Single chain Fragments of variable regions in the treatment of Familial ALS

描述(申请人提供)：大约10%的肌萎缩侧索硬化症(ALS)病例是家族性的(FAL)，其中20%的病例是由一种名为超氧化物歧化酶1(SOD1)的酶的突变引起的。许多证据表明，FALS运动神经元死亡的原因不是由于突变的SOD1蛋白缺乏酶活性，而是由于突变的(MT)酶的毒性。一个吸引人的假说是，SOD1蛋白的突变导致它错误折叠，错误折叠导致SOD1在细胞内聚集，并与对细胞生存重要的正常蛋白质-蛋白质相互作用相关的干扰。与这一建议相关的是最近的一项证明，即针对MTSOD1的单抗在转基因的FALS小鼠模型中可以起到治疗作用。抗MTSOD1抗体的可变区单链片段(ScFv)可以补充抗MTSOD1的单抗，因为scFv提供了细胞内(作为体内)表达的机会。我们已经分离出一些针对SOD1抗体的单链抗体，包括针对A4V MTSOD1的单链抗体，这是在北美看到的最常见的SOD1突变，通常在不到一年的时间内导致死亡。我们现在计划：制备更多针对野生型和MTSOD1的scFv；测试通过转染或病毒载体传递的scFv是否减少MTSOD1在MN细胞系和原代MN中的聚集形成和/或毒性；测试通过复制缺陷的重组腺相关病毒(AAV)传递到FALS转基因小鼠的scFv是否减少MTSOD1的聚集，延迟或延缓疾病的进展这些研究可能阐明细胞死亡的原因以及确定对具有SOD1突变的FALS患者的可能的治疗方法。了解FAL运动神经元死亡的机制及其治疗可能有助于我们理解非遗传性ALS--特别是最近的信息表明SOD1的生化变化可能有助于非遗传性ALS的疾病。 公共卫生相关性：肌萎缩侧索硬化症(ALS)是一种无法治愈或有效治疗的绝症。这项建议涉及提高针对一种蛋白的抗体，这种蛋白在某些家族性ALS病例中发生突变，并与散发性ALS的发展有关。我们提出的抗体可能有助于了解ALS的病因，并可能为遗传性ALS和散发性ALS的治疗提供方向。

DESCRIPTION (provided by applicant): Approximately 10% of cases of amyotrophic lateral sclerosis (ALS) are familial (FALS), and 20% of these cases are caused by mutations in an enzyme called superoxide dismutase type 1 (SOD1). Several lines of evidence have made it clear that the reason for motor neuron death in FALS is not due to a deficiency in enzymatic activity by the mutated SOD1 protein, but due to toxicity of the mutant (MT) enzyme. One attractive hypothesis is that the mutations in the SOD1 protein cause it to misfold, and the misfolding leads to aggregation of SOD1 within the cell and an associated interference with normal protein-protein interactions important for cell survival. Relevant to this proposal is the recent demonstration that monoclonal antibodies that are MTSOD1-specific can be therapeutically effective in a MTSOD1 transgenic mouse model of FALS. It may be that single chain fragments of variable region (scFvs) of antibodies directed against MTSOD1 can complement anti-MTSOD1 monoclonal antibodies since scFvs provide the opportunity for expression intracellularly (as intrabodies). We have isolated a number of scFvs of antibodies directed against SOD1, including an scFv that is directed against A4V MTSOD1 - the most common mutation of SOD1 seen in North America, and one that usually causes death in less than a year. We now plan to: prepare additional scFvs against wild type and MTSOD1; test whether scFvs delivered by means of transfection or virus vector delivery decrease aggregate formation and/or toxicity of MTSOD1 expression in a MN cell line and primary MNs; test whether scFvs delivered by means of a replication-deficient recombinant adeno-associated virus (AAV) into FALS transgenic mice decrease MTSOD1 aggregation, delay the onset or slow the progression of disease These studies may clarify the reasons for cell death as well as identify possible therapeutic approaches for FALS patients with SOD1 mutations. The knowledge of the mechanisms responsible for motor neuron death in FALS and its treatment may guide us in our understanding of non-inherited ALS - especially since recent information suggests that biochemical changes in SOD1 may contribute to disease in non-inherited ALS. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a desperate disease in which there is no cure or effective treatment. This proposal involves raising antibodies directed against a protein that is mutated in some cases of familial ALS and has been implicated in the development of sporadic ALS. The antibodies that we raise may help understand the cause of ALS and potentially provide a direction for treatment of inherited ALS as well as sporadic ALS.