Fine-Tuning of Hydrophobicity in Amphiphilic Polyaspartamide Derivatives for Rapid and Transient Expression of Messenger RNA Directed Toward Genome Engineering in Brain
Fine-Tuning of Hydrophobicity in Amphiphilic Polyaspartamide Derivatives for Rapid and Transient Expression of Messenger RNA Directed Toward Genome Engineering in Brain
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DOI：
10.1021/acscentsci.9b00843
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发表时间：
2019-11-27
影响因子：
18.2
通讯作者：
Miyata, Kanjiro
中科院分区：
文献类型：
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作者：
Kim, Hyun Jin;Ogura, Satomi;Miyata, Kanjiro
体外转录mRNA （IVT mRNA）在靶细胞中的快速瞬时表达是当前基因组工程治疗的主要挑战。为了提高mRNA的传递效率，我们合成了一系列两亲性聚天冬酰胺衍生物，这些衍生物含有不同的疏水基团，其侧链中含有阳离子二乙烯三胺（DET）基团，并系统地比较了它们作为mRNA传递载体（或mRNA负载复合物）的性能。结果表明，聚阿斯巴胺衍生物的侧链结构对多聚物的mRNA传递效率至关重要。有趣的是，当mRNA传递效率（或培养细胞中的荧光素酶表达水平）与辛醇-水分配系数（log P）作为疏水性指标进行对比时，可以清楚地观察到log P阈值可以获得高水平的mRNA表达。事实上，在-2.45和-2.31的log P中，mRNA的表达水平有3.5个数量级的差异。mRNA转染效率的log P阈值与复合体稳定性和细胞摄取效率的阈值明显相关。在log P b> -2.31的聚天冬酰胺衍生物中，侧链上含有11个2-环己基乙基（CHE）和15个DET基团的聚天冬酰胺衍生物在培养细胞中mRNA表达量最高。优化后的复合体进一步实现了脑室内和鞘内注射后小鼠脑内高效、快速、瞬时的IVT mRNA表达。最终，在报告小鼠模型中，通过在脑室室管膜层表达化脓性链球菌Cas9核酸酶编码IVT mRNA，这种复合体实现了高效的靶基因缺失。这些结果证明了log P驱动聚合物设计用于体内IVT mRNA递送的实用性。
Rapid and transient expression of in vitro transcribed mRNA (IVT mRNA) in target cells is a current major challenge in genome engineering therapy. To improve mRNA delivery efficiency, a series of amphiphilic polyaspartamide derivatives were synthesized to contain various hydrophobic moieties with cationic diethylenetriamine (DET) moieties in the side chain and systematically compared as mRNA delivery vehicles (or mRNA-loaded polyplexes). The obtained results demonstrated that the side chain structures of polyaspartamide derivatives were critical for the mRNA delivery efficiency of polyplexes. Interestingly, when the mRNA delivery efficiencies (or the luciferase expression levels in cultured cells) were plotted against an octanol-water partition coefficient (log P) as an indicator of hydrophobicity, a log P threshold was clearly observed to obtain high levels of mRNA expression. Indeed, 3.5 orders of magnitude difference in the expression level is observed between -2.45 and -2.31 in log P. This threshold of log P for the mRNA transfection efficiency apparently correlated with those for the polyplex stability and cellular uptake efficiency. Among the polyaspartamide derivatives with log P > -2.31, a polyaspartamide derivative with 11 residues of 2-cyclohexylethyl (CHE) moieties and 15 residues of DET moieties in the side chains elicited the highest mRNA expression in cultured cells. The optimized polyplex further accomplished highly efficient, rapid, and transient IVT mRNA expression in mouse brain after intracerebroventricular and intrathecal injection. Ultimately, the polyplex allowed for the highly efficient target gene deletion via the expression of Streptococcus pyogenes Cas9 nuclease-coding IVT mRNA in the ependymal layer of ventricles in a reporter mouse model. These results demonstrate the utility of log P driven polymer design for in vivo IVT mRNA delivery.