Modified ARCA analogs providing enhanced translational properties of capped mRNAs

Abstract

Nowadays gene manipulation techniques ("DNA therapy") undergo progressive development and become widely used in industry and medicine. Since new advances in mRNA technologies are capable for obtaining particles with increased stability and translational efficiency, RNA become an attractive alternative for advancement of DNA therapy. For the past years studies have been conducted to explore different modification in mRNA cap structure and its effect on RNA properties. Recently we have shown that modification of the cap structure at the N2 position of 7-methylguanosine leads to an enhancement in translation inhibition. Currently, we have decided to exploit translational properties of mRNA capped with the ARCA (anti-reversed cap) analogs modified within N2 position of purine moiety s. We designed and synthesized three new dinucleotide cap analogs and investigated them in the rabbit reticulocyte lysate (RRL) and the human embryonic kidney derived HEK293 cell line, in vitro translational model systems. The obtained data indicate that, in both translational assays, the cap analogs synthesized by us when incorporated into mRNA improved its translational properties compared to the ARCA capped transcripts. Furthermore, the introduced modifications enhanced stability of the capped transcripts in HEK293 cells, which become higher compared to that of the transcripts capped with regular cap or with ARCA. Additionally one of the synthesized cap analogs revealed strong translation inhibition potency in RRL system, with IC value 1.7 µM.

Opportunities and Challenges in the Delivery of mRNA-Based Vaccines

“…Three different classes of m7GpppG cap analogs are used [38]: (i) anti-reverse cap analogs (ARCAs) [39], (ii) 3'-O-Me-m7GpppG [40], and (iii) modified ARCAs [41]. Initial mRNA research was performed using mRNA containing the m7 cap analog (GpppG) [42], and it is currently the most commonly used mRNA cap in clinical trials.…”

“…ARCAs juxtaposing the traditional cap analogues have been shown to exhibit more than four times RNA transcription efficiency [44]. In addition, the duration and the levels of protein expression have been found to be enhanced in cells transfected with ARCA-capped IVT RNA [41]. Recently, new types of chemically-modified cap analogues have been introduced, e.g., of phosphorothioate, phosphorothiolate [35], imidiphosphate [45], locked nucleic acid [46], boranophosphate bonds [47] and other types of modifications, which provide the mRNA with resistance to decapping by the mRNA-decapping enzyme 2, eventually resulting in a longer half-life of the mRNA [48].…”

Gene Therapy for Heart Disease: Modified mRNA Perspectives

“…However, Cap 1 can sometimes attach the mRNA strand in a reverse orientation, i.e., Gpppm7 instead of m7Gppp, which reduces translation efficiency. Thus, the second-generation capping system of anti-reverse cap analogs (ARCA) 3′-O-Mem7G(5′)ppp(5′)G, which cannot incorporate in the reverse orientation, is frequently used in vitro and in vivo to ensure correct capping orientation and higher translational efficiency [29,30]. One downside of this chemical capping strategy is that the need for higher-ratio ARCA and GTP concentrations to produce a high percentage of capped mRNA significantly elevates the cost of modRNA production.…”

The multifaceted eukaryotic cap structure

“…Numerous cap analogs have been tested as inhibitors of cap‐dependent translation and an interesting group of these contain modifications to the exocyclic amine group (Cai et al, 1999; X. Chen et al, 2012; Jia et al, 2010). Aromatic substituents (benzyl (bn), p‐methoxybenzyl, and triazole) at N2 were found to exhibit superior inhibitory properties (Kocmik et al, 2018). The incorporation of the benzyl and methoxybenzyl features within the mRNA cap structures was also undertaken to assess the consequences on mRNA function (Kocmik et al, 2018).…”