Chemo‐Enzymatic Modification of the 5′ Cap Maintains Translation and Increases Immunogenic Properties of mRNA

Abstract

Eukaryotic mRNAs are emerging modalities for protein replacement therapy and vaccination. Their 5' cap is important for mRNA translation and immune response and can be naturally methylated at different positions by Sadenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). We report on the cosubstrate scope of the MTase CAPAM responsible for methylation at the N 6 -position of adenosine start nucleotides using synthetic AdoMet analogs. The chemo-enzymatic propargylation enabled production of site-specifically modified reporter-mRNAs. These cap-propargylated mRNAs were efficiently translated and showed % 3fold increased immune response in human cells. The same effects were observed when the receptor binding domain (RBD) of SARS-CoV-2-a currently tested epitope for mRNA vaccination-was used. Site-specific chemo-enzymatic modification of eukaryotic mRNA may thus be a suitable strategy to modulate translation and immune response of mRNAs for future therapeutic applications.Scheme 1. mRNAs and chemo-enzymatic modifications used in this study. A) 5'-Clean Cap (5'-CC) mRNA is produced via IVT. B,C) N 6 -Modification of adenosine at the transcription start nucleotide using CAPAM and AdoMet (S-adenosyl-l-methionine) or AdoMet analogs yields methylated Clean Cap (5'-CC-N 6 mA m ) or propargylated Clean Cap (5'-CC-N 6 pA m ), respectively. D) Modification of the poly(A) tail of 5'-CC-mRNA using poly(A) polymerase and 2'-azido ATP followed by labeling with Cy5 yields 5'-CC-3'-Cy5-mRNA. E) 5'-ARCA (5'-AC) mRNA is produced via IVT. F) N 2 -Modification of m 7 G using GlaTgs and AdoMet results in N 2 methylated ARCA (5'-N 2 m-AC)-mRNA. G) Modification of the poly(A) tail of 5'-AC-mRNA using poly(A) polymerase and 2'-azido ATP followed by labeling with Cy5 yields 5'-AC-3'-Cy5-mRNA.

Quantification of mRNA cap-modifications by means of LC-QqQ-MS

“…Nucleosides and cap standards were obtained from NEB (m 7 GpppG, GpppG), Trilink (m 7 GpppA m G), Biomol (A m ), Carbosynth (m 6 A m ) and Merck (A). N 6 pAm was synthesized as described previously [ 20 ].…”

“…The enzymes Ecm1, CAPAM and MTAN were produced and purified as previously described [ 19 , 20 , 34 ].…”

“…To this end, N 6 pA m was synthesized in four steps from 6-chloropurine riboside ( Scheme 4 ) as described recently by our group [ 20 ]. Briefly, 2′- O -methylation required 3′- and 5′-protection with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPSDSiCl 2 ) followed by methylation with methyl iodide and NaH [ 46 ].…”

“…Introduction of a photocleavable group into the methyltransferase Ecm1 enabled light-controlled terminal N 7-guanosine methylation of GpppG-capped RNAs and thus provided a means to trigger translation at the time point of choice ( N 7-methylation by Ecm1 shown in Scheme 1A ) [ 19 ]. We also investigated CAPAM’s cosubstrate promiscuity and found that N 6 -propargylation of 2′ - O -methyl adenosine at the TSN ( Scheme 1B ) altered the immunogenicity of the transcript while translation remained high [ 20 ].…”

Messenger RNA vaccines for cancer immunotherapy: progress promotes promise

“…2022;132(6):e156211 https://doi.org/10.1172/JCI156211 transformation. Recent studies have shown that methylation of adenosine start nucleotides using synthetic S-adenosyl-L-methionine analogs significantly improves protein expression and vaccine immunogenicity in in vitro systems (101). Understanding the role of cap binding proteins in transcript translation within cancer cells or immune cells should further inform the rational design of mRNA vectors with improved stability and translation efficiency.…”