Polyamine‐Mediated Stoichiometric Assembly of Ribonucleoproteins for Enhanced mRNA Delivery

Abstract

Messenger RNA (mRNA) represents a promising class of nucleic acid drugs. Although numerous carriers have been developed for mRNA delivery, the inefficient mRNA expression inside cells remains a major challenge. Inspired by the dependence of mRNA on 3′ terminal poly adenosine nucleotides (poly A) and poly A binding proteins (PABPs) for optimal expression, we complex synthetic mRNA containing a poly A tail with PABPs in a stoichiometric manner and stabilize the ribonucleoproteins (RNPs) via a family of polypeptides bearing different arrangements of cationic side groups. We find that the molecular structure of these polypeptides modulates the degree of PABP-mediated enhancement of mRNA expression. This strategy elicits an up to 20-fold increase in mRNA expression in vitro and ~4-fold increase in mice. These findings suggest a set of new design principles for gene delivery via synergistic co-assembly of mRNA with helper proteins. Graphical AbstractCo-assembly of mRNA/PABP nanoplexes with polyamines enhances mRNA translation via improvement of both mRNA stability and protein translation. [1][2][3][4] Unlike protein-based biologics, mRNA co-opts natural biological processes to express proteins and thereby achieve a desired therapeutic effect. [5][6] In contrast to DNA, however, mRNA does not need to enter the nucleus to be functional, which allows for transfection of non-dividing cells with potentially high efficiency. [7] Additionally, mRNA does not integrate into the genome and hence has little risk of insertional mutagenesis. [8][9] These advantages enable the potential treatment of a broad spectrum of diseases, many of which cannot be addressed with current technologies. [1][2] Nevertheless, inefficient transfection of exogenous mRNA remains a key barrier to broad applications of mRNA-based drugs. [10] Endogenous mRNA associates with specific proteins at different stages when trafficking from the nucleus to cytoplasmic ribosomes for maximal protein production. In contrast, the existing approach towards transfecting exogenous mRNA remains direct complexation of mRNA with cationic carriers. [11][12][13] These carriers are designed to protect mRNA from degradation, enable uptake by cells, and facilitate endosomal escape. [10] However, it was recently reported that certain polycation-based delivery vehicles can partially block mRNA from effectively recognizing the complementary protein responsible for mRNA translation. [14] To circumvent this problem, we recently demonstrated that preloading the 5′ end of mRNA with recombinant cap-binding protein, eIF4E, increases the formation of the mRNA translation initiation complex and leads to enhanced mRNA transfection via improved mRNA stability and expression inside cells. [15] Nevertheless, the 3′ poly A tail of mRNA is susceptible to de-adenylation (an enzyme-mediated hydrolysis of poly A), which can trigger RNA degradation by 3′-5′ RNA exonucleases in the cytoplasm. [16] Additionally, fundamental biology studies have shown that the poly A tail of endogenous mRNA i...

Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference

“…Therefore, keeping the same backbone while using distinct side-chain structures enables in-depth characterization of synthetic gene carrierassisted assembly and delivery of siRNA/Ago2 complexes. Moreover, these polypeptides have demonstrated superior transfection efficacy for mRNA and DNA delivery with minimal cytotoxicity in vitro and in vivo (20,(27)(28)(29).…”

Nanomaterial Delivery Systems for mRNA Vaccines

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Toward new design principles for superior gene silencing

“…To study the design parameters of the synthetic polyamine delivery vehicle that govern the coassembly and the resulting gene-silencing efficiency of siRNA/Ago2, Li et al synthesized a series of synthetic polypeptides derived from N-carboxyanhydride polymerization of an L-benzyl aspartate backbone, with varying aminoethylene side chain substitutions and additional methylene groups between the amines. This polymer backbone has been shown to be relatively safe in vitro and in vivo, indicating that appropriate manipulation of the side chains may enable superior delivery without negatively affecting toxicity (10). For a fixed N/P ratio of 20:1 (ratio of the number of protonatable amine groups in the polyamine side chain to that in the siRNA), Li et al (8) show that the gene-silencing efficacy was higher: (i) when the extent of amine protonation at the cytoplasmic pH (∼7.4) was higher, and the number of amine groups in the side chain was higher, due to the increased colocalization between siRNA and Ago2 inside cells; and (ii) when there is more spacing between the protonatable amine side groups.…”