Rznomics has reported a significant improvement in the manufacturing of long circular RNA molecules, addressing a technical bottleneck that could determine how broadly the emerging RNA modality can be used for vaccines, protein replacement and engineered cell therapies. In a peer-reviewed Nucleic Acids Research study, company researchers optimized their Self-Targeting and Splicing platform through target-site selection and engineering of a P1 construct involved in the circularization reaction. For long RNA sequences approaching eight kilonucleotides, the optimized system achieved as much as twice the self-circularization efficiency of the conventional Permuted Intron-Exon method.
The strongest demonstration used Factor VIII RNA of approximately 7.8 kilonucleotides, where Rznomics reported roughly twofold greater circularization efficiency than PIE. Within its own STS architecture, sequential addition of a polyA10 sequence and an antisense component improved efficiency by as much as sevenfold compared with the original design. The results are a manufacturing and molecular-engineering advance rather than a patient-treatment milestone. No therapeutic circRNA produced with the method has been shown safe or effective in people as a result of this paper.
Why is circular RNA interesting for medicines in the first place?
Ordinary messenger RNA is linear. It has a 5′ end and a 3′ end, and cellular enzymes can attack those exposed ends, contributing to degradation.
Circular RNA joins those ends into a covalently closed loop. Without free termini, the molecule can be more resistant to exonuclease-mediated degradation and potentially persist longer inside cells.
That stability creates a potentially valuable property for therapeutic use. If an RNA molecule encodes a protein, longer persistence may allow cells to continue producing that protein for more time from one dose.
Vaccines could theoretically achieve more durable antigen production. Protein-replacement approaches could potentially require less frequent administration. Engineered immune-cell applications might use circular RNA to provide transient but sustained expression without introducing permanent DNA modifications.
Circularity alone is not sufficient to make a successful therapy. RNA still needs to enter the right cells, avoid excessive immune activation and translate efficiently.
Manufacturing is another problem, and that is the one Rznomics’ paper addresses.
Why does circular RNA become harder to manufacture as the sequence gets longer?
RNA molecules fold into complex three-dimensional structures. As their length increases, the number of possible internal interactions expands, making it harder for the ends and catalytic components needed for circularization to align correctly.
The Rznomics study found that self-circularization efficiency depended strongly on which target site was selected within the gene-of-interest sequence. Even different positions inside the same RNA could produce markedly different yields.
This becomes commercially important for large therapeutic payloads. A short antigen sequence may be relatively easy to circularize, whereas a long protein-coding sequence approaching eight kilonucleotides can form far more elaborate secondary structures.
Poor circularization means more precursor RNA remains linear or becomes undesirable byproduct. Manufacturers then have to purify the intended circular product away from unwanted material, reducing yield and increasing cost.
A platform that preserves efficiency as payload length increases expands the range of proteins potentially accessible to circRNA.
How does Rznomics’ Self-Targeting and Splicing method work?
STS uses a self-splicing ribozyme derived from the Tetrahymena group I intron. The RNA precursor is engineered so that the catalytic reaction joins the gene-of-interest ends during in-vitro transcription and forms a circular product.
A key attraction of the method is that it can generate what researchers describe as scarless circular RNA, meaning the finished molecule does not need to retain unnecessary spacer sequences introduced solely for the circularization process.
Extra sequences can potentially affect translation, stability, immune recognition or product consistency, so minimizing nonfunctional material can be advantageous.
The new paper did not replace STS with a completely different technology. Researchers systematically optimized two critical elements: where the self-circularization reaction targets the RNA and how the P1 structure brings the necessary regions together.
What did target-site screening reveal?
Researchers found that circularization efficiency changed significantly depending on target-site position even within the same gene-of-interest sequence. That suggests developers cannot assume that any theoretically compatible site will perform equally well.
RNA folding can hide one target site and expose another. Long-range interactions elsewhere in the molecule can also alter accessibility.
The team therefore screened potential sites and identified locations that supported stronger self-circularization.
This sounds like a straightforward optimization, but it has practical consequences. Instead of viewing poor yield from a long RNA as an unavoidable property of the payload, developers can systematically search for a more favorable circularization site before changing the therapeutic sequence itself.
The limitation is labor. Extensive screening can become time-consuming when every new therapeutic construct requires a separate optimization campaign.
That is why the P1 engineering component of the paper may ultimately be just as important.
What did adding polyA10 and an antisense sequence accomplish?
Rznomics modified the P1 construct by introducing a short sequence of ten adenosines upstream of the internal guide sequence and adding an antisense region complementary to the target site and neighboring RNA. The goal was to strengthen the productive interaction required for the self-splicing reaction.
Combining these engineering changes increased self-circularization efficiency by as much as approximately sevenfold compared with the original STS architecture in selected experiments.
The improvement is important because it offers a more general engineering strategy rather than relying only on finding one unusually favorable site in every gene.
In long RNAs, the engineered interaction may help overcome structural constraints that otherwise prevent the catalytic regions from aligning effectively.
Why did the researchers use Factor VIII RNA as an important test?
Factor VIII is a large protein involved in blood coagulation, and its coding sequence provides a demanding long-payload challenge. The study tested an RNA of approximately 7.8 kilonucleotides, close to the eight-kilobase range that is particularly difficult for efficient circularization.
With this construct, optimized STS achieved approximately twice the circularization efficiency of the conventional PIE approach.
The result does not mean Rznomics has created an approved circular-RNA Factor VIII therapy for hemophilia. Factor VIII served primarily as a large gene-of-interest model demonstrating that the method can accommodate therapeutically relevant long sequences.
If the platform remains efficient with other large proteins, it could widen circRNA’s addressable therapeutic space beyond relatively short antigens or signaling proteins.
What is the PIE method and why is it an important benchmark?
Permuted Intron-Exon circularization is one of the established strategies for generating synthetic circRNA. It uses rearranged intron and exon elements that drive a self-splicing reaction, joining the ends of the desired RNA.
PIE has been widely used in circRNA research, making it a useful reference method for evaluating newer manufacturing approaches.
Rznomics’ paper reports that STS can outperform PIE particularly for long genes of interest while generating circular products without extraneous sequences.
That comparison matters because claiming a sevenfold improvement over an earlier internal design does not reveal how the technology performs against an external industry-standard approach. The approximately twofold advantage over PIE for the long Factor VIII sequence gives the paper a more meaningful benchmark.
Whether that advantage persists under industrial GMP manufacturing conditions remains another question.
Why can manufacturing efficiency determine whether an RNA therapy becomes commercially viable?
A therapeutic platform can work beautifully in a laboratory and still fail as a drug if it cannot be produced reproducibly at large scale.
Poor reaction efficiency increases raw-material use and creates more impurities that must be removed. Every additional purification step can reduce yield and increase cost.
For RNA therapeutics, manufacturers also need extremely consistent product identity because linear precursors, truncated molecules and abnormal circles may behave differently biologically.
A more efficient circularization reaction can therefore affect cost, batch consistency and the practicality of scaling production.
This becomes especially important for vaccines or chronic medicines where commercial volumes may be large.
The new Rznomics method remains a research-stage manufacturing platform rather than a validated commercial GMP process, but it attacks a bottleneck that would eventually become unavoidable if long circRNA medicines move into late-stage trials.
How could circular RNA be used in CAR-T cell therapy?
Rznomics says it plans to explore its platform across therapeutic applications including CAR-T.
Traditional autologous CAR-T products often use viral vectors to introduce DNA encoding the chimeric antigen receptor into patient T cells, creating durable receptor expression. RNA-based approaches can instead provide temporary expression without permanently integrating genetic material.
Linear mRNA has already been used experimentally for transient CAR expression, but its relatively short intracellular lifetime can limit duration.
Circular RNA could theoretically sustain CAR or other therapeutic protein production longer than linear RNA while remaining nonintegrating. That might be attractive when developers want potent activity for a defined period without permanent genetic modification.
This remains a prospective application. The Rznomics manufacturing paper does not present clinical CAR-T results using its circular RNA.
How does this research fit with Rznomics’ existing RNA business?
Rznomics is already a clinical-stage RNA biotechnology company, but its better-known therapeutic platform involves trans-splicing ribozymes rather than circRNA manufacturing. Its lead oncology program, RZ-001, has entered clinical development, and the company has an RNA-editing collaboration with Eli Lilly.
The circRNA work therefore expands rather than replaces its existing platform strategy.
For a biotechnology company, a manufacturing technology can also have partnership value independent of an internally owned drug. Other developers may need efficient ways to manufacture circular constructs encoding their own therapeutic proteins.
That possibility will depend on intellectual property, reproducibility and whether Rznomics can translate the academic reaction into robust industrial processes.
What are the main limitations of the new study?
First, it is an in-vitro manufacturing study. No patients were treated.
Second, higher circularization efficiency does not automatically mean the finished RNA translates more protein, lasts longer in vivo or is safer.
Third, the best improvements were construct dependent. RNA structure varies considerably, so developers need to know how reliably the engineering rules generalize to completely different sequences.
Fourth, commercial manufacturing introduces scale, purity and quality-control constraints that are not identical to research-scale experiments.
Finally, the authors include Rznomics employees and the company’s chief executive, and Rznomics supported the open-access publication fee. The work is peer reviewed and its data are available with the publication, but independent replication would add confidence.
What should the RNA therapeutics field watch next?
The next step is demonstrating reproducibility across a larger variety of clinically relevant payloads and at increasing manufacturing scale.
Researchers will also need to compare translation efficiency, stability, innate immune activation and protein-expression duration between optimized STS circles and competing circRNA architectures.
Eventually, a drug candidate manufactured with the process needs to enter animal and human testing before the technology can be linked to clinical benefit.
The significance of the September paper is consequently more industrial than therapeutic. Circular RNA promises longer-lived expression than ordinary linear RNA, but that advantage becomes less useful if large molecules cannot be manufactured efficiently.
Rznomics has shown that smarter target-site selection and a redesigned self-splicing construct can materially improve that reaction, including roughly doubling efficiency against PIE for an approximately 7.8-kilobase payload.
That does not produce a new medicine today. It potentially removes one obstacle standing between circular RNA and the larger proteins developers may want it to produce tomorrow.