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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

脆弱的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最普遍的自闭症原因。 FXS是由200个扩展引起的或FMR1基因的5'非翻译区（UTR）中的CGG三重态，该区域导致FMR1甲基化和转录沉默。 FMR1基因产物FMRP的损失导致过量蛋白质合成在大脑中，这可能导致该疾病的几种表现。我们还对FXS个体的白细胞（WBC）进行了深入的RNA测序作为年龄匹配的人通常是发展（TD）个体。我们发现数百个RNA上下与TD相比，在FXS WBC中受到调节。我们还检测到数百个在FXS中被删除的RNA 与TD相比。这些未调节的RNA事件具有统计学意义，可能构成强大的 FXS个人的生物标志物。令我们惊讶的是，我们还发现在50％的FXS WBC样品中，FMR1 RNA为合成。在这些情况下，FMR1 RNA被遗漏，以使外显子被剪接到“伪外的” 在FMR1内含子中。该遗漏的RNA是聚腺苷酸化的，可以编码一个小多肽功能（如果有）是未知的。我们产生了2'-O-甲氧亚乙基（2'-MoE）反义寡核苷酸（ASO）铺在内含子，内含子 - 外观交界处，并进入伪外的瓷砖。当转染FXS WBC时线，我们发现两个ASO阻断了不当的FMR1剪接，恢复了适当的FMR1剪接，并且大多数重要的是，将FMRP恢复到TD水平。我们还检测到FXS验尸大脑中的FMR1误解，表明FXS个体中FMR1错过的宽度性质。这些和其他数据表明 FMR1错过的ASO校正和FMRP的恢复可能会提供一种创新的治疗FXS的疗法。为了评估ASO治疗FXS的治疗潜力，我们将研究CGG- 依赖的FMR1失误切换，确定具有不同链接，长度或序列的其他ASO是否更有效地抑制fMR1失误并恢复FMRP，检查ASO救援是否发生在IPSC- 衍生的FXS神经元以及评估ASO毒性，药代动力学和大脑靶向以下脑室内（ICV）注射小鼠。