Optimizing Functional-Domain Integrity of Circular Ribonucleic Acid With Improved Vaccine Immunogenicity by circDesign Algorithm.

Circular RNA Translation Modulation via Small Ligand-Mediated Protein Stabilization.

Circular RNAs (circRNAs) have emerged as a promising platform for RNA-based therapeutics, ascribed to their excellent stability and prolonged protein translation capacity. However, modulation of circRNA translation remains a great challenge. Here, we develop a new chemically regulated circRNA translation system based on small ligand-induced stabilization of RNA-binding proteins (RBPs). The system is engineered by incorporating RNA motifs into an internal ribosomal entry site and fusing RBPs with small ligand-stabilized conditional proteins. We show that this design allows small ligand-modulated translation of circRNAs with a wide dynamic range and dose dependency. It is demonstrated that this strategy is modular and versatile to different RBPs and small-ligand-stabilized conditional proteins. We further employ this strategy to control the translation of functional proteins, including a guanine nucleotide exchange factor for modulating signaling pathways and a bispecific engager for regulating T cell activities. This system is highly modular and versatile, highlighting its great potential for basic research and therapeutic applications.

Circular Logic: Engineering Next-Generation Circular RNA Therapeutics for Precision Oncology.

The topology of RNA therapeutics is emerging as a critical design dimension in precision oncology. Unlike linear mRNA, circular RNA (circRNA) lacks free ends, conferring exceptional resistance to exonuclease degradation and enabling sustained protein expression for days to weeks. Beyond their use as engineered therapeutics, endogenous circRNAs exhibit cancer-associated expression patterns and persistence in biofluids, supporting complementary roles in tumor biology and as candidate biomarkers for diagnosis and longitudinal disease monitoring. This review argues that circular topology should be viewed as an active pharmacologic variable, not merely a stability enhancement. We dissect recent advances in cap-independent translation initiation, including IRES elements and m6A-driven mechanisms, rolling-circle translation for multi-epitope vaccine design, and programmable stability circuits that integrate tumor-microenvironment cues such as miRNA signatures. Delivery innovations are equally transformative: antibody-guided lipid nanoparticles and engineered extracellular vesicles enable increasingly selective RNA delivery, while local depot formulations and organ-selective systemic routes expand therapeutic reach. Safety considerations are re-evaluated as double-edged tools-innate immunogenicity can serve as a self-adjuvant for cancer vaccines, whereas back-splice-junction neoantigens offer both vaccine opportunities and tolerance risks. Recent advances in scarless circularization, topology-sensitive purification, dsRNA depletion, and lyophilized formulations have begun to address key manufacturing bottlenecks, although clinical-scale recovery and process scalability remain insufficiently characterized. Key applications include circRNA cancer vaccines, transient CAR-T/NK cell engineering, tumor-suppressor replacement, and circRNA-encoded bispecific T-cell engagers. The field now requires real-time pharmacokinetic tracking, reproducible and scalable manufacturing, validated liquid-biopsy assays, and indication-specific regulatory pathways to translate circRNA from bench to bedside.

Split intron-exon system for circular RNA synthesis

Ribozyme-based approaches have been extensively employed for in vitro circular RNA (circRNA) synthesis. However, several critical challenges are impeding the progress in this field, including the relatively low ribozyme utilization efficiency, limited synthetic flexibility, and complicated downstream purification procedures. Here, we introduce a split intron-exon (SIE) system for circRNA synthesis by physically separating the ribozyme from its substrates. This design ensures efficient circRNA synthesis by overcoming the inherent limitations of current ribozyme-based techniques, in which each ribozyme can only catalyse the synthesis of at most one circRNA molecule. Moreover, the SIE system enables the efficient synthesis of both unmodified and chemically modified circRNAs. Furthermore, the recyclability and immobilization of the ribozyme within the SIE system reduce reaction impurities and improves circRNA enrichment and yield. Overall, the SIE system holds great potential to advance the diversification and large-scale development of circRNA therapeutics. Current ribozyme-based methods enable efficient circular RNA synthesis but suffer from inflexibility. Here, the authors introduce the split intron-exon (SIE) system, which separates ribozymes from substrates to facilitate the efficient production of modified circular RNAs, optimizing production process.

Deoxyribonucleotide incorporation reshapes mRNA design beyond canonical ribonucleotide boundaries

Measuring Live-Cell mRNA Translational Dynamics with Split Luminescent Tagging in HEK293 Cells.

The swift vaccine development to combat COVID-19 illustrated the potential for messenger RNA (mRNA) therapeutics to transform drug development. Like mature mRNA, in vitro transcribed mRNA possesses the same elements including a 5' cap, untranslated regions (UTRs), coding sequence and a poly(A) tail. Previous work studying the effects these components have on mRNA translation has primarily utilized highly engineered reporter proteins which exhibit efficient translation and protein stability. With the structural elements of each mRNA differentially affecting their translation, it is imperative to identify the optimal design relevant to the therapeutic protein of interest (POI). To enable POI translation characterization, a split luciferase complementation system was employed. A short peptide tag (HiBiT), which can be fused to either terminus of the POI, associates with its complementary heterodimer (LgBiT) to reconstitute enzymatic activity in the presence of a cell-permeable substrate. To date, split luminescent tagging has been primarily used for high-throughput protein turnover studies. We have previously demonstrated how split luminescent tagging can be employed to enable high-throughput quantification of mRNA translation temporally in cellulo in HEK293 cells constitutively expressing the complementary heterodimer. Here, we further demonstrate the versatility of the assay and detail how this assay can be employed for optimizing in vitro transcription to reduce costs. The assay system can uniquely distinguish alterations in structural components whilst highlighting the effects of coding sequence optimization using non-engineered genes. Additionally, we demonstrate that a 4-fold reduction in 5' cap concentration for in vitro transcription results in equivalent translation in cellulo. These findings illustrate 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 thereby offering a versatile method for the advancement of mRNA-based therapeutics.

Transgene Sequence Codon Optimization and Composition Determines Replication Competence of Self-Amplifying RNA.

Self-amplifying RNA (saRNA) is an emerging RNA therapeutic modality that can facilitate higher magnitude and more durable protein expression at substantially lower doses than non-replicating mRNA. Unlike conventional mRNA, alphavirus derived saRNA must support a replicase driven RNA amplification step in addition to translation, raising the possibility that transgene coding sequences impose sequence level constraints on replication. Here, saRNA replication was found to be dependent on the codon composition of the transgene; multiple therapeutic transgenes were replication-defective despite an intact VEEV derived saRNA backbone. Replication defects were rescued by synonymous codon re-optimization of the same transgenes, indicating that nucleotide level features of the coding sequence, rather than the encoded protein, govern replication competence. Comparative compositional analyses identified a distinct signature associated with productive replication, characterized by elevated GC (>53%) and GC3 (>63%) content, higher codon adaptation to human (>0.75), and reduced UpA (<43/kb), UpU (<41/kb) dinucleotide density. Moreover, deliberate compositional perturbation of an otherwise replication-competent transgene shifted these features and abolished replication, supporting a causal and combinatorial role for sequence composition in defining saRNA replication outcome. These findings define an underappreciated constraint in saRNA therapeutics and motivate saRNA-specific payload design frameworks that incorporate alphavirus associated compositional biases during transgene sequence optimization.