Chemically Modified mocRNAs for Highly Efficient Protein Expression in Mammalian Cells

Abstract

mRNA has recently been established as a new class of therapeutics, due to its programmability and ability to produce proteins of interest rapidly in vivo. Despite its demonstrated utility, mRNA as a protein expression platform remains limited by its translational capacity and RNA stability. Here, we introduce messenger-oligonucleotide conjugated RNAs (mocRNAs) to enable site-specific, robust, and modularized encoding of chemical modifications for highly efficient and stable protein expression. In mocRNA constructs, chemically synthesized oligonucleotides are ligated to the 3′ terminus of mRNA substrates to protect poly(A) tails from degradation, without compromising their potency in stimulating translation. As a proof-of-concept, mocRNAs modified by deadenylase-resistant oligonucleotides result in augmented protein production by factors of 2−4 in human HeLa cells and by 10-fold in primary rat cortical neuronal cultures. By directly linking enzymatic and organic synthesis of mRNA, we envision that the mocRNA design will open new avenues to expand the chemical space and translational capacity of RNA-based vectors in basic research and therapeutic applications.

iCodon customizes gene expression based on the codon composition

“…Finally, we envision that iCodon can contribute to the design of RNA-based therapeutics (e.g., mRNA vaccines) (Fig. 7 ), in which increased stability and expression may correlate with stronger efficacy (e.g., stronger immune response) and/or smaller doses 47 – 50 .…”

The Pivotal Role of Chemical Modifications in mRNA Therapeutics

“…Recently, our group has demonstrated the generation of chimeric mRNAs, formed by the enzymatic ligation of an IVT synthesized mRNA transcript with a chemically synthesized oligonucleotide ( Aditham et al, 2022 ) ( Figure 2A ). Termed mRNA-oligonucleotide conjugated RNA (mocRNA), this platform presents a novel method of circumventing translational restrictions on incorporating modified nucleotides and expands the possible space of synthetic transcripts for therapeutics.…”

Chemically Modified Poly(A) Analogs Targeting PABP: Structure Activity Relationship and Translation Inhibitory Properties

“…The binding of PABP to the poly(A) tail promotes translation initiation and protects the poly(A) tail from degradation by deadenylases [17,18] . Recently, chemical and genetically‐encoded poly(A) modifications have been investigated intensively for mRNA labeling and to increase the translational efficiency of in vitro‐transcribed mRNAs for therapeutic use [19–22] . However, the impact of these modifications on PABP binding has not yet been systematically studied.…”