Second Generation Gene Therapy for APOE4 Homozygous Individuals at High Risk for Alzheimer's Disease

阿尔茨海默病（AD）是一种退行性脑部疾病，也是痴呆症最常见的原因，目前影响着580万美国人和全球5000万人。AD症状包括认知和功能能力的进行性下降以及脑病理学，包括细胞外β-淀粉样蛋白斑块、细胞内tau蛋白缠结、慢性炎症和脑萎缩。晚发性AD易感性的最强遗传危险因素涉及载脂蛋白E（APOE）等位基因的多态性。在AD患者中发现APOE 4的频率很高，纯合子遗传与AD发生风险增加14.5倍相关。相比之下，APOE 2可降低AD发展的风险并延迟疾病的发作。先前的研究表明，使用腺相关病毒（AAV）载体将APOE 2直接递送至AD小鼠模型的CNS可提供显著的APOE 2表达，并降低可溶性和不溶性淀粉样蛋白-β肽和淀粉样蛋白负荷的水平。然而，即使补充了APOE 2，APOE 4的存在仍然构成增加的风险，因为APOE 2/4杂合子患AD的风险增加2.6倍。我们建议开发第二代基于AAV的基因疗法，以引入保护性APOE 2的表达，同时降低APOE 4纯合子中有害的内源性APOE 4的水平。我们的策略是将靶向内源性APOE 4的人工microRNA（miRNA）与人APOE 2基因的cDNA（hAPOE 2-mirAPOE 4）一起编码到AAV表达盒中沿着。AAV 9血清型衣壳将用于包装表达盒，因为它介导星形胶质细胞（APOE的主要生产者）以及小胶质细胞和神经元的有效转导。已建立的表达突变型人tau和人APOE 4并具有高磷酸化tau负荷、慢性炎症和广泛神经变性的P301 S/E4 AD小鼠模型将用于评估治疗疗效。为了用AAV 9-hAPOE 2-mirAPOE 4评估这种第二代策略，我们提出了两个具体目标：目标1。在体外测试AAV构建体沉默内源性APOE 4表达并递送APOE 2编码序列。目标2.确定APOE 2的增加与内源性APOE 4的减少是否在体内保护免于tau病理、神经变性和神经炎症。

Alzheimer’s disease (AD), a degenerative brain disease and the most common cause of dementia, currently affects 5.8 million Americans and 50 million people worldwide. AD symptoms include progressive decline of cognitive and functional abilities and brain pathology, including extracellular beta-amyloid plaques, intracellular tau tangles, chronic inflammation, and brain atrophy. The strongest genetic risk factor for susceptibility to late-onset AD concerns polymorphisms in the apolipoprotein E (APOE) allele. APOE4 is found at high frequency in AD patients and homozygous inheritance is associated with a 14.5-fold increased risk of developing AD. In contrast, APOE2 decreases risk of AD development and delays onset of disease. A previous study demonstrated that delivery of APOE2 directly to the CNS of AD murine models using an adeno-associated virus (AAV) vector could provide significant APOE2 expression and decrease the levels of soluble and insoluble amyloid-β peptide and amyloid burden. However, even with APOE2 supplementation, the presence of APOE4 still constitutes an increased risk as APOE2/4 heterozygotes have a 2.6-fold increased risk of developing AD. We propose to develop a second-generation AAV-based gene therapy to introduce expression of the protective APOE2 while simultaneously decreasing the levels of deleterious endogenous APOE4 in APOE4 homozygotes. Our strategy is to encode artificial microRNAs (miRNA) targeting the endogenous APOE4 into the AAV expression cassette along with the cDNA for the human APOE2 gene (hAPOE2-mirAPOE4). The AAV9 serotype capsid will be used to package the expression cassette because it mediates efficient transduction of astrocytes, the main producers of APOE, as well as microglia and neurons. The established P301S/E4 AD mouse model that expresses mutant human tau and human APOE4 and has high phosphorylated tau burden, chronic inflammation, and extensive neurodegeneration will be used to assess therapy efficacy. To evaluate this second-generation strategy with AAV9-hAPOE2-mirAPOE4, we propose two specific aims: Aim 1. Test an AAV construct to silence endogenous APOE4 expression and deliver the APOE2 coding sequence in vitro. Aim 2. Determine if augmentation of APOE2 with reduction of endogenous APOE4 protects against tau pathology, neurodegeneration, and neuroinflammation in vivo.