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

摘要 卡纳万病是一种罕见的遗传性神经系统疾病，其特征是空泡状（海绵状） 由ASPA突变引起的脑白质营养不良，该突变降低了脑乙酰化酶的活性。 新生儿/婴儿卡纳万病是最常见和最严重的形式的条件。 受影响的婴儿在出生后的头几个月看起来正常，但发育问题变得越来越严重。 在3到5个月大的时候就很明显了。这些婴儿通常不会发展运动技能，如翻身， 控制头部运动，无支撑地坐着。这种情况的其他特征包括 张力减退、大头畸形和易怒。没有治愈方法，也没有标准的治疗过程。 这种神经退行性疾病的症状支持包括饮食控制， 施用柠檬酸锂和抗惊厥剂，以及AAV介导的脑实质ASPA基因 然而，这些治疗并不能逆转或阻止神经功能缺损的进展， 影响婴儿和儿童。为了满足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，包括通过子宫内递送固体脂质纳米颗粒（LNP）/mRNA复合物， 脑室内注射将在出生前靶向和编辑少突胶质细胞，纠正ASAP突变， 并恢复受点突变影响的患者的乙酰化酶表达。基因编辑技术 子宫期有很大的潜力治愈神经系统疾病，包括卡纳万病之前， 症状不明显。我们的初步研究表明，在子宫内脑室内输送LNP mRNA复合物可以有效地将mRNA递送到脑和脊髓组织。这些实验 是首次在子宫内展示基于mRNA的非病毒基因编辑， 考虑到mRNA的巨大多功能性，这个问题的核心假设是 LNP将在子宫内为基因编辑酶提供mRNA，并将拯救小鼠， 出生时和出生后的Canavan病。这个假设是基于我们的初步数据， 含CRE的LNPs能安全、有效地抑制Ai 9小鼠脑内少突胶质细胞 在子宫内脑室内分娩后。本研究的中心目标是开发LNP制剂 它可以在子宫内有效地编辑少突胶质细胞，并开发出治疗卡纳万病的策略。

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.