Circular RNA (circRNA) has rapidly emerged as a paradigm-shifting modality in nucleic acid therapeutics, offering critical advantages over traditional linear messenger RNA (mRNA). Distinguished by its covalently closed-loop structure, circRNA naturally resists degradation by cellular exonucleases, leading to significantly enhanced in vivo stability and prolonged protein expression. This superior kinetic profile positions circRNA as the ideal expression vehicle for next-generation vaccines, protein replacement therapies, and precision medicine applications. At CD BioGlyco, we leverage deep biological expertise and proprietary synthesis platforms to deliver highly pure circRNA. We specialize in overcoming the historically significant hurdles of inefficient cyclization and purification, ensuring that our clients receive circRNA that maximizes therapeutic potency and minimizes immunogenic risk.
CD BioGlyco's capacity for delivering high-performance circRNA relies on integrating advanced molecular engineering with optimized chemical manufacturing. Our approach focuses on high-efficiency cyclization and meticulous purification to maximize yield and biological function.
We utilize advanced self-splicing technologies, primarily based on highly optimized permuted intron-exon (PIE) systems. This proprietary in vitro transcription (IVT) approach facilitates robust, scarless cyclization of the RNA transcript. By promoting autocatalytic splicing and ligation, the PIE system dramatically increases the ratio of functional circular transcripts, ensuring high purity and reducing undesirable linear RNA contaminants that can compromise downstream applications and trigger innate immunity.
Unlike linear mRNA, circRNA translation is independent of a 5' cap structure. Instead, efficient protein production is enabled by incorporating a proprietary, highly optimized internal translation initiation element within the circular sequence. Our platform leverages advanced sequence engineering to refine this key element, ensuring it achieves maximum ribosome engagement and supports sustained translation. This directly results in higher yields of functional protein in vivo.
To further enhance stability and minimize the immunogenic potential inherent in IVT-produced RNA, we integrate strategically selected modified nucleotides (such as N1-methylpseudouridine or 5-methylcytosine) during synthesis. Furthermore, critical impurities like double-stranded RNA (dsRNA), a potent trigger of innate immunity, are removed using multi-stage purification protocols, including specialized ion-pair reversed-phase liquid chromatography (IP-RP).
Consultation on therapeutic goal; sequence engineering for coding regions (ORF), UTRs, and PIE intron placement; selection of optimized backbones; synthesis and cloning of the high-purity DNA template.
High-yield IVT using optimized reaction buffer and controlled Mg2+ concentrations to promote high transcription efficiency; integration of modified nucleotides to reduce immunogenicity and enhance translation.
Enzymatic processing step employing proprietary PIE-based technology to drive efficient formation of the covalently closed circular RNA structure.
Targeted removal of the remaining linear RNA precursor and other linear byproducts using specific enzymatic treatment (e.g., RNase R) and highly scalable chromatography methods to ensure the final product is highly enriched for the desired circRNA.
Multi-step chromatography (e.g., IP-RP) purification to eliminate enzyme residuals, DNA template traces, and critical dsRNA impurities, ensuring pharmaceutical-grade purity suitable for preclinical testing.
Full analytical panel including gel electrophoresis (integrity and circularity check), LC-MS/HPLC analysis (purity), Qubit (quantification), and functional assays (translatability) to ensure batch consistency and performance.
Journal: Nature Biomedical Engineering
DOI: 10.1038/s41551-024-01306-3
Published: 2025
Results: In this study, the authors develop innovative trans-splicing-based methods, termed TRIC and TERIC, for highly efficient synthesis of long circRNAs exceeding 8,000 nucleotides, positioning them as robust candidates for next-generation therapeutics. These approaches circumvent the limitations of previous techniques by eliminating bacterial sequences, accommodating RNA modifications, and yielding circRNAs with low immunogenicity. A key advancement is the demonstration that circRNAs incorporating human ribosomal RNA sequences are translated more efficiently than those produced by conventional permuted intron–exon methods. Furthermore, by leveraging viral internal ribosome entry sites, the authors achieve a remarkable over 7,000-fold enhancement in rolling circle translation efficiency, enabling continuous protein production. The work also introduces practical analytical methods for circRNA characterization and highlights the potential of these optimized circRNAs for durable gene expression and therapeutic applications, marking a significant leap in nucleic acid biotechnology.
Next-Generation Vaccine Development
CircRNA vaccines offer superior stability and sustained antigen expression compared to conventional mRNA. This prolonged presentation leads to enhanced humoral and T-cell-mediated immune responses, making them ideal candidates for long-lasting immunity against infectious diseases (e.g., SARS-CoV-2) and highly mutation-prone pathogens.
Inborn Genetic Disorder Correction
For diseases requiring steady-state levels of functional protein (e.g., enzyme replacement therapy), the sustained expression profile of circRNA minimizes the frequency of dosing compared to transient mRNA delivery, improving patient compliance and therapeutic outcome.
Transient In Vivo Gene Editing (CRISPR/Cas)
Delivering components like Cas9 mRNA or gRNA as a circRNA construct provides the necessary transient expression without the risk of genomic integration associated with DNA vectors, offering a safer and more controllable method for high-precision genome editing applications.
Chronic Protein Replacement Therapy
CircRNA serves as an effective vehicle for continuous expression of therapeutic proteins (e.g., antibodies, growth factors) over extended periods, providing a non-viral alternative for chronic conditions where gene expression maintenance is required.
Exceptional Stability for Extended Efficacy
Our circRNA is inherently resistant to exonuclease degradation due to its closed-loop structure, delivering significantly sustained protein expression in vivo compared to linear mRNA, crucial for chronic disease treatment.
High Functionality
We utilize empirically validated internal translation initiation elements and advanced sequence optimization methods. This approach ensures efficient ribosome recruitment and robust protein production, which is essential for achieving the desired therapeutic effect at low dose levels.
Proprietary High-Yield Cyclization
Utilization of our optimized group I and group II self-splicing PIE methodologies ensures maximal conversion of linear precursor to functional circular transcript, boosting yield and reducing purification complexity.
Unmatched Purity Standards
Our multi-step purification workflow, including specialized IP-RP, achieves industry-leading purity standards, drastically reducing regulatory friction during transition to clinical studies.
We appreciated the end-to-end integration CD BioGlyco offered. Not only did they synthesize our complex circRNA construct efficiently, but their associated LNP formulation service packaged it perfectly. The robust in vivo expression validation they provided was the perfect final step before our submission. Highly recommended.
— Senior Researcher O'Connell, Molecular Biology Department
The specialized purification protocols CD BioGlyco employs are top-tier. We needed ultra-high purity circRNA for a sensitive cell therapy application, and CD BioGlyco delivered consistent, high-integrity batches that passed all our internal quality benchmarks. Their service is truly pharmaceutical-grade.
— Principal Investigator Patel, Oncology Research
CD BioGlyco's expertise in minimizing dsRNA impurities was crucial for our vaccine program. The resulting product showed remarkably low innate immune activation while delivering the prolonged antigen expression needed for potent, durable immunity. Their technical consultation during the template design phase was invaluable.
— Director Williams, Vaccine Development Unit
Achieving therapeutic success with circRNA often requires robust delivery technology and comprehensive ancillary support. CD BioGlyco offers an integrated suite of complementary services designed to optimize the performance and utility of your circRNA construct. CircRNA efficacy is heavily dependent on efficient and targeted cellular delivery. We offer specialized conjugation services to enhance tissue specificity, maximizing therapeutic index while minimizing systemic exposure.
Integrating GalNAc conjugation directly onto the surface of custom-formulated LNPs for dual-mechanism targeting and enhanced in vivo performance.
Employing highly specific bioorthogonal chemistry to rapidly and efficiently functionalize circRNA with GalNAc, suitable for complex structures requiring minimal harsh chemical exposure.
CD BioGlyco is your trusted partner for high-purity, scalable circRNA synthesis and integrated delivery solutions. We are dedicated to accelerating your therapeutic development pipeline from discovery through to preclinical readiness. If you are interested in our commitment to proprietary synthesis technology, rigorous quality control, and expert consultation that ensures that your project benefits from maximum stability, optimal expression, and minimal immunogenicity, contact us!
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