mRNA递送脂质纳米颗粒在新生小鼠内耳中的生物分布探索

文献信息

中文摘要

背景： 听力损失影响全球数百万人，但助听器和人工耳蜗存在重大局限。基于mRNA的基因治疗可能解决潜在的分子病因，但脂质纳米颗粒（LNP）在内耳中的递送仍研究不足，其生物分布尚未被系统表征。方法：在此，我们基于预定义的关键质量属性，采用实验设计（DoE）方法建立并放大了一种包裹mRNA的LNP制剂，并绘制了报告基因mRNA表达在新生小鼠耳蜗中依赖于给药途径的分布图。结果：在HEI-OC1听觉细胞中，与阳离子试剂Lipofectamine在匹配mRNA剂量下相比，负载mRNA的LNP产生更高比例的报告基因表达细胞，且表达分布更均匀，而总报告蛋白相当，代谢活力未降低。随后我们比较了经圆窗膜（RWM）递送与经蜗管（scala media）给药后的耳蜗分布。RWM递送在外淋巴暴露区室——螺旋韧带、前庭阶和鼓阶区域、血管纹和螺旋神经节区域——产生广泛的报告基因表达，在毛细胞中未检测到表达。蜗管注射产生更局限的模式，主要局限于内毛细胞和外毛细胞的表皮板以及部分外沟，在螺旋神经节区域或听神经中无表达，表明该途径不会分布到内淋巴腔室之外。结论：这些结果界定了LNP介导的mRNA递送可到达的耳蜗区室，以及这种可达性对给药途径的依赖性，并为内耳中基于LNP的mRNA策略设计提供了所需的递送图谱。

英文摘要

Hearing loss affects millions worldwide, yet hearing aids and cochlear implants have major limitations. mRNA-based gene therapy could address the underlying molecular causes, but lipid nanoparticle (LNP) delivery in the inner ear remains underexplored and its biodistribution has not been systematically characterized. Here we establish and scale up an mRNA-encapsulated LNP formulation using a design-of-experiments (DoE) approach built on predefined critical quality attributes, and map the route-dependent distribution of reporter mRNA expression in the neonatal mouse cochlea. In HEI-OC1 auditory cells, mRNA-loaded LNPs produced a higher proportion of reporter-expressing cells and a more uniform distribution of expression than the cationic reagent Lipofectamine at a matched mRNA dose, while total reporter protein was comparable and metabolic viability was not reduced. We then compared cochlear distribution following round window membrane (RWM) delivery versus scala media administration. RWM delivery produced widespread reporter expression in perilymph-exposed compartments-the spiral ligament, the scala vestibuli and scala tympani regions, the stria vascularis and the spiral ganglion region-with no detectable expression in hair cells. Scala media injection produced a more restricted pattern, largely confined to the cuticular plate of inner and outer hair cells and part of the outer sulcus, with no expression in the spiral ganglion region or auditory nerve, indicating that this route does not distribute beyond the endolymphatic compartment. These results define the cochlear compartments accessible to LNP-mediated mRNA delivery and the dependence of that access on administration route, and provide the delivery map required for the design of LNP-based mRNA strategies in the inner ear.