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

阿尔茨海默病（AD）是一种退行性脑疾病，也是痴呆症的最常见原因，目前影响着580万美国人和全球5000万人。阿尔茨海默病的症状包括认知和功能能力的进行性下降以及脑病理，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。对迟发性阿尔茨海默病易感性最强的遗传风险因素与载脂蛋白E （APOE）等位基因的多态性有关。APOE4在阿尔茨海默病患者中发现频率很高，纯合遗传与患阿尔茨海默病的风险增加14.5倍相关。相反，APOE2降低了AD发展的风险并延缓了疾病的发作。先前的研究表明，使用腺相关病毒（AAV）载体将APOE2直接递送到AD小鼠模型的中枢神经系统可以显著提高APOE2的表达，并降低可溶性和不可溶性淀粉样蛋白-β肽和淀粉样蛋白负荷的水平。然而，即使补充了APOE2， APOE4的存在仍然会增加患AD的风险，因为APOE2/4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，引入保护性APOE2的表达，同时降低APOE4纯合子中有害的内源性APOE4的水平。我们的策略是将针对内源性APOE4的人工microRNAs （miRNA）与人类APOE2基因（hAPOE2-mirAPOE4）的cDNA一起编码到AAV表达盒中。AAV9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的主要产生者）以及小胶质细胞和神经元的有效转导。已建立的P301S/E4 AD小鼠模型表达突变的人tau和人APOE4，具有高磷酸化tau负荷、慢性炎症和广泛的神经变性，将用于评估治疗效果。为了评估AAV9-hAPOE2-mirAPOE4的第二代策略，我们提出了两个具体目标：测试AAV结构沉默内源性APOE4表达并在体外传递APOE2编码序列。目标2。确定APOE2的增加与内源性APOE4的减少是否在体内保护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.