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，是对公众健康的严重威胁。阿尔茨海默病患者在记忆、运动、说话和理解、性格改变和定向障碍方面都有障碍。伴随的神经病理特征是由淀粉样蛋白- β (Ab)肽组成的老年斑，神经原纤维缠结，以及海马和新皮层区域的神经元丢失。Ab在AD的发病机制中起着关键作用；特别是有证据表明，早老素1、2和淀粉样蛋白前体蛋白的基因突变可以有效地增加淀粉样蛋白原a1 -42，这被认为是导致家族性AD的原因。最近，Ab的可溶性低聚物形式，以及其他淀粉样蛋白的低聚物，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疗法来清除或中和实质空间内的abo。我们寻求向大脑传递重组病毒载体，该载体将表达针对各种abo的人单链片段变量（scFv）抗体，目的是促进清除和防止突触功能障碍和神经元毒性。目标1：开发针对abo构象表位的人单链抗体，靶向最近被证明与记忆缺陷相关的特定寡聚物种。我们将在不同的淀粉样蛋白上筛选人类scFv噬菌体展示文库。噬菌体文库将在聚集的α -突触核蛋白上筛选，扩增结合物，然后在abo上筛选富集的噬菌体。目的2：将scFv基因克隆到重组腺相关病毒（rAAV）载体中，包装成病毒，评价其表达生物活性scFv抗体的效果。目的3：通过海马立体定向注射将rAAV scFv注入AD小鼠模型的大脑。通过行为学测量，巴恩斯迷宫，以及组织学和生化分析来测量大脑中的Ab水平，评估表达的scFv的影响。阿尔茨海默病是一种神经退行性疾病，目前估计有400万至500万美国人受到影响，每年的医疗费用超过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。无论多么昂贵，家庭和照顾者所承担的情感负担是无法用金钱来衡量的。我们希望我们的研究能够更好地阐明其病理机制，并为开发有效的治疗AD患者的方法迈出一大步。

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.