Self-amplifying RNA (saRNA) Synthesis Service
In the rapidly evolving landscape of nucleic acid therapeutics, self-amplifying RNA (saRNA) has emerged as a revolutionary "next-generation" messenger RNA (mRNA) technology. Unlike conventional mRNA, which provides a transient template for protein translation, saRNA is engineered to replicate itself once inside the host cell. By encoding a viral-derived RNA-dependent RNA polymerase (RdRp) complex, a single saRNA molecule generates thousands of copies of the subgenomic transcript. At CD BioGlyco, we provide a comprehensive saRNA synthesis service that addresses the inherent technical challenges of these large, complex molecules—typically ranging from 9 to 13 kilobases (kb). Our platform enables researchers to achieve sustained protein expression and superior immunogenicity at doses significantly lower than traditional mRNA (often 10 to 100 times lower), effectively reducing manufacturing costs and potential toxicity.
Key Technologies
Viral Replicon Engineering
We utilize optimized alphavirus-derived backbones (such as Venezuelan equine encephalitis virus (VEEV) or Sindbis virus) to encode the non-structural proteins (nsP1-4). This ensures high-fidelity replication and robust subgenomic promoter activity.
High-Fidelity In Vitro Transcription (IVT)
Given the exceptional length of saRNA (up to 13 kb), we employ specialized T7 RNA polymerase variants and optimized buffer systems to minimize premature termination and ensure the production of full-length, intact RNA transcripts.
Advanced Capping and Tailing Systems
We utilize enzymatic capping and co-transcriptional capping technologies alongside precise enzymatic polyadenylation to maximize the stability and translation efficiency of the saRNA once it enters the cytoplasm.
Precision Replication, Infinite Potential: Leading the Charge in saRNA Innovation
As a core component of our therapeutic nucleic acid development platform, the therapeutic oligonucleotide synthesis service at CD BioGlyco offers a diverse range of synthesis solutions tailored to specific therapeutic goals. Our service scope includes:
- Custom saRNA Vector Design: We provide a variety of replicon sequences, including wild-type and mutant variants, to tune the level of immunogenicity and duration of expression.
- Large-Fragment Synthesis and Assembly: Our platform is optimized for the synthesis of long-chain RNA (8-13 kb). We handle the complex assembly of the RdRp genes and your gene of interest (GOI) into a single, cohesive construct.
- Modified Nucleoside Integration: While saRNA replication is sensitive to certain modifications, we offer specialized synthesis using 5-methylcytosine (5mC) or other compatible modified nucleotides that help evade innate immune sensing while maintaining RdRp activity.
- Sub-substance Synthesis: We provide synthesis of related components, including double-stranded RNA (dsRNA) standards for quality control (QC) and specific untranslated regions (UTRs) for enhanced tissue-specific expression.
Workflow
Sequence Design and Optimization
We perform codon optimization for the GOI and secondary structure analysis of the saRNA backbone. This step ensures optimal translation and minimizes RNA folding issues that could impede the replication machinery.
Template Plasmid Construction
The optimized sequence is cloned into a high-copy plasmid DNA (pDNA) vector. This template includes a T7 promoter, the alphavirus replicon, the GOI, and a defined poly(A) tail sequence.
Plasmid Linearization and Purification
To ensure clean IVT results, the pDNA is linearized using high-fidelity restriction enzymes. We then perform stringent purification to remove any traces of circular DNA or enzymatic proteins.
Large-Scale In Vitro Transcription
Using our optimized IVT platform, we generate the saRNA transcripts. This stage is closely monitored to maintain the integrity of the long RNA strands and to ensure high yields.
Stringent Purification and Polishing
We employ tangential flow filtration (TFF) and high-performance liquid chromatography (HPLC) to remove dsRNA byproducts, residual DNA templates, and shorter RNA fragments. This is critical for reducing unwanted interferon (IFN) responses.
Comprehensive Quality Control
Each batch undergoes a battery of tests, including capillary electrophoresis (CE) for integrity, LC-MS for capping efficiency, and enzyme-linked immunosorbent assay (ELISA) for dsRNA residue levels, ensuring the product meets all therapeutic specifications.
Publication Data
DoI: 10.3390/vaccines13101062.
Journal: Vaccines
IF: 3.4
Published: 2025
Results: This study optimizes the IVT process of saRNA vaccines using Design of Experiment (DoE) under the Quality by Design (QbD) framework to address poor integrity caused by saRNA's long sequence. Five key parameters were analyzed, with Mg2+ concentration identified as the most critical factor (non-linear effect) affecting saRNA integrity and yield. Through response surface methodology and Monte Carlo simulation, an optimal IVT parameter set and design space were established, achieving saRNA integrity >85% and yield ≥600 μg/100 μL. This system is applicable to longer saRNA constructs (9.2k-11.5k nt). Murine immunization experiments confirmed that higher saRNA integrity (80% vs. 37%/64%) significantly enhanced antigen-specific antibody titers, pseudovirus neutralization activity, and IFN-γ-secreting T-cell responses. The findings provide a robust technical basis for the industrialization and quality standardization of saRNA vaccines.
Fig.1 Construction of saRNA expressing receptor-binding domain (RBD). (Hu, et al., 2025)
Applications
Infectious Disease Vaccines
Achieving high antigen titers with microgram-level doses for rapid pandemic response.
Cancer Immunotherapy
Sustained expression of tumor-associated antigens or cytokines to modulate the tumor microenvironment.
Protein Replacement Therapy
Long-term expression of therapeutic proteins for the treatment of metabolic or genetic disorders.
In Vivo CAR-T/TCR Generation
Utilizing saRNA to transiently but powerfully reprogram immune cells directly within the mice.
Advantages
Exceptional Integrity
Our optimized processes consistently achieve >90% RNA integrity for sequences exceeding 10 kb.
Ultra-Low dsRNA Contamination
We utilize specialized purification and enzymatic strategies to reduce dsRNA levels to <0.1 ng/µg, minimizing innate immune over-activation.
High Potency at Low Doses
Our constructs are designed for maximum dose-sparing, allowing for effective results at 1/10th the mass of conventional mRNA.
Customizable Backbones
Access to multiple viral replicon types allows for fine-tuning of the expression kinetics (fast peak vs.sustained plateau).
Frequently Asked Questions
Customer Review
"The saRNA synthesis service from CD BioGlyco was instrumental in our recent vaccine study. The integrity of the 11 kb construct was remarkable, and we observed sustained antigen expression in our murine models for over 14 days with a single low dose."
– S.E., Principal Investigator
"We were concerned about the dsRNA-induced toxicity often associated with long RNA molecules. However, the purification process at CD BioGlyco provided such a clean product that we saw minimal inflammatory markers while maintaining high protein output."
– A.D., Senior Scientist
"Their team's expertise in viral replicon engineering helped us optimize our GOI for much better results than our previous in-house attempts. The technical support throughout the project was exceptional."
– E.V., R&D Director
Associated Services
CD BioGlyco stands at the forefront of saRNA technology, providing a high-end synthesis platform that combines viral engineering expertise with advanced RNA chemistry. By delivering long-chain, high-purity saRNA constructs, we enable our clients to explore the full potential of self-amplifying nucleic acids in vaccines, oncology, and beyond. Please feel free to contact us to assist with sequence design, synthesis, and characterization.
Reference
- Hu, C.; et al. Optimization of in vitro transcription by design of experiment to achieve high self-amplifying rna integrity. Vaccines. 2025, 13(10): 1062. (Open Access)