Development of DNAzyme Gene Therapy for Huntington's Disease

延缓遗传性神经退行性疾病进展的药物治疗的临床试验 亨廷顿病(HD)尚未确定有效的药物。部分是因为这一点，也是因为减少 水平的致病突变亨廷顿蛋白很可能是最有效的治疗方法，基因疗法 被许多研究人员和临床医生追踪，通常使用反义寡核苷酸 (ASOS)针对突变的Huntingtin mRNA，或针对干扰其基因的突变Huntingtin的RNA 表达(RNAi)。因为ASO和RNAi传递结构(通常是表达RNAi的AAV)不能 跨越血脑屏障，因为任何一种治疗方法的有效性都会随着时间的推移而减弱，因此直接使用中枢神经系统 为了维持疗效，一年需要注射几次。这种重复注射是安全的 在关键的大脑区域达到足够高的水平以获得治疗益处的风险和担忧。我们 一直在测试一种替代方法，即在R6/2 HD转基因中系统输送DNAzyme 老鼠，并发现它们有相当大的疗效和前景。脱氧核糖核酸酶是人工合成的具有催化活性的酶 结合并切割靶向信使核糖核酸的DNA分子。它们具有一个中心催化结构域，包括 15个脱氧核苷酸的特定序列和两个可变的侧翼结构域，它们可以设计成 将脱氧核酶分子与特定的靶基因杂交。MRNA切割后，DNAzyme解离 切割产物被细胞内的核糖核酸酶进一步降解。脱氧核酶 与其他基因疗法相比，它们具有优势，因为：1)它们具有催化活性，导致更好的 剂量-反应效应；2)它们是稳定的，可以穿过血脑屏障，因此可以传递。 反复全身注射，从而避免需要直接注射中枢神经系统；3)全身注射 给药使DNAzyme同时输送到外周器官和大脑，从而治疗外周和 疾病的主要表现；以及4)在出现不良副作用的情况下可以停止治疗。在……里面 在拟议的研究中，我们将进一步开发系统DNAzyme疗法作为HD治疗，通过 用快速发展的R6/2小鼠模型表征其有效性的程度和持续时间 表达截短突变的人亨廷顿蛋白(Aim 1)和进展较慢的BACHD小鼠 表达全长突变体人亨廷顿蛋白的模型(目标2)。这两个小鼠品系的研究将评估 在每天一个月的腹膜内分娩后，突变的猎人假期会持续多久(Out 在R6/2小鼠中为一个月后，在BACHD小鼠中为两个月后)，并根据突变程度评估收益 大脑和外周器官中Huntingtin mRNA和蛋白质的敲除(qPCR和Western blots)， 行为测试(旋转和开阔场地)，以及脑神经化学和病理学。我们还将评估 任何可能的DNZ6毒性或炎症效应，并确定DNA酶(S)在多大程度上 体外击倒人WT亨廷顿蛋白，体内击倒内源性小鼠WT亨廷顿蛋白。

Clinical trials of pharmacotherapies to slow progression of the hereditary neurodegenerative disorder Huntington's disease (HD) have not identified effective drugs. Partly because of this and because reducing levels of the pathogenic mutant huntingtin protein is likely to be the most effective treatment, gene therapy is being pursued by many investigators and clinicians, typically using either antisense oligonucleotides (ASOs) against mutant huntingtin mRNA, or RNA against mutant huntingtin mRNA that interferes with its expression (RNAi). Because ASOs and RNAi delivery constructs (typically AAV expressing RNAi) cannot cross the blood-brain-barrier and because efficacy of any one treatment wanes with time, direct CNS delivery several times a year will be required to maintain efficacy. Such repeated injections pose safety risks and concerns about achieving high enough levels in the critical brain areas for therapeutic benefit. We have been testing an alternative approach using systemic delivery of DNAzymes in R6/2 HD transgenic mice, and found they have considerable efficacy and promise. DNAzymes are synthetic catalytically active DNA molecules that bind to and cleave targeted mRNA. They possess a central catalytic domain consisting of a specific sequence of 15 deoxynucleotides, and two variable flanking domains that can be designed to hybridize a DNAzyme molecule to a specific target mRNA. After mRNA cleavage, DNAzymes dissociate from the mRNA, and the cleavage products are further degraded by intracellular RNases. DNAzymes possess advantages over other gene therapies because: 1) they are catalytically active, leading to a better dose-response efficacy; 2) they are stable and cross the blood-brain barrier, and thus can be delivered repeatedly by systemic injections, thereby avoiding the need for direct CNS injection; 3) systemic administration yields DNAzyme delivery to both peripheral organs and brain, thus treating peripheral and central manifestations of disease; and 4) therapy can be discontinued in the event of adverse side effects. In the proposed studies, we will further develop systemic DNAzyme therapy as an HD treatment, by characterizing the extent and duration of its effectiveness using the rapidly progressing R6/2 mouse model expressing truncated mutant human huntingtin (Aim 1) and the more slowly progressing BACHD mouse model expressing full-length mutant human huntingtin (Aim 2). Studies in both mouse lines will evaluate how long the mutant huntingtin holiday lasts after a one-month daily intraperitoneal delivery period (out to a month post in R6/2 mice and two months post in BACHD mice), and assess benefit by extent of mutant huntingtin mRNA and protein knockdown in brain and peripheral organs (qPCR and Western blots), behavioral testing (rotarod and open field), and brain neurochemistry and pathology. We will also evaluate any possible DNZ6 toxicity or inflammatory effects, and determine the extent to which the DNAzyme(s) we test knock down human WT huntingtin in vitro and endogenous mouse WT huntingtin in vivo.