Gene therapy for glycogen storage disease type III

摘要 糖原贮积症III型(GSD III)是一种常染色体隐性遗传性疾病，由缺乏引起 糖原去分支酶(GDE)导致异常糖原过度堆积(限制 糊精)在肌肉和肝脏组织中。大多数患者(~85%)同时有肌肉和肝脏受累。 (GSD IIIa)，而其他人的疾病仅限于肝脏(GSD IIIb)。在缺乏有效治疗的情况下，患者 患有GSD III的患者正在经历进行性肝功能衰竭和肌肉损伤，同时发病率增加 和死亡率。腺相关病毒(AAV)介导的基因治疗已显示出治疗遗传性疾病的前景 肌肉和肝脏疾病，并成功转化为临床试验。然而，这种方法并没有 高级用于GSD III，因为AAV不能传递大(4.6kb)的人GDE cDNA，因为它 包装容量小。我们已经开发了一种在小鼠身上使用AAV9的创新基因治疗方法 GSD IIIa模型，包含两个关键组件：1)一个小型细菌GDE类似物，以克服 2)一种新型免疫耐受双启动子，可预防细胞毒性T细胞 淋巴细胞(CTL)对细菌酶的反应并使受影响的ALL能够长期表达普鲁兰酶 纸巾。这个项目的总体目标是确定一条前进的道路，以便在临床上翻译这个有希望的 心理治疗。众所周知，AAV载体在小鼠模型中的治疗效果并不总是 翻译成人类。目前的AAV血清型，特别是AAV9，可在高水平的小鼠中转导肌肉和肝脏 然而，在人类患者中，需要高剂量的AAV9来转导骨骼肌可能导致 不良的肝毒性，甚至肝功能衰竭。在这项建议中，我们的目标是确定一种主要的治疗候选AAV 高效跨物种相容AAV衣壳蛋白(CcAAVs)在GSD IIIa小鼠中的载体 免疫转基因表达盒以最大限度地减少基因治疗相关的免疫反应，并减少 有效媒介剂量(目标1a)。我们将在GSD IIIa患者肌肉细胞和人类中验证Lead AAV载体 提高肝嵌合小鼠的临床可译性(目标1b)。然后，我们将检查长期疗效 在GSD IIIa小鼠体内构建AAV先导载体(AIM 2)，并检测其在GSD IIIa犬体内的安全性和有效性(AIM 3)。数据 将为将这一创新的基因疗法应用于患者奠定基础 使用细菌酶通过基因疗法治疗人类疾病的概念可能会打开 为大基因缺陷引起的代谢紊乱的治疗开发提供新的替代方案。这个 免疫耐受双启动子技术也可广泛用于治疗其他影响 用基因疗法治疗多种组织。

ABSTRACT Glycogen storage disease type III (GSD III) is an autosomal recessive inherited disorder caused by deficiency of glycogen debranching enzyme (GDE) that leads to excessive accumulation of abnormal glycogen (limit dextrin) in muscle and liver tissues. The majority of patients (~85%) have both muscle and liver involvement (GSD IIIa) while others have disease limited to the liver (GSD IIIb). In absence of an effective therapy, patients with GSD III are experiencing progressive liver failure and muscle damage accompanied by increased morbidity and mortality. Adeno-associated virus (AAV) mediated gene therapy has shown promise for treating inherited muscle and liver disorders with successful translation to clinical trials. However, this approach has not been advanced for GSD III because AAV is not capable of delivering the large (4.6 kb) human GDE cDNA, due to its small packaging capacity. We have developed an innovative gene therapy approach with AAV9 in a mouse model of GSD IIIa with two key components incorporated 1) a small bacterial GDE analog to overcome the limitation of small AAV carrying capacity; and 2) a novel immunotolerizing dual promoter to prevent cytotoxic T lymphocyte (CTL) response to the bacterial enzyme and enable long-term Pullulanase expression in all affected tissues. The overall objective of this project is to identify a path forward for clinical translation of this promising therapy. It is commonly known that the therapeutic outcomes of AAV vectors in mouse models does not always translate into human. Current AAV serotypes, especially AAV9, transduce muscle and liver in mice with high efficiency; however, high doses of AAV9 required to transduce skeletal muscles in human patients can led to adverse hepatotoxicity or even liver failure. In this proposal, we aim to identify a lead therapeutic candidate AAV vector in GSD IIIa mice using high potency cross-species compatible AAV capsids (ccAAVs) containing a de- immunized transgene expression cassette to minimize gene therapy related immune responses and reduce the effective vector dose (Aim 1a). We will validate the lead AAV vector in GSD IIIa patient muscle cells and human liver chimeric mice to increase its clinical translatability (Aim 1b). We will then examine the long-term efficacy of the lead AAV vector in GSD IIIa mice (Aim 2), and test its safety and efficacy in GSD IIIa dogs (Aim 3). Data generated from the proposed studies will lay the foundation for translating this innovative gene therapy to patients with GSD III. The concept of using a bacterial enzyme to treat human diseases through gene therapy may open up new alternatives for therapeutic development for metabolic disorders caused by defects in large genes. The immunotolerizing dual promoter technology can also be broadly used for treating other conditions that affect multiple tissues with gene therapy.