Novel Gene Therapy Strategies for Canavan Disease

摘要 Canavan病（CD）是一种罕见的儿童脑白质营养不良，由常染色体隐性突变引起， 乙酰化酶（ASPA）基因。Canavan患者缺乏ASPA导致N- 乙酰天冬氨酸（NAA），导致脑内白色物质肿胀和海绵状变性。的 这种致命疾病的临床表现包括精神发育迟滞，肌肉张力减退， 大头畸形头迟滞癫痫和早逝NAA的合成在线粒体中进行， 神经元通过N-乙酰转移酶-8-样（NAT 8L）和ASPA水解少突胶质细胞。基因 ASPA缺乏的替代疗法是目前治疗CD最有希望的策略。值得注意的是， 最近在Aspa敲除（CD-KO）中实现了Canavan表型的完全治疗校正 小鼠模型单次静脉注射重组腺相关病毒包装的人 ASPA转基因（rAAV-hASPA）在早期完全解决了神经病理学， 在运动功能测试中表现优于野生型。然而，基于强有力的初步证据， 假设CD表型是与代谢功能障碍相关的次要病因。在 此外，我们最近发现ASPA在体外野生型细胞中的过表达导致了异常的细胞凋亡。 线粒体的形状和功能。这些发现需要进一步的临床前研究，重点是：1） 表征ASPA过表达在CNS和外周器官的细胞类型中的可能毒性，2） 开发能够模拟ASPA的内源生理水平的ASPA调节盒， 避免由于治疗而可能存在的副作用，以及3）确定生理和 使用临床相关的非人灵长类动物模型研究ASPA过表达的行为效应。我们的新 研究战略现在建立在我们目前有希望的进展和推进我们的目标，为一个安全， 有效基因治疗CD。

ABSTRACT Canavan disease (CD) is a rare childhood leukodystrophy caused by autosomal recessive mutations in the aspartoacylase (ASPA) gene. Deficiency of ASPA in Canavan patients leads to the accumulation of N- Acetyl-Aspartic Acid (NAA), resulting in swelling and spongy degeneration of white matter in the brain. The clinical manifestations of this fatal disease include psychomotor retardation, muscular hypotonia, macrocephaly, head lag, seizures, and early death. Synthesis of NAA is carried out in the mitochondria of neurons by N-acetyltransferase-8-like (NAT8L) and hydrolyzed in oligodendrocytes by ASPA. Gene replacement therapy for ASPA deficiency is currently the most promising strategy for treating CD. Notably, we have recently achieved full therapeutic correction of the Canavan phenotype in the Aspa knockout (CD-KO) mouse model. A single intravenous injection of recombinant adeno-associated virus packaged with the human ASPA transgene (rAAV-hASPA) at early ages completely resolves neuropathology, resulting in treated animals that outperform wild-types in motor function tests. However, based on strong preliminary evidence, we now hypothesize that the CD phenotype presents a secondary etiology related to metabolism dysfunction. In addition, we recently revealed that overexpression of ASPA in wild type cells in vitro resulted in abnormal mitochondrial shape and function. These findings necessitate further preclinical investigations that focus on: 1) characterizing the possible toxicity of ASPA overexpression in cell types of the CNS and peripheral organs, 2) developing ASPA regulatory cassette(s) that can mimic endogenous physiological levels of ASPA to circumvent adverse effects that may exist due to treatment, and 3) determining the physiological and behavioral effects of ASPA overexpression using a clinically relevant non-human primate model. Our new research strategy now builds on our current promising progress and advances our goals for a safe and effective gene therapy for CD.