CRISPR/Cas9-based gene therapy for Angelman syndrome

基于 CRISPR/Cas9 的 Angelman 综合征基因疗法

• 批准号: 10490828
• 负责人: Mark J. Zylka
• 金额: $ 54.07万
• 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/10490828
• 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; efficacy evaluation; efficacy validation; 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 In unpublished studies, we generated hundreds of S. pyogenes (Sp)Cas9 guide RNAs (gRNAs) that target regions throughout UBE3A-ATS.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.

项目摘要 Angelman综合征（AS）是由缺失或突变引起的严重神经发育障碍 Ube3a的母亲等位基因。 Ube3a在身体几乎所有细胞中均以双重表达 神经元，父亲等位基因被一种非常长的非编码RNA沉默，称为ube3a-ats。在 这种生物学的光，这是治疗与无限相关的行为功能障碍的最直接方法 完整的父亲Ube3a等位基因。 CRISPR/CAS9技术可用于针对特定区域 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，我们产生了 靶向整个UBE3A-ATS的数百个链球菌（SP）CAS9引导RNA（GRNA）。一些 在这些grnas中，当与SPCAS9一起转染时，在培养的小鼠中有效未灌输的父亲Ube3a 皮质神经元。我们最有效的GRNA针对的是ube3a-ats的区域 在小鼠和人类之间，可以将我们的发现转化为人类神经元。在这里，我们将测试 CNS定向CAS9和针对UBE3A-ATS的GRNA的中心假设可以 持久的含父子UBE3A并治疗与Angelman相关的行为表型 综合征。我们将使用腺相关病毒（AAV）进行输送，因为它可以驱动基因表达 在大脑中多年。较小的Cas9变体的Aureus（SA）Cas9的试点研究表明我们的基因 治疗方法可用于至少三个月的小鼠中的父亲UBE3A。推进这一点 对诊所的创新基因疗法，我们将评估功效，靶向效果和机制 针对ube3a-ats的候选治疗性sacas9 grnas的作用。我们将使用培养的神经元 作为模型小鼠和衍生的人类神经元。我们将把sacas9和优化的grna包装到一个 单个AAV矢量，然后在小鼠中评估长达两年的未递送功效和寿命 生物分布和毒性。最后，我们将评估AAV介导的此次交付的程度 基于CRISPR/CAS9的基因疗法将AS Model小鼠的行为表型处理。