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，是最常见的遗传形式 智力障碍和自闭症最常见的单基因原因。 FXS是200扩展引起的 FMR1 基因 5' 非翻译区 (UTR) 中存在多个 CGG 三联体，这会导致 FMR1 甲基化 和转录沉默。 FMR1基因产物FMRP的丢失导致蛋白质合成过多 在大脑中，这可能导致该疾病的多种表现。 我们还对 FXS 患者白细胞 (WBC) 的 RNA 进行了深度测序 作为年龄匹配的典型发育（TD）个体。我们发现数百个 RNA 上升或下降 与 TD 相比，FXS WBC 中受到监管。我们还检测到 FXS 中数百个错误剪接的 RNA 与TD相比。这些错误调控的 RNA 事件具有统计显着性，可能构成一个强有力的证据。 FXS 个体的生物标志物。令我们惊讶的是，我们还发现在 50% FXS WBC 样本中，FMR1 RNA 合成的。在这些情况下，FMR1 RNA 被错误剪接，导致外显子被剪接到“伪外显子” FMR1 内含子内。这种错误剪接的 RNA 是聚腺苷酸化的，可以编码一种小多肽，其 功能（如果有的话）是未知的。我们生成了 2'-O-甲氧基乙基 (2'-MOE) 反义寡核苷酸 (ASO) 平铺穿过内含子、内含子-外显子连接处，并进入伪外显子。当转染到 FXS WBC 中时 行中，我们发现两个 ASO 阻断了不正确的 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 死后大脑中检测到 FMR1 错误剪接， 表明 FMR1 错误剪接在 FXS 个体中广泛存在。这些和其他数据表明 ASO 纠正 FMR1 错误剪接和恢复 FMRP 可能为治疗 FXS 提供创新疗法。 为了评估 ASO 治疗 FXS 的治疗潜力，我们将研究 CGG-的机制 依赖的 FMR1 错误剪接，确定是否有具有不同连接、长度或序列的其他 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救援- 衍生的 FXS 神经元，并评估 ASO 毒性、药代动力学和脑靶向性 小鼠脑室内（ICV）注射。