Personalized messenger RNA (mRNA) neoantigen vaccines hold immense potential in cancer immunotherapy. However, their customized manufacturing pipeline typically spans more than three months, which risks missing the optimal therapeutic window for patients. The primary bottleneck causing this delay is that current mRNA vaccine production relies heavily on plasmid fermentation and in vitro transcription (IVT), involving numerous complex steps.
Chemically synthesized RNA oligonucleotides, such as antisense oligonucleotides (ASOs), circumvent the need for DNA templates or IVT during synthesis, thereby enabling rapid manufacturing. However, ASOs are restricted in length and cannot encode proteins.
Recently, researchers from Fudan University in China published a research paper online in PNAS titled "IVT-free, chemically synthesized protein-encoding RNA oligonucleotides for rapid production of personalized cancer vaccines." The study introduces a 39-nucleotide cap-independent translation enhancer (CITE) element, designated BBV, which is capable of driving RNA translation. Furthermore, BBV is compatible with highly efficient rolling circle translation (RCT).
The authors chemically synthesized RNA oligonucleotides harboring BBV and a gene of interest (GOI), featuring characteristic 5'-OH and 3'-P termini. These oligonucleotides undergo intracellular circularization mediated by endogenous RtcB RNA ligase and efficiently encode proteins via RCT in mammalian cells. The authors named these constructs protein-encoding RNA oligonucleotides (PEOs).
Notably, compared with IVT-produced RNA, PEOs exhibit undetectable levels of pro-inflammatory double-stranded RNA (dsRNA), demonstrating remarkably low immunogenicity. The authors further confirmed that PEO-OVA (encoding the OVA antigen) achieved tumor growth inhibition comparable to that of a standard mRNA vaccine. In an orthotopic glioma model, the PEO vaccine combined with immune checkpoint blockade therapy also demonstrated clear therapeutic efficacy. This study establishes an IVT-free RNA vaccine platform that enables the rapid, safe, and highly druggable production of personalized cancer vaccines, holding significant potential for clinical translation.
Figure 1. Short CITE elements enabled translation initiation of RNA. (Pan Q, et al., 2026)
Background and Technological Breakthrough
RNA oligonucleotides, such as ASOs or small interfering RNAs (siRNAs), are produced entirely via solid-phase chemical synthesis without requiring DNA templates or IVT. Consequently, they offer rapid manufacturing turnaround alongside potential advantages in safety and druggability. Such oligonucleotides can be designed in silico and directly synthesized, dramatically shortening vaccine production timelines while avoiding enzymatically generated impurities like dsRNA.
However, the restricted length of chemically synthesized RNA oligonucleotides (typically under 150 nucleotides) severely hampers their protein-encoding capacity. Linear mRNA requires essential translational elements—including a 5' cap, a GOI, and a 3' poly(A) tail—whereas circular RNAs (circRNAs) typically require an internal ribosome entry site (IRES) element of approximately 500 to 700 nucleotides (nt) in addition to the GOI sequence.
The inclusion of a poly(A) tail or a long IRES causes the total construct length to exceed the maximum threshold achievable through pure chemical synthesis. As a result, chemically synthesized RNA therapeutics have been predominantly restricted to ASOs, which function through non-coding transcriptional or post-transcriptional mechanisms and are incapable of encoding proteins.
Recently, several research groups have attempted to engineer protein-encoding RNA oligonucleotides via solid-phase chemical synthesis. For instance, Hiroshi Abe and colleagues chemically synthesized RNA oligonucleotides capped at the 5' end; however, increased synthetic complexity and lower translational efficiency remain obstacles to broader application. Canonical linear mRNA translation requires the 5' cap and 3' poly(A) tail to be brought into physical proximity (via non-covalent 5'-to-3' circularization) mediated by eukaryotic translation initiation factor 4G (eIF4G)–PABP interactions to initiate translation.
To overcome this constraint, two independent teams utilized enzymatic (T4 RNA ligase) or chemically catalyzed ligation, respectively, to covalently circularize chemically synthesized linear RNA in vitro, thereby improving translational efficiency to some degree. Nevertheless, additional enzymatic or chemical circularization steps—along with subsequent purification—prolong manufacturing timelines and complicate industrial scaling due to stability and quality-control challenges.
Moreover, chemical circularization strategies may introduce non-canonical P–N chemical bonds in certain contexts, potentially interfering with normal protein translation from the synthesized RNA oligonucleotides. Although cap analogs have been introduced to bypass the requirement for long IRES sequences in circRNA, these methods still depend on IVT and necessitate enzymatic or chemical ligation between the synthesized capped oligonucleotide and the IVT-derived RNA, further increasing complexity and production lead time.
A recent study demonstrated that flanking RNA with Twister ribozyme sequences generates 5'-OH and 2',3'-cyclic phosphate termini through self-cleavage. Upon delivery into mammalian cells, these processed RNAs are covalently circularized by endogenous RtcB RNA ligase to form circRNA. Furthermore, circRNA containing covalent phosphodiester bonds supports highly efficient rolling circle translation (RCT)—a mechanism involving single-site initiation and continuous elongation that yields significantly higher protein output than classical linear translation.
Therefore, developing a rapid, one-step chemically synthesized RNA oligonucleotide platform with robust protein-coding capacity that requires no post-synthesis processing is immensely attractive. Such a strategy preserves all the inherent benefits of chemically synthesized oligonucleotides while enabling the rapid manufacturing of personalized cancer neoantigen RNA vaccines.
Reference
- Pan Q, et al. IVT-free, chemically synthesized protein-encoding RNA oligonucleotides for rapid production of personalized cancer vaccines. Proceedings of the National Academy of Sciences of the United States of America, 2026, 123(29): e2529799123.