CRISPR/Cas9-based gene therapy for Angelman syndrome

项目总结 Angelman综合征(AS)是由基因缺失或突变引起的一种严重的神经发育障碍 UBE3A的母系等位基因。UBE3A在体内除成熟细胞外的几乎所有细胞中双等位表达 其中父亲的等位基因被一种名为UBE3A-ATS的超长非编码RNA沉默。在……里面 从这一生物学角度来看，治疗与强直性脊柱炎相关的行为障碍的最直接方法是解除沉默 完整的父系UBE3A等位基因。CRISPR/Cas9技术可用于针对 用于突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 数百个化脓性链球菌(Sp)Cas9指导RNAs(GRNAs)，靶向整个UBE3A-ATS区域。几个 在这些gRNA中，当与SpCas9一起转染时，在培养的小鼠中有效地沉默了父亲的Ube3a 皮质神经元。我们的一些最有效的gRNA针对的是Ube3a-ATS的保守区域 在老鼠和人类之间，使我们的发现有可能转化为人类神经元。在这里，我们将测试 中枢假设，即CNS导向的Cas9和靶向Ube3a-ATS的gRNA可以 持久不让父亲UBE3A沉默并治疗与Angelman相关的行为表型 综合症。我们将使用腺相关病毒(AAV)进行传递，因为它可以驱动基因表达 在大脑里呆了几年。对金黄色葡萄球菌(Sa)Cas9的初步研究表明，我们的基因 治疗方法可用于解除小鼠父亲Ube3a至少三个月的沉默。为了推进这一进程 创新的基因治疗将走向临床，我们将评估疗效、靶上和靶外效应以及机制 靶向Ube3a-ATS的候选治疗性SaCas9 gRNA的作用。我们将使用培养的神经元 作为模型小鼠和AS来源的人类神经元。我们将把SaCas9和优化后的gRNA打包成 单个AAV载体，然后评估小鼠的去沉默效果和寿命长达两年，以及 生物分布和毒性。最后，我们将评估AAV介导的交付在多大程度上 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 In unpublished studies, we generated hundreds of S. pyogenes (Sp)Cas9 guide RNAs (gRNAs) that target regions throughout UBE3A-ATS.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.