Oligo(serine ester) Charge-Altering Releasable Transporters: Organocatalytic Ring-Opening Polymerization and their Use for in Vitro and in Vivo mRNA Delivery

Abstract

RNA technology is transforming life science research and medicine, but many applications are limited by the accessibility, cost, efficacy, and tolerability of delivery systems. Here we report the first members of a new class of dynamic RNA delivery vectors, oligo(serine ester)-based chargealtering releasable transporters (Ser-CARTs). Composed of lipid-containing oligocarbonates and cationic oligo(serine esters), Ser-CARTs are readily prepared (one flask) by a mild ring-opening polymerization using thiourea anions and, upon simple mixing with mRNA, readily form complexes that degrade to neutral serine-based products, efficiently releasing their mRNA cargo. mRNA/Ser-CART transfection efficiencies of >95% are achieved in vitro. Intramuscular or intravenous (iv) injections of mRNA/Ser-CARTs into living mice result in in vivo expression of a luciferase reporter protein, with spleen localization observed after iv injection.Messenger RNA (mRNA) is advancing fundamental research and medicine through its ability to induce the transient catalytic expression of target proteins in vitro, in vivo, and ex vivo. Applications of mRNA include protein replacement therapy, gene editing, vaccination, and cancer immunotherapy. 1 However, the challenge of developing synthetically accessible, affordable, safe, and effective delivery vectors that extracellularly protect and intracellularly release mRNA have hampered applications, driving demand for improved delivery systems. 2,3 Current delivery strategies focus on mechanical methods and viral and nonviral vectors. [4][5][6] Mechanical methods that temporarily render the cellular membrane permeable are limited to accessible tissues and ex vivo techniques, often suffer from poor cell viability, and *

Synthesis and mechanistic investigations of pH-responsive cationic poly(aminoester)s

“…1) that are effective for the delivery of mRNA and pDNA to a variety of cell types in cell culture and live animals. 33,[45][46][47][48][49] These cationic amphiphilic oligomers, when complexed with negatively-charged oligonucleotides, form polyelectrolyte complexes that exhibit dynamic, charge-altering behavior that facilitates the intracellular release of the oligonucleotides. 33 This dynamic charge-altering behavior was attributed to the unique structural features of the cationic a-aminoester sequences 44 of these amphiphilic oligomers which exhibit a pH-dependent selective degradation to neutral diketopiperazines.…”

“…48,49 As the pH-induced charge-canceling degradation of the cationic poly(a-aminoester) backbone was implicated in the intracellular release of mRNA, 33 herein we describe a detailed mechanistic study directed at elucidation of the structural and environmental factors that lead to the selective degradation processes for the cationic poly(ammonium ester) homopolymers in aqueous buffers. While not directly comparable to the polyelectrolyte environments in which the amphiphilic CARTs degrade, 33,[45][46][47] these studies provide fundamental insights on the structural factors that contribute to the degradation behavior of poly(amino ester)s in aqueous buffers. We report the synthesis of a family of structurally related cationic polyaminoester homopolymers and investigate how their structural variations inuence the mechanism, product distribution, and pH dependence of their degradation.…”

“…[36][37][38][39][40][41][42][43] We recently reported a novel class of materials based on cationic poly(a-aminoester) backbones that undergo a pHsensitive rapid and selective degradation to neutral amide and amino acid byproducts in water. 44 We have leveraged this behavior in the design of novel cationic amphiphilic oligomers, charge-altering releasable transporters (CARTs), 33,[45][46][47] that bind, complex, and deliver oligonucleotides of wide ranging size (mRNA, pDNA) into cells, in vitro and in vivo. 33,[45][46][47][48][49] At low pH, these oligomeric amphiphiles are cationic and associate in water with polyanionic oligonucleotides to form polyelectrolyte complexes.…”

“…44 We have leveraged this behavior in the design of novel cationic amphiphilic oligomers, charge-altering releasable transporters (CARTs), 33,[45][46][47] that bind, complex, and deliver oligonucleotides of wide ranging size (mRNA, pDNA) into cells, in vitro and in vivo. 33,[45][46][47][48][49] At low pH, these oligomeric amphiphiles are cationic and associate in water with polyanionic oligonucleotides to form polyelectrolyte complexes. At elevated pH these polyelectrolyte complexes release the oligonucleotides, which was attributed to the charge-altering degradative behavior of the cationic oligo(aamino ester)s (Fig.…”

“…1). These charge-altering releasable transporters (CARTs) 33,[45][46][47] were shown to be effective for mRNA and pDNA delivery to a variety of cell types in cell culture and live animals, enabling multiple therapeutic vaccination strategies for eradication of established tumors in mice. 48,49 As the pH-induced charge-canceling degradation of the cationic poly(a-aminoester) backbone was implicated in the intracellular release of mRNA, 33 herein we describe a detailed mechanistic study directed at elucidation of the structural and environmental factors that lead to the selective degradation processes for the cationic poly(ammonium ester) homopolymers in aqueous buffers.…”