Targeting Amyloid Beta Oligomers in the 3xTg-AD Mouse Model with scFv Antibodies

描述（由申请人提供）：阿尔茨海默病（AD）是美国痴呆症的主要原因，对公共健康构成严重威胁。AD患者经历记忆、运动、说话和理解、人格改变和定向障碍的障碍。伴随的神经病理学标志是由淀粉样蛋白-β（Ab）肽组成的老年斑、神经纤维缠结以及海马和新皮质区域内的神经元损失。Ab已成为AD发病机制中的关键参与者;特别是由于早老素1、2和淀粉样前体蛋白中的基因突变（其有效地增加淀粉样蛋白原Ab 1 -42）被认为是导致家族性AD的证据。最近，可溶性低聚形式的Ab（AbO）以及其他淀粉样蛋白的低聚物已经被认为是认知缺陷和突触功能障碍的毒性介质。我们的实验室有兴趣开发一种免疫系统来清除或中和实质空间内的AbO。我们寻求将重组病毒载体递送到大脑，该载体将表达针对各种AbO的人单链片段可变区（scFv）抗体，目标是促进清除并预防突触功能障碍和神经元毒性。目标1：开发针对针对最近显示与记忆缺陷相关的特定寡聚物种类的AbO构象表位的人scFv抗体。我们将筛选不同淀粉样蛋白的人源单链抗体噬菌体展示文库。将在聚集的α-突触核蛋白上淘选噬菌体文库，扩增结合物，随后在AbO上淘选富集的噬菌体原液。目标2：将单链抗体基因克隆到重组腺相关病毒（rAAV）载体中，包装成病毒，并对其表达活性单链抗体的效果进行评价。目的3：通过立体定向注射入海马将rAAV单链抗体递送到AD小鼠模型的脑中。通过使用行为测量、巴恩斯迷宫以及组织学和生物化学分析来评估表达的scFv的作用，以测量脑中的Ab水平。阿尔茨海默病是一种神经退行性疾病，目前影响着估计400 - 500万美国人，每年的医疗保健费用超过1000亿美元，但随着婴儿潮一代的年龄增长，这个数字将增加两倍以上。无论代价多么高昂，都不能为家庭和照顾者所承担的情感负担付出代价。我们希望我们的研究能更好地阐明其病理机制，并为开发有效的AD患者治疗方法迈出重要一步。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the leading cause of dementia in the United States and a serious threat to public health. AD patients experience impairments in memory, movement, speaking and comprehension, personality changes and disorientation. The accompanying neuropathological hallmarks are senile plaques comprised of the amyloid-beta (Ab) peptide, neurofibrillary tangles, and neuronal loss within the hippocampus and neocortical areas. Ab has emerged as a key player in the pathogenesis of AD; particularly due to the evidence that genetic mutations in presenilin 1, 2 and amyloid precursor protein, which effectively increase the pro-amyloidogenic Ab1-42, are thought to cause familial AD. Most recently soluble oligomeric forms of Ab (AbOs), as well oligomers of other amyloidogenic proteins, have been implicated as toxic mediators of cognitive deficits and synaptic dysfunction. Our laboratory is interested in developing an immunotherapeutic to clear or neutralize AbOs within the parenchymal space. We seek to deliver to the brain recombinant viral vectors which will express a human single chain fragment variable (scFv) antibody directed against various AbOs with the goal of facilitating clearance and preventing synaptic dysfunction and neuronal toxicity. AIM 1: Develop human scFv antibodies directed against conformational epitopes of AbOs targeting particular oligomeric species recently shown to be correlated with memory deficits. We will screen a human scFv phage display library on different amyloid proteins. The phage library will be panned on aggregated alpha-synuclein, binders amplified, and the enriched phage stock subsequently panned on AbOs. AIM 2: Clone scFv genes into recombinant adeno-associated viral (rAAV) vectors, package into virus and evaluate for its efficacy to express bio-active scFv antibody. AIM 3: Deliver the rAAV scFv into the brains of an AD mouse model by stereotaxic injections into the hippocampi. Evaluate the effects of the expressed scFv by using a behavioral measure, the Barnes maze, as well as histological and biochemical analyses to measure the Ab levels in the brain. Alzheimer's Disease is a neurodegenerative disease currently affecting an estimated 4-5 million Americans with health care costs over $100 billion annually, yet as the baby boomer generation ages this number will more than triple. No matter how costly, a price can not be put on the emotional burden that family and care- givers undertake. We hope our research better elucidates the pathological mechanisms and will be a great step toward developing an effective therapeutic for AD patients.