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 亨廷顿氏病（HD）尚未确定有效的药物。部分原因在于， 水平的致病突变亨廷顿蛋白可能是最有效的治疗，基因治疗 正被许多研究者和临床医生所追求，通常使用反义寡核苷酸， 针对突变亨廷顿蛋白mRNA的反义寡核苷酸（ASO），或针对干扰其表达的突变亨廷顿蛋白mRNA的RNA。 表达（RNAi）。因为ASO和RNAi递送构建体（通常是表达RNAi的AAV）不能 由于任何一种治疗的疗效都随着时间的推移而减弱，直接CNS 需要每年数次给药以保持疗效。这种重复注射的安全性 风险和对在关键大脑区域达到足够高的水平以获得治疗益处的担忧。我们 我一直在测试一种替代方法，该方法使用在R6/2 HD转基因小鼠中全身递送DNA酶， 小鼠，并发现它们具有相当大的功效和前景。DNA酶具有合成催化活性 结合并切割靶mRNA的DNA分子。它们具有一个中心催化结构域， 15个脱氧核苷酸的特定序列，和两个可变侧翼结构域，可以设计为 使DNA酶分子与特定靶mRNA杂交。mRNA裂解后，DNA酶解离 裂解产物被细胞内RNA酶进一步降解。dna酶 具有优于其他基因疗法的优势，因为：1）它们具有催化活性，导致更好的 剂量-反应功效; 2）它们是稳定的并且穿过血脑屏障，并且因此可以被递送 通过全身注射重复，从而避免直接CNS注射的需要; 3）全身 施用产生DNA酶递送至外周器官和脑两者，从而治疗外周和 疾病的中枢表现;和4）在不良副作用的情况下可以中断治疗。在 在拟议的研究中，我们将进一步开发作为HD治疗的全身性DNAzyme治疗， 使用快速进展的R6/2小鼠模型表征其有效性的程度和持续时间 表达截短的突变型人亨廷顿蛋白（Aim 1）和进展更慢的BACHD小鼠 表达全长突变型人亨廷顿蛋白的模型（Aim 2）。两种小鼠品系的研究将评价 在一个月的每日腹膜内分娩期（out）后，突变的亨廷顿假期持续多长时间 在R6/2小鼠中为一个月后，在BACHD小鼠中为两个月后），并通过突变体的程度评估益处。 脑和外周器官中亨廷顿蛋白mRNA和蛋白质敲低（qPCR和蛋白质印迹）， 行为测试（旋转棒和开放领域），以及脑神经化学和病理学。我们还将评估 任何可能的DNZ 6毒性或炎症作用，并确定DNA酶在多大程度上被激活， 在体外测试敲低人WT亨廷顿蛋白和在体内测试敲低内源性小鼠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.