Nanoliposome-based Treatment of Amyloid Protein (AL) Toxicity

描述（由申请人提供）： 淀粉样蛋白错误折叠疾病如阿尔茨海默病（AD）和轻链淀粉样变性（AL）仍然是具有高发病率和死亡率的难治性疾病。AL是一种浆细胞恶性肿瘤，由于免疫球蛋白轻链蛋白（LC）的克隆性过度产生，导致其错误折叠并存款在心脏、血管和其他组织中，如果不治疗，中位生存期为4个月。AL是一种恶性肿瘤，退伍军人事务部认为与橙子剂暴露有关，指出其在退伍军人中的具体相关性。化疗和干细胞移植是唯一可用的治疗方法，具有很高的治疗相关死亡率和不完全成功率。我们目前缺乏直接治疗或保护细胞免受淀粉样蛋白损伤的选择。淀粉样蛋白疾病（AL和AD）具有共同的病理生理学，涉及在暴露于可溶性前原纤维淀粉样蛋白的微血管和组织中早期发生的氧化应激和凋亡损伤（现在被认为是损伤的主要途径）。纳米脂质体（nanoliposomes，NLs）是直径小于100 nm的人工磷脂囊泡，其具有治疗淀粉样蛋白疾病的巨大潜力。与其他纳米颗粒不同，NL是人类治疗的理想选择，因为它们无毒、无免疫原性、完全可生物降解且结构多样。它们可以在体内以高亲和力结合淀粉样蛋白，减少细胞暴露于淀粉样蛋白。它们的表面可以进行化学修饰，以允许组织靶向和细胞内甚至细胞器特异性递送治疗货物。在许多可以与NL缀合的治疗性货物中，丛生蛋白（也称为载脂蛋白J）具有保护免受淀粉样蛋白细胞损伤的有希望的潜力。胆固醇是一种组成型表达的分子伴侣糖蛋白，具有转运异常蛋白的双重作用，也是一种重要的促生存蛋白。它在AL和AD中都有异常代谢。我们的总体目标是测试基于纳米脂质体的淀粉样蛋白疾病的治疗，通过开发和测试单独的纳米脂质体（未缀合的）或用丛生蛋白功能化的纳米脂质体（缀合的）作为针对LC诱导的对人血管组织的毒性的保护剂。我们将检验由胆固醇、鞘磷脂（SM）、磷脂酰胆碱（PC）和磷脂酸（PA）组成的NL将结合源自AL患者的LC从而降低对离体人脂肪小动脉的毒性的假设。第二，我们将测试以下假设：功能化以携带丛生蛋白的NL（通过非共价和共价方法）将允许细胞周和细胞内递送丛生蛋白，这也将保护LC免受损伤。为了测试这些假设，在目标1中，我们将开发将有效结合LC的NL并量化NL对LC诱导的毒性的保护作用。目的二是通过与clusterin偶联的方法开发功能化的NLs，用于细胞内的clusterin转运，并检测偶联NLs对LC的保护作用 毒性在这两个目标中，我们将探讨NLs保护作用的关键机制。该提案代表了一种使用纳米颗粒治疗淀粉样蛋白疾病的新方法，该方法可能用于治疗退伍军人和AL淀粉样变性和其他淀粉样疾病（如阿尔茨海默病）患者。

DESCRIPTION (provided by applicant): Amyloid protein-misfolding diseases such as Alzheimer's (AD) and light chain amyloidosis (AL) remain intractable diseases with high morbidity and mortality. In AL, a plasma cell malignancy from clonal overproduction of immunoglobulin light chain proteins (LC) that misfold, and deposit in the heart, vessels and other tissues, median survival is 4 months if left untreated. AL is a malignancy that the VA recognizes as being related to Agent Orange exposure, pointing to its specific relevance in the veteran population. Chemotherapy¿stem cell transplantation, the only treatment available, has high treatment- related mortality and incomplete success rate. We currently lack options to directly treat or protect cells against amyloid protein injury. Amyloid diseases (AL and AD) share common pathophysiology involving oxidative stress and apoptotic injury that occur early in microvessels and tissues exposed to soluble prefibrillar amyloid proteins (now recognized as a major path to injury). Nanoliposomes (NLs) are artificial phospholipid vesicles with a diameter of <100 nm that have great potential for treating amyloid protein disease. Unlike other nanoparticles, NLs are ideal for human treatment because they are non- toxic, non-immunogenic, fully biodegradable and structurally versatile. They can bind amyloid proteins with high affinity in vivo, reducing cell exposure to amyloid proteins. Their surface can be chemically modified to allow tissue targeting and intracellular and even organelle-specific delivery of therapeutic cargo. Among many therapeutic cargos that can be conjugated to NLs, clusterin (also known as apolipoprotein J) has promising potential to protect against amyloid protein cell injury. Clusterin is a constitutively expressed chaperone glycoprotein that has major dual roles of transporting abnormal proteins and is also an important pro-survival protein. It has abnormal metabolism in both AL and AD. Our overall goal is to test nanoliposome-based treatment of amyloid protein disease by developing and testing nanoliposomes alone (unconjugated) or functionalized with clusterin (conjugated) as protective agents against LC-induced toxicity to human vascular tissue. We will test the hypothesis that NLs composed of cholesterol, sphingomyelin (SM), phosphatidylcholine (PC) and phosphatidic acid (PA) will bind LC derived from AL patients thereby reducing toxicity to ex-vivo human adipose arterioles. Second, we will test the hypothesis that NLs functionalized to carry clusterin (both through non-covalent and covalent methods) will allow pericellular and intracellular delivery of clusterin that will also protect against LC injury. To test these hypotheses, in aim 1 we will develop NLs that will efficiently bind LC and quantify NLs' protective effect against LC induced toxicity. In aim 2, we will develop functionalized NLs through conjugation with clusterin for use in intracellular clusterin delivery and test protective effect of conjugated NLs against LC toxicity. In both aims, we will probe key mechanisms underlying the protective effects of NLs. The proposal represents a novel approach to the treatment of amyloid protein disease using nanoparticles that might be useful for the treatment of veterans and patients with AL amyloidosis and other amyloid diseases such as Alzheimer's disease in the future.