mRNA is a new medical modality as vaccine and raises hopes to become a protein replacement agent. Despite notable advances in manufacturing, stabilization and modification, its short half-life remains a major limitation. Circular RNA bears potential to solve this issue, as degradation of mRNA proceeds primarily from the 5' and 3' ends. However, circular RNAs normally lack a 5' cap and are less efficiently translated. We present a strategy to circularize mRNA covalently via click chemistry by modifying the 5' cap and the 3' poly(A) tail with bioorthogonal functional groups. The resulting Cyclic Click Cap-containing mRNA (CyCliCap-mRNA) is translationally muted by a photocleavable protecting group. Upon irradiation by light, the photocleavable protecting group is removed and the respective linear Cap0-mRNA released. We show that CyCliCap-mRNA can be activated for efficient translation at late timepoints (24-48 h post transfection) in HeLa cells, which is not possible with the respective linear mRNA. The light-mediated opening releases linear mRNA with a native Cap0 and thus overcomes the problem of inefficient translation of circular RNAs.
A new chemically regulated circRNA translation system based on small ligand-induced stabilization of RNA-binding proteins (RBPs), engineered by incorporating RNA motifs into an internal ribosomal entry site and fusing RBPs with small ligand-stabilized conditional proteins is developed.
The versatility of the assay is demonstrated and a 4-fold reduction in 5' cap concentration for in vitro transcription results in equivalent translation in cellulo, illustrating how split luminescent tagging can be easily integrated into the mRNA therapeutic workflow, enabling monitoring of real-time mRNA-driven protein expression dynamics in cellulo.
C. Batho, Camilla Ascanelli, Megan L. Maple et al.· Journal of Visualized Experi...· 0 citations
A series of disulfide-based, self-immolative acyl conjugates have been designed and tested for reversibly protecting the ribose 2'-hydroxy position of RNA. These modifications were found to be responsive to endogenous glutathione levels for efficient RNA recovery both in solution and cultured cells. Through this modification strategy, we achieve shielding of the biomolecule from both nuclease enzymes and translational machinery, allowing for a tunable release of RNA with improved cellular stability and minimal innate immune activation. The self-immolative RNA deprotection includes a cyclization step which exhibits differing kinetic profiles based on ring size and electronic properties. Moreover, we demonstrate sustained target protein production for both green-fluorescent protein and nanoluciferase in cell models. Both acylated mRNA models resulted in significant increases in protein expression compared to the unmodified mRNA control, with increases in protein expression of up to 600% and sustained higher protein expression of modified mRNA samples observed over 120 h in comparison to unmodified RNA. These exciting results highlight the versatility of our approach in the design of next-generation RNA-based prodrugs.
Mary E. Flood, Sathishkumar Kurusamy, Mark Berney et al.· Angewandte Chemie· 0 citations
Messenger RNA (mRNA) technology has emerged as a cornerstone in vaccine development and therapeutic applications, offering key benefits such as high potency, rapid scalability, and cost-effectiveness. The success of COVID-19 mRNA vaccines has underscored their efficacy and safety. However, residual byproducts generated during mRNA synthesis, such as unincorporated caps, nucleoside triphosphates (NTPs), DNA templates, enzymes, abortive transcripts, and double-stranded RNA (dsRNA), pose significant challenges to the clinical application of the RNA therapy. Among these, dsRNA is particularly problematic as it can activate various innate immune responses, suppress mRNA translation and potentially compromise the therapeutic efficacy of mRNA. Therefore, effectively removing dsRNA from in vitro synthesized mRNA is essential before its used in preclinical or clinical settings. In this review article, we provide a comprehensive overview of current mRNA development pipelines and ongoing clinical trials, and recent advances in mRNA purification techniques. Specifically, we focus on strategies for dsRNA removal, which can be broadly categorized into two approaches: (1) separating or removing dsRNA from in vitro transcription (IVT) mRNA products using methods such as RP-HPLC chromatography and cellulose-based purification; and (2) minimizing dsRNA formation during IVT by employing engineered RNA polymerase mutants, chaotropic agents, and magnetic beads, as well as modifying/optimizing DNA templates or RNA molecules to reduce dsRNA generation. We also discuss the advantages and limitations of these purification methods, the factors influencing the selection of purification strategies, and explore potential future directions for improving dsRNA purification technologies and their applications in mRNA-based therapeutics.
Jin-He Liu, Jian Zang, Jingru Xu et al.· Journal of Biomedical Scienc...· 0 citations
Recombinant in vitro-transcribed mRNA is broadly used for vaccination and is evaluated in numerous clinical studies for multiple indications. Typical features of mRNA are the 5' cap, 5' untranslated region, start and stop codons, 3' untranslated region and 3' poly(A) tail. Here, contrary to current dogma, we show that short, chemically synthesized RNA oligonucleotides lacking some or all of these features are efficiently translated when they encode epitopes recognized by CD8+ T cells. In particular, one design that we termed ChemRNA with the structure 5'-OH-AUG-coding sequence-3'-OH strongly stimulates antigen-specific CD8+ T cells both in vitro and in vivo. Our results challenge the current understanding of canonical mRNA structure and introduce the possibility that defective or supposedly non-coding RNA may encode human and murine major histocompatibility complex class I-associated peptides. Moreover, ChemRNA could help overcome challenges associated with the design and purification of individualized anti-cancer vaccines.
Julia Frei, D. Eisel, N. Jarzębska et al.· Nature Biomedical Engineerin...· 0 citations
Significance Personalized mRNA cancer vaccines hold great therapeutic promise, yet their production depends on multistep in vitro transcription (IVT) that generates immunogenic dsRNA impurities and prolongs manufacturing to nearly three months, often missing the optimal therapeutic window. Here, we introduced a cap-independent translation enhancer BBV and developed protein-encoding RNA oligonucleotides (PEOs), which were chemically synthesized RNAs bearing 5’-OH and 3’-P termini that drive rolling circle translation after endogenous circularization. PEOs contain undetectable dsRNA and exhibit minimal immunogenicity. In melanoma and orthotopic glioma models, PEO vaccines significantly inhibited tumor growth and synergized with anti-PD-1 therapy. In conclusion, this proof-of-concept study built an IVT-free platform for rapid, safe, and highly druggable vaccines.
Qian Pan, Chi Zhang, Xinyue Wang et al.· Proceedings of the National...· 0 citations
