Jacek Jemielity

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5′ mRNA cap analogs on the kinetics of the encoded protein, we found that m27,2′−OGppSpG (β-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as β-S-ARCA(D1)-capped RNA species is superior for protein expression and for efficient priming and expansion of naïve antigen-specific T cells in mice. Our findings establish 5′ mRNA cap analogs as yet another module for tuning immunopharmacological properties of recombinant antigen-encoding RNA for vaccination purposes.

Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

mRNA degradation predominantly proceeds through two alternative routes: the 5′→3′ pathway, which requires deadenylation followed by decapping and 5′→3′ hydrolysis; and the 3′→5′ pathway, which involves deadenylation followed by 3′→5′ hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp3G, m7Gp3G, or m27,3′-O Gp3G) and 3′ termini (A31,A60, or G16) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5′ terminus to bind eIF4E, not merely the presence of a blocking group at the 5′-end. Introducing a stem-loop in the 5′-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. To test the relative contributions of the 5′→3′ versus 3′→5′ pathways, we designed and synthesized two new cap analogs, in which a methylene group was substituted between the α- and β-phosphate moieties, m27,3′-OGppCH2pG and m27,3′-OGpCH2ppG, that are predicted to be resistant to cleavage by Dcp1/Dcp2 and DcpS, respectively. These cap analogs were recognized by eIF4E and conferred cap-dependent translation to mRNA both in vitro and in vivo. Oligonucleotides capped with m27,3′-OGppCH2pG were resistant to hydrolysis by recombinant human Dcp2 in vitro. mRNAs capped with m27,3′-OGppCH2pG, but not m27,3′-OGpCH2ppG, were more stable in vivo, indicating that the 5′→3′ pathway makes a major contribution to overall degradation. Luciferase mRNA containing a 5′-terminal m27,3′-OGppCH2pG and 3′-terminal poly(G) had the greatest stability of all mRNAs tested.

Synthetic mRNA cap analogs with a modified triphosphate bridge – synthesis, applications and prospects

The cap structure was discovered at the 5′-end of the eukaryotic mRNA over three and a half decades ago. Since then, hundreds of chemically modified cap analogs have been synthesized and applied in numerous studies on the elucidation of cap-related physiological processes in the cell, and became important members of the biophosphate family. In this Perspective, we present recent developments in the synthesis of cap analogs modified within 5′,5′-triphosphate bridges and their utility for interdisciplinary studies on cap-dependent processes in gene expression and its regulation, as well as biotechnological applications and perspectives in medicine.

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization.

Potential therapeutic applications of RNA cap analogs

Cap analogs are chemically modified derivatives of the unique cap structure present at the 5´ end of all eukaryotic mRNAs and several non-coding RNAs. Until recently, cap analogs have served primarily as tools in the study of RNA metabolism. Continuing advances in our understanding of cap biological functions (including RNA stabilization, pre-mRNA splicing, initiation of mRNA translation, as well as cellular transport of mRNAs and snRNAs) and the consequences of the disruption of these processes - resulting in serious medical disorders - have opened new possibilities for pharmaceutical applications of these compounds. In this review, the medicinal potential of cap analogs in areas, such as cancer treatment (including eIF4E targeting and mRNA-based immunotherapy), spinal muscular atrophy treatment, antiviral therapy and the improvement of the localization of nucleus-targeting drugs, are highlighted. Advances achieved to date, challenges, plausible solutions and prospects for the future development of cap analog-based drug design are described.

Structural analysis of 5'-mRNA-cap interactions with the human AGO2 MID domain.

In RNA silencing, microRNA (miRNA)‐mediated translational repression occurs through mechanisms that do not invoke messenger‐RNA (mRNA) target cleavage by Argonaute proteins. The nature of these mechanisms is unclear, but several recent studies have proposed that a direct interaction between the mRNA–cap and the middle (MID) domain of Argonautes is involved. Here, we present crystallographic and NMR data demonstrating that cap analogues do not bind significantly to the isolated MID domain of human Argonaute 2 (hAGO2) and are found in the miRNA 5′‐nucleotide binding site in an implausible binding mode. Additionally, in vitro pull‐down experiments with full‐length hAGO2 indicate that the interaction with cap analogues is nonspecific.

Synthesis and biochemical properties of novel mRNA 5' cap analogs resistant to enzymatic hydrolysis

A series of new dinucleotide cap analogs with methylene groups replacing oxygens within the pyrophosphate moieties have been synthesized. All the compounds were resistant to the human scavenger decapping hydrolase, DcpS. Binding constants of the modified caps to eIF4E are comparable to those obtained for m7GpppG. This suggests these methylene modifications in the pyrophosphate chain do not significantly affect cap-binding at least for eIF4E. These cap analogs are also good inhibitors of in vitro translation. mRNAs capped with novel analogs were translated similarly to the mRNA capped with the parent m7GpppG.

Recognition of different nucleotidyl-derivatives as substrates of reactions catalyzed by various HIT-proteins

Proteins that have a histidine triad in their active sites belong to the HIT-protein superfamily. They are ubiquitous, are involved in the metabolism of different nucleotides and catalyze their hydrolysis and/or phosphorolysis liberating either the corresponding 5′-NMP or 5′-NDP, respectively. We studied substrate specificity of nine recombinant HIT-proteins with adenosine 5′-phosphosulfate (1), adenosine 5′-phosphoramidate (2), adenosine 5′-phosphorothioate (3), adenosine 5′-phosphorofluoride (4), diadenosine 5′,5′′′-P1,P3-triphosphate (5), di(7-methylguanosine) 5′,5′′′-P1,P3-triphosphate (6) and adenosine 5′-hypophosphate (7). Preferences for the recognition of these compounds as substrates by individual proteins differed. All the proteins hydrolyzed (1) but the Arabidopsis thaliana Hint1 did it very slowly. None of the proteins cleaved (7). Only A. thaliana Hint1 and Escherichia coli HinT hydrolyzed (3). Three proteins known as dinucleoside triphosphatases, human and A. thaliana Fhit-proteins and Trypanosoma brucei HIT-45, cleaved (1), (2), (4), (5) and (6). Caenorhabditis elegans decapping protein DcpS degraded (1), (5), (6) and poorly (4). A. thaliana aprataxin-like protein and Hint4 hydrolyzed only (1), (2) and (4), in that order of efficiency. Velocities of those reactions and some Km values were determined. Applicability of this study to the metabolism of certain nucleotidyl-derivatives is discussed.

Phosphoroselenoate Dinucleotides for Modification of mRNA 5′ End

er, this structure has a disproportionately large effect on mRNA function since it represents an anchoring point for a variety of proteins involved in mRNA-related physiological processes, as well as protecting mRNA from 5’!3’ exonucleolytic degradation. 2] Hence, synthetic cap analogues are invaluable tools for studying mRNA translation and turnover. Moreover, dinucleotide cap analogues can be easily incorporated into the mRNA 5’ end during in vitro transcription; this makes them useful for biotechnological purposes (protein production) and, potentially, for medical applications related to mRNA-mediated gene therapy. 5] Here, we describe the synthesis and properties of two P diastereomers of a cap analogue modified with phosphoroselenoate moiety at the b position of the triphosphate bridge (m2 GppSepG, D1 and D2; Figure 1 B). The compounds additionally contain a 2’-O-methyl group in the mGuo moiety to ensure their incorporation into mRNA during in vitro transcription exclusively in the correct orientation (antireverse cap analogues, ARCAs), and hence they can be referred to as b-SeARCAs. The idea of synthesising b-Se-ARCAs arose from our previous finding that mRNAs with a cap structure modified with a phosphorothioate moiety at the b position of the triphosphate bridge, m2 GppSpG (D2), were resistant to Dcp2 pyrophosphatase, which is a decapping enzyme essential for triggering 5’!3’ mRNA decay. Hence, such mRNAs were more stable as well as fiveand twofold more efficiently translated in vivo (mammalian cells) than mRNAs capped with mGpppG and m2 GpppG, respectively. Phosphoroselenoates (PSe) are closely related to phosphorothioates (PS) in terms of chemical and biochemical properties, thus, we expected that also PSe cap analogues would share these interesting characteristics. One important superiority of nucleotide phosphoroselenoate analogues over phosphorothioate analogues is their usefulness in nucleic acid X-ray crystallography for multiwavelength anomalous diffraction (MAD) phasing technique. This fact combined with the expected resistance of m2 GppSepG to Dcp2, led us to the conclusion that such compounds (or mRNA fragments capped with such compounds) could serve as perfect ligands for co-crystallisation with Dcp2, as well as other proteins involved in mRNA 5’-end recognition. To the best of our knowledge, b-Se-ARCAs also represent the first examples of nucleotide analogues that are modified with a PSe moiety not at the a position with respect to nucleoside. The synthesis of m2 GppSepG is depicted in Scheme 1. The selenophosphate (1) was prepared by using a modification of previously reported procedures. 12] Tris(trimethylsilyl)phosphite was treated with a suspension of selenium in pyridine to give tris(trimethylsilyl)phosphoroselenoate, which was then desilylated by methanol in the presence of triethylamine to give selenophosphate triethylammonium (TEA) salt. In agreement with previous reports, 13] we found selenophosphate to be extremely labile and prone to oxidation. However, despite its instability, a freshly prepared selenophosphate TEA salt was efficiently coupled with nucleoside 5’-monophosphate P-imidazolide derivatives to produce the corresponding nucleoside 5’O-(2-selenodiphosphates) under similar conditions to those we developed previously for the synthesis of the analogous PS nucleotides. Figure 1. A) Structure of eukaryotic mRNA 5’ end (cap). B) Structure of the phosphoroselenoate mRNA cap analogue synthesised in this work.

Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma

Adenovirus dodecahedron (Dd), a nanoparticulate proteinaceous biodegradable virus-like particle (VLP), was used as a vector for delivery of an oncogene inhibitor to hepatocellular carcinoma (HCC) rat orthotopic model. Initiation factor eIF4E is an oncogene with elevated expression in human cancers. Cell-impermeant eIF4E inhibitor, cap structure analog (cap) and anti-cancer antibiotic doxorubicin (Dox) were delivered as Dd conjugates. Dd-cap and Dd-dox inhibited cancer cell culture proliferation up to 50 and 84%, respectively, while with free Dox similar results could be obtained only at a 5 times higher concentration. In animal HCC model the combination treatment of Dd-cap/Dd-dox caused 40% inhibition of tumor growth. Importantly, the level of two pro-oncogenes, eIF4E and c-myc, was significantly diminished in tumor sections of treated rats. Attachment to Dd, a virus-like particle, permitted the first demonstration of cap analog intracellular delivery and resulted in improved doxorubicin delivery leading to statistically significant inhibition of HCC tumor growth.