Fine-Tuning of Hydrophobicity in Amphiphilic Polyaspartamide Derivatives for Rapid and Transient Expression of Messenger RNA Directed Toward Genome Engineering in Brain.

Rapid and transient expression of 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 ) as an indicator of hydrophobicity, a log 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 . This threshold of log for the mRNA transfection efficiency apparently correlated with those for the polyplex stability and cellular uptake efficiency. Among the polyaspartamide derivatives with log > -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 Cas9 nuclease-coding IVT mRNA in the ependymal layer of ventricles in a reporter mouse model. These results demonstrate the utility of log driven polymer design for IVT mRNA delivery.