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.

中文翻译：

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两亲聚天冬酰胺衍生物疏水性的微调，用于脑中基因组工程的信使RNA的快速和瞬时表达。

在靶细胞中体外转录的mRNA（IVT mRNA）的快速和瞬时表达是基因组工程治疗中的当前主要挑战。为了提高mRNA的输送效率，合成了一系列两亲性的聚天冬酰胺衍生物，在侧链中含有各种带有阳离子二亚乙基三胺（DET）的疏水部分，并系统地比较了它们作为mRNA的运载工具（或载有mRNA的多链体）。获得的结果表明，聚天冬酰胺衍生物的侧链结构对于多聚体的mRNA递送效率至关重要。有趣的是，当将mRNA输送效率（或培养细胞中的萤光素酶表达水平）相对于辛醇-水分配系数作图时（log P）作为疏水性的指标，清楚地观察到log P阈值以获得高水平的mRNA表达。实际上，在log P中在-2.45和-2.31之间观察到表达水平的3.5个数量级差异。对于mRNA转染效率，log P的这个阈值显然与多聚体稳定性和细胞摄取效率的log P阈值相关。在具有log P的聚天冬酰胺衍生物中> -2.31，在侧链中具有11个2-环己基乙基（CHE）部分残基和15个DET部分残基的聚天冬酰胺衍生物在培养细胞中诱导了最高的mRNA表达。优化的复合物进一步在脑室内和鞘内注射后在小鼠脑中实现了高效，快速和短暂的IVT mRNA表达。最终，该复合物通过在报告小鼠模型的心室室管膜层中表达化脓性链球菌Cas9核酸酶的IVT mRNA的表达，实现了高效靶基因的删除。这些结果证明了log P驱动的聚合物设计在体内IVT mRNA递送中的实用性。