CRISPR/Cas9-based gene therapy for Angelman syndrome

项目摘要 Angelman综合征（AS）是一种严重的神经发育障碍， UBE 3A的母系等位基因。UBE 3A在除了成熟的细胞外的几乎所有细胞中都有双等位基因表达。 在神经元中，父本等位基因被称为UBE 3A-ATS的超长非编码RNA沉默。在 根据这种生物学，治疗与AS相关的行为功能障碍的最直接方法是打破沉默， 完整的父亲UBE 3A等位基因。CRISPR/Cas9技术可用于靶向肿瘤细胞的特定区域。 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， 几百个S。化脓性链球菌（Sp）Cas9引导RNA（gRNA），其靶向整个UBE 3A-ATS的区域。几 当这些gRNA与SpCas 9一起沿着转染时，在培养的小鼠中有效地不沉默父本Ube 3a 皮质神经元我们的一些最有效的gRNA靶向Ube 3a-ATS的保守区域， 在小鼠和人类之间的差异，使我们的发现转化为人类神经元成为可能。在这里，我们将测试 CNS指导的Cas9和靶向Ube 3a-ATS的gRNA的递送的中心假设可以 非沉默父亲UBE 3A和治疗与Angelman相关的行为表型 综合征我们将使用腺相关病毒（AAV）进行递送，因为它可以驱动基因表达， 岁月在脑中与S.金黄色葡萄球菌（Sa）Cas9，一种较小的Cas9变体，表明我们的基因 治疗方法可用于在小鼠中使父本Ube 3a不沉默至少三个月。推进这一 创新的基因治疗走向临床，我们将评估疗效，对和脱靶效应，和机制 靶向Ube 3a-ATS的候选治疗性SaCas 9 gRNA的作用。我们将使用培养的神经元 AS模型小鼠和AS衍生的人神经元。我们将把SaCas 9和优化的gRNA包装成一个 单个AAV载体，然后在小鼠中评估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介导的这种递送的程度。 基于CRISPR/Cas9的基因疗法治疗AS模型小鼠的行为表型

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 In unpublished studies, we generated hundreds of S. pyogenes (Sp)Cas9 guide RNAs (gRNAs) that target regions throughout UBE3A-ATS.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.