Gene Therapy Rescue of Angelman Syndrome with Reelin

Reelin 基因疗法拯救天使综合症

• 批准号: 10317654
• 负责人: Kevin Ron Nash
• 金额: $ 41.11万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10317654
• 关键词: Acids; Address; Agonist; Alleles; Angelman Syndrome; Animal Model; Animals; Ataxia; Behavioral; Biochemical; Biological; Birth; Brain; Cerebrospinal Fluid; Chronic; Clinical; Cognition; Cognitive; Cognitive deficits; Data; Dendritic Spines; Dependovirus; Diffuse; Disease; EGF gene; Effectiveness; Electrophysiology (science); Exhibits; Extracellular Matrix Proteins; Fragile X Syndrome; Genes; Genetic Diseases; Grant; Hippocampus (Brain); Histocytochemistry; Human; Impaired cognition; Impairment; Inherited; Injections; Lead; Learning; Length; Long-Term Depression; Long-Term Effects; Long-Term Potentiation; Memory; Modeling; Molecular; Motor; Mus; Mutation; N-Methyl-D-Aspartate Receptors; Neurodevelopmental Disorder; Neurons; Pathology; Patients; Phenotype; Process; Protease Domain; Proteins; Publishing; Rattus; Recombinant adeno-associated virus (rAAV); Recovery of Function; Reelin Signaling Pathway; Rodent; Seizures; Signal Transduction; Signaling Protein; Site; Supplementation; Synapses; Synaptic Transmission; Synaptic plasticity; Testing; Therapeutic; Therapeutic Intervention; UBE3A gene; Uniparental Disomy; Vertebral column; Viral Vector; Western Blotting; adeno-associated viral vector; analog; behavior test; calmodulin-dependent protein kinase II; cellular transduction; cognitive testing; density; extracellular; gene replacement therapy; gene therapy; imprint; improved; in vivo; innovation; motor deficit; mouse model; nonhuman primate; novel; novel strategies; novel therapeutics; protein biomarkers; rare genetic disorder; receptor function; reelin protein; restoration; severe intellectual disability; small molecule; therapeutic target; tool; ubiquitin-protein ligase; vector

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.

摘要：Angelman综合征（AS）是一种遗传疾病，每15,000个出生大约发生。 它的特征是严重的智力残疾，癫痫发作，难以说话和共济失调。负责的基因 对于AS被识别为UBE3A，并为E6AP（E3泛素连接酶）编码。该基因的独特特征是 它以神经元特异性的方式经历产妇基因。在大多数AS情况下，有一个 母体遗传的UBE3A基因中的突变或缺失，尽管其他情况是单亲的结果 孕产妇等位基因的疾病或甲基化。目前，E6AP作用的特定分子机制 这导致认知破坏AS未定义，这可能导致缺乏治疗剂。 已显示为患者和AS小鼠，细胞外基质蛋白reelin（其中 调节几乎所有上述分子成分）显着降低。对此更重要 研究，补充reelin可以从单个脑室内（ICV）中恢复小鼠的表型 蛋白质注射。但是，由于缺乏用于reelin信号的小分子激动剂，因此没有 研究探索长期reelin信号传导的效果是AS的潜在治疗方法。为了 解决这个问题，我们生成了一种新型的小型活性reelin构造，可以包装到腺相关的 病毒（AAV）载体可以实现大脑内长期升高的reelin信号。我们证明了这一点 片段表现出与全长reelin相同的生物学效应，如行为救援在小鼠中所表现出的 使用蛋白质和AAV递送方法的脆弱X综合征（FXS）模型。 我们先前表明，RAAV UBE3A基因替代疗法成功地营救了一些 AS小鼠的认知障碍，但没有完全恢复长期增强（LTP）缺陷或改善电动机 缺陷。使用当前病毒载体，在人类甚至动物模型中都不可行，可以转导所有神经元 在大脑内，这可能会限制基因治疗使用细胞蛋白（例如E6AP）的有效性。我们的新 方法提供了使用分泌的reelin构建体的明显优势，可以从子集中扩散 转导的细胞以实现大脑内部更广泛的分布。我们假设这种reelin基因治疗 方法将挽救AS大鼠的突触可塑性和认知缺陷。我们将通过 以下目标。 AIM 1：RAAV9-R36挽救了作为大鼠的认知缺陷。 AIM 2：RAAV9-R36改善了AS大鼠的电生理缺陷。 由于目前缺乏治疗性干预措施，我们独特的Reelin Analog提供了一种新颖的治疗性 改善认知的观点。