Gene Therapy Rescue of Angelman Syndrome with Reelin

翻译后摘要：Angelman综合征（AS）是一种遗传性疾病，大约每15，000名新生儿中发生一个。 其特征是严重的智力残疾、癫痫发作、说话困难和共济失调。致病基因 AS的编码基因为UBE 3A，编码E3泛素连接酶E6 AP。这种基因的一个独特特征是 它以神经元特异性的方式经历母体基因印记。在大多数AS病例中， 突变或缺失在母系遗传的UBE 3A基因，虽然其他情况下是单亲的结果 母体等位基因的二体或甲基化错误。目前，E6 AP作用的具体分子机制 导致AS认知障碍的因素尚未明确，这可能导致缺乏治疗方法。 已经在AS患者和AS小鼠中显示，细胞外基质蛋白Reelin（其 调节几乎所有上面列出的分子组分）显著降低。更重要的是 在一项研究中，补充Reelin可以恢复AS小鼠的表型， 蛋白质注射然而，由于缺乏用于Reelin信号传导的小分子激动剂，因此不存在用于Reelin信号传导的小分子激动剂。 研究探索长期增加的Reelin信号传导作为AS的潜在治疗方法的效果。为了 为了解决这个问题，我们产生了一种新的小的活性Reelin结构，可以包装到腺相关病毒中， 病毒（AAV）载体来实现脑内长期升高的Reelin信号传导。我们证明，这 片段表现出与全长Reelin相同的生物学效应，如在小鼠中的行为拯救所表现出的 使用蛋白质和AAV递送方法的脆性X综合征（FXS）模型。 我们先前表明，rAAV Ube 3a基因替代疗法成功地挽救了一些人的生命。 AS小鼠的认知障碍，但没有完全恢复长时程增强（LTP）缺陷或改善运动 赤字使用目前的病毒载体，在人类甚至动物模型中覆盖所有神经元是不可行的。 这可能会限制用细胞蛋白质如E6 AP进行基因治疗的有效性。我们的新 这种方法提供了使用分泌的Reelin构建体的明显优势，其可以从一个子集扩散 以实现脑内更广泛的分布。我们假设这种Reelin基因疗法 这种方法将挽救AS大鼠的突触可塑性和认知缺陷。我们将测试我们的假设与 的目标。 目的1：rAAV 9-R36挽救AS大鼠的认知缺陷。 目的2：rAAV 9-R36改善AS大鼠的电生理功能。 由于目前缺乏AS的治疗干预，我们独特的Reelin类似物提供了一种新的治疗方法， 改善AS认知的前景。

Abstract: Angelman syndrome (AS) is a genetic disorder occurring approximately one in every 15,000 births. It is characterized by severe intellectual disability, seizures, difficulty speaking and ataxia. The gene responsible for AS was identified as UBE3A and encodes for E6AP, an E3 ubiquitin ligase. A unique feature of this gene is that it undergoes maternal gene imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal allele. Currently, the specific molecular mechanisms of E6AP action that lead to cognitive disruption in AS is not defined and this has likely contributed to lack of therapeutics. It has been shown in AS patients and the AS mouse, that the extracellular matrix protein Reelin (which modulates nearly all of the molecular components listed above) is significantly reduced. More important to this study, Reelin supplementation can recover the phenotype of AS mice from a single intracerebroventricular (ICV) protein injection. However, due to a lack of small molecule agonists for Reelin signaling, there is an absence of studies exploring effects of long-term increased Reelin signaling as a potential therapeutic for AS. In order to address this, we generated a novel small active Reelin construct that can be packaged into an adeno-associated virus (AAV) vector to achieve long-term elevated Reelin signaling within the brain. We demonstrate that this fragment exhibits the same biological effects as full-length Reelin, as exhibited by behavioral rescue in a mouse model of Fragile X syndrome (FXS) using both protein and AAV delivery approaches. We previously shown that a rAAV Ube3a gene replacement therapy was successful in rescuing some cognitive impairments in AS mice but did not fully recover long-term potentiation (LTP) deficits or improve motor deficits. With current viral vectors it is not feasible in humans or even animal models to transduce all neurons within the brain, which may limit the effectiveness of gene therapy with cellular proteins such as E6AP. Our new approach offers the distinct advantage of using the secreted Reelin construct, which can diffuse from a subset of transduced cells to achieve a broader distribution within the brain. We hypothesize this Reelin gene therapy approach will rescue synaptic plasticity and cognitive deficits in the AS rat. We will test our hypothesis with the following aims. Aim 1: rAAV9-R36 rescues cognitive deficits in AS rats. Aim 2: rAAV9-R36 improves electrophysiology deficits in AS rats. With therapeutic interventions currently lacking for AS, our unique Reelin analog offers a novel therapeutic perspective to improve cognition in AS.