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

Abstract

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.

mRNA vaccines for infectious diseases: principles, delivery and clinical translation

“…Poly(aspartamide)s conjugated to ionizable aminoethylene side chains are protonated at the acidic pH inside endosomes, facilitating RNA delivery. The hydrophobicity and length of the side chain influence poly(aspartamide) protonation and delivery efficacy 93 . For example, PEGylated poly(aspartamide) with an ethylenediamine side chain delivers mRNA to liver 94 , brain 95 , spinal cord 96 , knee joint 97 and olfactory nerves 98 .…”

Indole, Phenyl, and Phenol Groups: The Role of the Comonomer on Gene Delivery in Guanidinium Containing Methacrylamide Terpolymers

“…[ 12,13 ] The cation‐π interactions between the side chains of arginine and tryptophan, tyrosine, or phenylalanine play an important role in these processes. [ 14,15 ] Considering the hydrophobic nature of the π‐faces in guanidinium ions, such stacking against hydrophobic side chain in a protein could reduce the entropic cost of hydrophobic hydration. Jobin et al.…”

Designing peptide nanoparticles for efficient brain delivery

“…Mice pre-treatment with NEP mRNA micelles increased NEP concentration and almost doubled its proteolytic activity in brain and diminished almost to half the amount of exogenous Aβ 1-40 injected thereafter as compared to other groups. The authors recently reported an upgrade of these polypeptide nanocarriers by fine-tuning the hydrophobicity (logP) of the P(Asp) residues in the side chains [271]. A direct correlation among logP and polyplex stability and cell internalisation efficiency was found.…”