Nanoparticle-mediated delivery of a base editor for in utero treatment of Canavan Disease

摘要 Canavan病是一种罕见的遗传性神经系统疾病，以空泡(海绵状)为特征。 脑白质营养不良是由于ASPA突变导致大脑天冬氨酸氨基转移酶活性降低而引起的。 新生儿/婴儿卡纳万病是最常见和最严重的疾病形式。 受影响的婴儿在生命的头几个月看起来是正常的，但发育方面的问题 在3到5个月大的时候很明显。这些婴儿通常不会发展翻身等运动技能， 控制头部运动，在没有支撑的情况下坐着。这种情况的其他特征包括 低眼压、大头畸形和易怒。没有治愈的方法，也没有标准的疗程。 对这种神经退行性疾病的症状支持包括饮食控制， 柠檬酸锂和抗惊厥药物的给药以及AAV介导的脑实质ASPA基因 治疗，然而，这些治疗不能逆转或阻止神经功能障碍的进展。 受影响的婴儿和儿童。为了解决这一未得到满足的医疗需求，我们建议开发一种创新的 治疗包括宫内递送固体脂质纳米粒(LNP)/信使核糖核酸复合体 脑室注射，将在出生前靶向和编辑少突胶质细胞，纠正尽快突变， 并恢复受点突变影响的患者的天冬氨酸酯酶表达。In的基因编辑 宫内期在治疗神经系统疾病方面有很大的潜力，包括以前的Canavan病 症状未出现。我们的初步研究表明，在宫内脑室交付LNP 信使核糖核酸复合体可以有效地将信使核糖核酸传递到脑和脊髓组织。这些实验 是第一个在子宫内进行基于信使核糖核酸的非病毒基因编辑的示范，并开启了许多 治疗应用，给出了信使核糖核酸的巨大多功能性。这一点的中心假设是 提议是：LNPs将在子宫内传递基因编辑酶的mRNA，并将把小鼠从 出生时和出生后的卡纳万病。这一假设是基于我们的初步数据证明的 含Cre的LNPs可安全高效地转导Ai9小鼠脑内少突胶质细胞 宫内脑室分娩后。这项研究的中心目标是开发LNP配方 这可以有效地编辑子宫内的少突胶质细胞，并开发一种治疗Canavan病的策略。

ABSTRACT Canavan disease is a rare genetic neurological disorder characterized by a vacuolar (spongiform) leukodystrophy caused by ASPA mutations that diminish brain aspartoacylase activity. Neonatal/infantile Canavan disease is the most common and most severe form of the condition. Affected infants appear normal for the first few months of life, but problems with development become noticeable by age 3 to 5 months. These infants usually do not develop motor skills such as turning over, controlling head movement, and sitting without support. Other characteristics of this condition include hypotonia, macrocephaly, and irritability. There is no cure, nor is there a standard course of treatment. The symptomatic support for this neurodegenerative disorder includes dietary manipulations, administration of lithium citrate and anticonvulsants, and AAV-mediated brain parenchymal ASPA gene therapy, yet, these treatments do not reverse or prevent the progression of neurological deficits in affected infants and children. To address this unmet medical need, we propose to develop an innovative treatment comprised of an in utero delivery of solid lipid nanoparticle (LNP)/mRNA complexes by intraventricular injection that will target and edit oligodendrocyte before birth, correct ASAP mutations, and restore aspartoacylase expression in patients influenced by point mutations. Gene editing at the in utero stage has great potential to cure neurological disorders, including Canavan disease before symptoms are unset. Our preliminary studies demonstrated that in utero intraventricular delivery LNP mRNA complexes can efficiently deliver mRNA to the brain and spinal cord tissue. These experiments are the first demonstration of mRNA based nonviral gene editing in utero and open up numerous therapeutic applications, given the tremendous versatility of mRNA. The central hypothesis of this proposal is that: LNPs will deliver mRNA for gene editing enzymes in utero and will rescue mice from Canavan disease at and after birth. This hypothesis is based upon our preliminary data demonstrating that LNPs containing CRE can safely and efficiently transfect oligodendrocyte in the brain of Ai9 mice after in utero intraventricular delivery. The central objective of this study is to develop LNP formulations that can efficiently edit oligodendrocyte in utero and develop a strategy for treating Canavan disease.