Cationic polypeptide design for polyion complex-mediated mRNA delivery

Recently, with the success of the COVID-19 vaccine, mRNA therapeutics have received a great deal of attention as a next-generation biopharmaceutical. One of the current key issues in mRNA therapeutics is the development of delivery vehicles with higher safety and targetability, excepting the liver. Herein, we introduce a systematic design strategy of cationic polypeptides and their polyplexes for enhanced mRNA delivery. Indeed, a series of cationic polypeptides were synthesized by the aminolysis reaction of poly（β-benzyl-L-aspartate） with varying amine compounds. First, cationic polyaspartamide derivatives were developed for efficient endosomal escape（or endosomal membrane destabilization） by highlighting the acidic pH-sensitivity of aminoethylene（-NHCH₂CH₂-） units. Notably, a polyaspartamide derivative（PAsp（DET）） bearing diethylenetriamine（DET） moieties allowed efficient endosomal escape of polyplexes with lowered cytotoxicity. Second, varying hydrophobic moieties were introduced into the side chains of polyaspartamide derivative with the DET moieties to enhance the stability of mRNA-loaded polyplexes. The results indicated that the derivatives with a hydrophobicity index（or logD）＞–2.4 elicited efficient mRNA transfection in cultured cells. As a result, a polyaspartamide derivative（PAsp（DET/CHE）） with DET and cyclohexylethyl（CHE） moieties achieved the most efficient mRNA transfection without marked cytotoxicity, allowing the topical mRNA delivery in the ventricle via intracranial/intrathecal administration and the systemic mRNA delivery into the lung via intravenous administration. Third, self-catalytic degradation of cationic polyaspartamide derivatives was investigated by slightly changing the spacer length in the side chains. The main-chain degradation was substantially affected by the spacer length；the loss of one methylene spacer resulted in the 5-fold higher degradation rate. A polyaspartamide derivative（PAsp（EDA）） bearing the shorter spacer showed higher mRNA transfection efficiency in cultured cells with reduced cytotoxicity with an increase in pre-incubation time, compared with those bearing the longer spacers. Altogether, it is demonstrated that the polyplex-mediated mRNA delivery can be dramatically improved by fine-tuning the chemical structure of cationic polypeptides.