Polyadenylation; Positioning Attribute; Protein Biosynthesis; RNA; RNA Splicing; Regimen; Ribonuclease H; Sampling; Spinal Muscular Atrophy; Testing; Therapeutic; Therapeutic Agents; Toxic effect; Transfection; Triplet Multiple Birth; Up-Regulation; Vertebral column; autism spectrum disorder; deep sequencing; demethylation; efficacy evaluation; gene product; induced pluripotent stem cell; novel therapeutics; nucleoside analog; polypeptide; prevent; restoration

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.

脆性X综合征(FXS)是一种神经发育障碍，是最常见的遗传性 智力障碍和最常见的导致自闭症的单基因。FXS是由200%的扩张引起的 或更多的CGG三联体位于FMR1基因的5‘非翻译区(UTR)，导致FMR1甲基化 和转录沉默。FMR1基因产物FMRP的缺失导致蛋白质合成过多 这可能导致了这种疾病的几种表现。 我们还对FXS患者的白细胞(WBC)中的RNA进行了深度测序 作为年龄匹配的典型发育中(TD)个体。我们发现有数百个RNA处于运行或关闭状态 与TD相比，FXS WBC中的监管。我们还检测到数百个在FXS中错误拼接的RNA 与TD相比。这些错误调节的RNA事件在统计上具有重要意义，可能构成 FXS患者的生物标志物。令我们惊讶的是，我们还发现在50%的FXS WBC样本中，FMR1 RNA 合成的。在这些情况下，FMR1RNA被错误拼接，导致外显子被拼接到“假外显子”上 在FMR1内含子内。这种错误剪接的RNA是多腺苷化的，可以编码一种小的多肽，其 函数(如果有)未知。我们合成了2‘-O-甲氧基乙基(2’-MoE)反义寡核苷酸(ASO)， 平铺跨越内含子、内含子-外显子连接，进入伪外显子。转染FXS WBC的实验研究 我们发现，两个ASO阻止了不正确的FMR1剪接，挽救了正确的FMR1剪接，并且大多数 重要的是，将FMRP恢复到TD水平。我们还在FXS死后脑中检测到FMR1错误剪接， 这表明FMR1错误剪接在FXS个体中广泛存在。这些数据和其他数据表明 ASO纠正FMR1错误剪接和恢复FMRP可能为治疗FXS提供一种创新的治疗方法。 为了评估ASO治疗FXS的潜力，我们将探讨CGG- 依赖的FMR1错误剪接，确定具有不同连接、长度或序列的额外ASO 更有效地抑制FMR1错误剪接并恢复FMRP，检查IPSC中是否发生ASO拯救- 衍生的FXS神经元，并评估ASO的毒性、药代动力学和脑靶向性 小鼠脑室注射(Icv)。