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（海绵状） 由 ASPA 突变引起的脑白质营养不良会降低大脑天冬氨酸酰化酶的活性。 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最初几个月表现正常，但发育问题变得严重 3至5个月大时明显。这些婴儿通常不会发展运动技能，例如翻身、 控制头部运动，以及在没有支撑的情况下坐着。这种情况的其他特征包括 肌张力低下、大头畸形和烦躁。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、 给予柠檬酸锂和抗惊厥药，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 受影响的婴儿和儿童。为了解决这一未满足的医疗需求，我们建议开发一种创新的 治疗包括在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天冬氨酸酰化酶表达。基因编辑在 子宫阶段具有治愈神经系统疾病的巨大潜力，包括之前的卡纳万病 症状尚未确定。我们的初步研究表明，在子宫内递送 LNP mRNA复合物可以有效地将mRNA递送至大脑和脊髓组织。这些实验 是子宫内基于 mRNA 的非病毒基因编辑的首次演示，并开辟了许多 鉴于 mRNA 的巨大多功能性，其在治疗方面的应用。这个假设的中心假设 提议是：LNP 将在子宫内传递基因编辑酶的 mRNA，并将小鼠从 出生时和出生后的卡纳万病。这个假设是基于我们的初步数据证明 含有CRE的LNP可以安全有效地转染Ai9小鼠大脑中的少突胶质细胞 宫内分娩后。本研究的中心目标是开发 LNP 制剂 可以在子宫内有效编辑少突胶质细胞并制定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.