In Vivo Messenger RNA Introduction into the Central Nervous System Using Polyplex Nanomicelle

Abstract

Messenger RNA (mRNA) introduction is a promising approach to produce therapeutic proteins and peptides without any risk of insertion mutagenesis into the host genome. However, it is difficult to introduce mRNA in vivo mainly because of the instability of mRNA under physiological conditions and its strong immunogenicity through the recognition by Toll-like receptors (TLRs). We used a novel carrier based on self-assembly of a polyethylene glycol (PEG)-polyamino acid block copolymer, polyplex nanomicelle, to administer mRNA into the central nervous system (CNS). The nanomicelle with 50 nm in diameter has a core-shell structure with mRNA-containing inner core surrounded by PEG layer, providing the high stability and stealth property to the nanomicelle. The functional polyamino acids possessing the capacity of pH-responsive membrane destabilization allows smooth endosomal escape of the nanomicelle into the cytoplasm. After introduction into CNS, the nanomicelle successfully provided the sustained protein expression in the cerebrospinal fluid for almost a week. Immune responses after mRNA administration into CNS were effectively suppressed by the use of the nanomicelle compared with naked mRNA introduction. In vitro analyses using specific TLR-expressing HEK293 cells confirmed that the nanomicelle inclusion prevented mRNA from the recognition by TLRs. Thus, the polyplex nanomicelle is a promising system that simultaneously resolved the two major problems of in vivo mRNA introduction, the instability and immunogenicity, opening the door to various new therapeutic strategies using mRNA.

Treatment of neurological disorders by introducing mRNA in vivo using polyplex nanomicelles

“…PAsp(DET) has a high capacity for enhanced endosomal escape due to pH-sensitive membrane destabilization as well as the unique characteristic of rapidly degrading into nontoxic forms under physiological conditions [16][17][18]. The nanomicelles [PEG-PAsp(DET) block copolymer/mRNA] were shown to be safe and stable mRNA carriers to allow in vivo mRNA introduction into the central nervous system [14] and other tissues and organs (unpublished data). In addition, they exhibited a pronounced effect for regulating immune responses that could be induced by exogenous mRNA [14].…”

“…For this purpose, we recently established a novel carrier that was applicable for in vivo mRNA delivery based on the self-assembly of polyethylene glycol (PEG)-polyamino acid block copolymer, i.e. polyplex nanomicelles [14]. These nanomicelles have a core-shell structure surrounded by a PEG outer layer, with the inner core of a functionalized polyamino acid, poly[N′-[N-(2-aminoethyl)-2-aminoethyl] aspartamide] [PAsp(DET)] [15].…”

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

“…To date, naked, chemically modified, or protamine-complexed mRNA have shown promise in phase I/II cancer trials (24)(25)(26). Recently, preclinical development of materials specific for mRNA delivery has resulted in cationic polymers such as polymethacrylates (27)(28)(29), poly(aspartamides) (30,31), and polypeptides (32), as well as multicomponent cationic lipid or lipid-like formulations (21,(33)(34)(35)(36). In many of these examples, however, transfection efficiencies can be quite low, ranging 20-80% in cells (18), with likely much lower efficiencies in vivo, which requires either high mRNA doses or hydrodynamic injections (32,37).…”

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“…One such attempt was made using alphacyclodextrins as the hydrophilic macromolecular host and PCL (Mw = 37,000) as the hydrophobic guest molecule 21 . Using this approach, supramolecular polymeric micelles with a mean diameter of 30 nm were made.…”