CRISPR/Cas9-based gene therapy for Angelman syndrome

基于 CRISPR/Cas9 的 Angelman 综合征基因疗法

• 批准号: 10011898
• 负责人: Mark J. Zylka
• 金额: $ 56.99万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10011898
• 关键词: Age; Alleles; Angelman Syndrome; Antisense Oligonucleotides; Behavioral; Binding; Biodistribution; Biology; Brain; CRISPR/Cas technology; Cells; Child; Childhood; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Dependovirus; Functional disorder; Gene Expression; Gene Transduction Agent; Genes; Genetic Transcription; Guide RNA; Human; Injections; Intervention; Introns; Knowledge; Light; Longevity; Mediating; Medical; Modeling; Mus; Mutagenesis; Mutation; Neurodevelopmental Disorder; Neurons; Phenotype; Pilot Projects; Pre-Clinical Model; Primates; RNA; Research; Site; Small Nucleolar RNA; Staphylococcus aureus; Streptococcus pyogenes; Testing; Therapeutic; Time; Topoisomerase Inhibitors; Topotecan; Toxic effect; Translating; UBE3A gene; Untranslated RNA; Variant; adeno-associated viral vector; autism onset; autism spectrum disorder; base; behavioral phenotyping; design; effective therapy; gene repression; gene therapy; in vivo; innovation; mammalian genome; mouse model; novel strategies; pre-clinical; relating to nervous system; side effect; therapeutic candidate; ubiquitin-protein ligase

PROJECT SUMMARY Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of UBE3A. UBE3A is biallelically expressed in nearly all cells of the body except in mature neurons, where the paternal allele is silenced by an extremely long non-coding RNA called UBE3A-ATS. In light of this biology, the most direct way to treat behavioral dysfunctions associated with AS is to unsilence the intact paternal UBE3A allele. CRISPR/Cas9 technology can be used to target specific regions of the mammalian genome for mutagenesis or transcriptional repression. In unpublished studies, we generated hundreds of S. pyogenes (Sp)Cas9 guide RNAs (gRNAs) that target regions throughout UBE3A-ATS. Several of these gRNAs, when transfected along with SpCas9, potently unsilenced paternal Ube3a in cultured mouse cortical neurons. Some of our most effective gRNAs targeted a region of Ube3a-ATS that is conserved between mice and humans, making it possible to translate our findings to human neurons. Here, we will test the central hypothesis that CNS-directed delivery of Cas9 and a gRNA that targets Ube3a-ATS can enduringly unsilence paternal UBE3A and treat behavioral phenotypes associated with Angelman syndrome. We will use adeno-associated virus (AAV) for delivery because it can drive gene expression for years in the brain. Pilot studies with S. aureus (Sa)Cas9, a smaller Cas9 variant, suggest that our gene therapy approach can be used to unsilence paternal Ube3a in mice for at least three months. To advance this innovative gene therapy towards the clinic, we will evaluate efficacy, on- and off-target effects, and mechanism of action of candidate therapeutic SaCas9 gRNAs that target Ube3a-ATS. We will use cultured neurons from AS model mice and AS-derived human neurons. We will package SaCas9 and an optimized gRNA into a single AAV vector, and then evaluate unsilencing efficacy and longevity for up to two years in mice, as well as biodistribution and toxicity. Lastly, we will evaluate the extent to which AAV-mediated delivery of this CRISPR/Cas9-based gene therapy treats behavioral phenotypes in AS model mice.

项目概要 安格曼综合征（AS）是一种严重的神经发育障碍，由基因缺失或突变引起 UBE3A 的母体等位基因。 UBE3A 在除成熟细胞外的几乎所有身体细胞中双等位表达 神经元中，父系等位基因被称为 UBE3A-ATS 的极长非编码 RNA 沉默。在 根据这种生物学原理，治疗与 AS 相关的行为功能障碍的最直接方法是消除 完整的父本 UBE3A 等位基因。 CRISPR/Cas9技术可用于靶向特定区域 用于诱变或转录抑制的哺乳动物基因组。在未发表的研究中，我们生成了 数百个化脓性链球菌 (Sp)Cas9 引导 RNA (gRNA) 靶向整个 UBE3A-ATS 区域。一些 这些 gRNA 与 SpCas9 一起转染时，可在培养的小鼠中有效地抑制父本 Ube3a 皮质神经元。我们的一些最有效的 gRNA 靶向 Ube3a-ATS 的保守区域 小鼠和人类之间的研究，使我们的发现能够转化为人类神经元。在这里，我们将测试 中心假设是 CNS 定向递送 Cas9 和靶向 Ube3a-ATS 的 gRNA 可以 持久沉默父系 UBE3A 并治疗与 Angelman 相关的行为表型 综合症。我们将使用腺相关病毒（AAV）进行递送，因为它可以驱动基因表达 大脑里的岁月。对金黄色葡萄球菌 (Sa)Cas9（一种较小的 Cas9 变体）的初步研究表明，我们的基因 治疗方法可用于使小鼠的父系 Ube3a 沉默至少三个月。为了推进这个 创新的基因治疗走向临床，我们将评估疗效、脱靶效应和机制 靶向 Ube3a-ATS 的候选治疗 SaCas9 gRNA 的作用。我们将使用来自的培养神经元 AS 模型小鼠和 AS 衍生的人类神经元。我们将 SaCas9 和优化的 gRNA 打包成 单个 AAV 载体，然后评估小鼠体内长达两年的非沉默功效和寿命，以及 生物分布和毒性。最后，我们将评估 AAV 介导的这种传递的程度 基于 CRISPR/Cas9 的基因疗法可治疗 AS 模型小鼠的行为表型。