AAV-mediated muscle-directed gene transfer for Alzheimer's disease

描述(申请人提供)：阿尔茨海默病(AD)是60岁后最常见的痴呆症病因。阿尔茨海默病的病理特征包括淀粉样肽(A？)沉积在神经炎斑块和脑血管中，神经原纤维缠结和神经元丢失。越来越多的证据支持A？及其前体在阿尔茨海默病的发病机制中发挥重要作用的观点。用合成A免疫阿尔茨海默病小鼠模型可以减少A的沉积，并减弱它们的记忆和学习缺陷。然而，最近的临床试验由于脑部炎症而暂停，推测是由T细胞和/或FC介导的免疫反应引起的。在AD小鼠模型中，外周注射抗A？抗体也可以清除先前存在的淀粉样斑块，这表明主动的T细胞介导的免疫反应是不必要的。局部应用F(ab‘)2而不使用抗A抗体Fc导致AD小鼠模型中淀粉样蛋白沉积的清除，表明非Fc介导的机制也参与了清除。我们已经证明，抗A？的人单链抗体(ScFv)也能有效地降低AD小鼠模型的脑A？负荷。虽然这些被动免疫方案在治疗AD患者时可能是有效的，但不会引起炎症反应等副作用，但这些方案会反复注射抗体，给AD患者带来巨大的经济和身体负担。我们推测，抗A的单链抗体在治疗AD患者和AD小鼠模型中是有效和安全的。我们建议使用新的免疫基因疗法来治疗阿尔茨海默病，即编码抗A？单链抗体的重组腺相关病毒(RAVV)载体被定向到骨骼肌，以便将单链抗体输送到血液循环中，并可能进入大脑。这项研究将作为原则的证明，以证明这种基因治疗方式是否能够将抗A？单链抗体输送到血液循环中，以减少脑A？负荷，并改善AD小鼠模型的学习和记忆障碍。在具体目标1中，我们将评估肌肉导向的基因治疗方式在预防AD小鼠模型中A沉积和行为缺陷方面的效果。在目的2中，我们将评估通过编码抗A？单链抗体的rAAV载体进行肌肉导向基因治疗在清除A？沉积以及改善AD模型小鼠行为缺陷方面的疗效。我们利用神经病理、生化、免疫学和行为分析来确定该方法的预防和治疗效果及安全性。长期目标是为利用rAAV和scFv开发安全有效的AD基因治疗方法奠定逻辑基础。

DESCRIPTION (provided by applicant): Alzheimer's Disease (AD) is the most common cause of dementia after the age of 60. The pathological hallmarks of Alzheimer's disease include deposition of amyloid ¿-peptide (A¿) in neuritic plaques and cerebral blood vessels, neurofibrillary tangles, and loss of neurons. Increasing evidence supports the notion that A¿ and its precursor play important roles in the pathogenesis of Alzheimer's disease. Immunization of mouse models of Alzheimer's disease with synthetic A¿ reduces A¿ deposits and attenuates their memory and learning deficits. Recent clinical trials, however, were halted due to brain inflammation, presumably induced by T-cell mediated and/or Fc-mediated immune responses. Peripheral administration of antibodies against A¿ also induced clearance of preexisting amyloid plaques in AD mouse models, indicating that an active T-cell-mediated immune response is unnecessary. Topical application of the F(ab')2 without Fc of antibodies against A¿ led to clearance of amyloid deposits in an AD mouse model, indicating that non-Fc-mediated mechanisms are also involved in the clearance. We have demonstrated that human single chain antibody (scFv) against A¿ was also effective in reducing brain A¿ load in an AD mouse model. Although these passive immunization modalities may be effective in treating AD patients without inducing side effects such as inflammatory responses, such modalities suffer from repeated administrations of antibodies, leading to a large financial and physical burden to AD patients. We hypothesize that scFvs against A¿ are effective and safe in treating AD patients as well as AD mouse models. We propose to use novel immune gene therapy for Alzheimer's disease, whereby recombinant adeno-associated virus (rAVV) vectors encoding anti-A¿ scFvs are directed to skeletal muscles in order to deliver the scFvs into the circulation and, probably, into the brain. This study will serve as a proof of principle to demonstrate if this gene therapy modality can deliver anti-A¿ scFvs into the circulation enough to reduce brain A¿ load and improve learning and memory deficits in an AD mouse model. In Specific Aim 1, we will evaluate prophylactic effects of muscle directed gene therapy modality by an rAAV vector encoding anti-A¿ scFv on prevention of A¿ deposits and behavioral deficits in an AD mouse model. In Aim 2, we will evaluate therapeutic effects of muscle-directed gene therapy modality by an rAAV vector encoding anti-A¿ scFv on clearance of A¿ deposits as well as improvement of behavioral deficits in an AD mouse model. We utilize neuropathological, biochemical, immunological, and behavioral analyses to determine the prophylactic and therapeutic effects and safety of the modality. The long-term goal is to establish the logical basis for developing safe and effective gene therapy modalities for AD utilizing rAAV and scFv.