Folks who have been dreaming happily like I have over the past decade that nucleic acids are the right substrate for engineering medicines, well, here is one more evidence that we might just be right with our obsession with this marvelous polymer of life!
Here is how I evangelized my obsession amongst colleagues.
Silicon Valley is silicon valley and not germanium valley — germanium just wasn’t the right substrate though the first transistor was made of germanium after all, see here for the first paper and here for a lovely history of the transistor.
Aren’t you glad? — Germanium Valley just doesn’t quite have the right euphony, does it?
Nucleic acids are the right substrate for genetic and gene-centric medicines and I don’t think either small molecules or proteins are. Those are the germanium of genetic medicines — they may work but the sooner you use silicon the sooner we will solve all human diseases. Yeah, I am opinionated!
Circularized RNA + cell-type targeting aptamer
A fascinating paper quietly appeared on BioRxiv1 about a month or so back. It’s a collaboration amongst multiple groups in China, with Weihong Tan as the PI.
They report first-in-human testing of very curious idea I had toyed with for a while now as a high-risk high-reward R&D project. They created aptamer-embedded circular RNAs (Apt-circRNAs). What’s wild is that they tested the concept in Phase 1 human trial right away from what would otherwise still be a marvelous proof-of-concept tested in ex vivo (blood) setting or in in vivo (humanized rodent) studies.
They got human clinical data. Wow!
The study combines two distinct and established ideas in nucleic acids—
- Circularizing of synthetic mRNA to enhance stability (the payload)
- Use of aptamers as a targeting molecule for cell-type specific delivery
This a totally crazy pace of testing out platform ideas. For those of you who do not work in the field — why is this significant?
Current mRNA vaccines like those for COVID-19 rely on LNPs for delivery, which can sometimes cause immunogenicity and predominantly accumulates in the liver. The Apt-circRNA platform is clever: the RNA molecule itself contains targeting information (receptor-targeting aptamers) to achieve cell-type-specific delivery, eliminating the need for synthetic carriers like LNPs to gift wrap the RNA.
The Three-Module Design
The Apt-circRNA platform elegantly integrates three functional modules into a single RNA molecule—
Targeting Module
The authors embedded dendritic cell (DC)-specific aptamers at precise locations within the circular RNA scaffold. They tested three targeting aptamers—nucleolin (nuc), transferrin receptor (waz), and DEC-205 (also called CD-205) (min2).
- Recall that DEC-205 (also called CD-205) is a cell surface-receptor (and endocytic receptor) highly expressed in immature Dendritic Cells (DCs).
- Transferrin receptor (TfR) is highly expressed in mature DCs and is crucial for iron uptake
- Nucleolin is also a cell surface receptor in endothelian cells and DCs. It can internalize from cell-surface to the nucleus
They used the Waz aptamer sequence for TfR—Waz was created by a Matt Levy whom I had hired at Creyon Bio, and who has lead the aptamer team and created a diversity of cell-type specific aptamers since. We know this aptamer well!234.
The Waz aptamer has chemical modifications as far as I remember—2’F modified C/Us and probably 2’OMe for some positions. The study uses native RNA—so there are no chemical modifications on the aptamer sequence. It’s an important distinction to keep in mind.
The DEC-205 aptamer min2 was also discovered my Matt’s group.5 The sequence from Fig.1 of Ref. 4 is —
The waz aptamer showed superior binding to both murine and human DCs. Through some optimization, the study determined that a bispecific combination of 5 waz and 4 min2 aptamers yielded optimal antigen presentation. Intersting! Thats a lot of aptamers decorating the RNA!
Stable expression framework
The circular RNA architecture provides inherent nuclease resistance by eliminating free 5’/3′ termini. The study demsntrates that the Apt-circRNA maintains structural integrity for over 24 hours and dramatically outperforming N1-methylpseudouridine-modified linear mRNA (m1Ψ-mRNA). This confirms older work that circularization of mRNA helps in extending half-life67. The construct also remains stable across pH 4.0-8.0 which critical for endosomal trafficking.
Antigen-Encoding Region
An Internal Ribosome Entry Site (IRES) enables cap-independent translation, while codon-optimized sequences encode tumor-specific antigens. The modular design permits flexible incorporation of diverse antigens, demonstrated with ovalbumin peptides ranging from 8 to 386 amino acids.
The Clever Circularization Strategy
Here’s where the molecular engineering gets quite ingenious! The team adapted permuted intron-exon (PIE) ribozyme systems from two sources: Anabaena pre-tRNA and T4 bacteriophage td intron. The key innovation: they engineered the aptamer’s stem-loop structure to serve as the circularization site without mutating the aptamer sequence itself!
The process works by introducing a cleavage site within the aptamer’s loop region, then engineering the group I intron’s P1 and P10 guide sequences to complement sequences flanking the aptamer cleavage site. This enables precise, ribozyme-catalyzed splicing at the predefined loop site, generating Apt-circRNA products free of residual intron sequences.
Cute!
What’s the bio-distribution of Apt-circRNA?
PET Imaging reveals precise lymph node targeting!
They used radio-labeled Apt-circRNA and positron emission tomography (PET) to track spatial and temporal distribution.
PET imaging showed predominant renal accumulation with no notable off-target accumulation in liver, spleen, heart, or other major organs. This specificity is striking and addresses a major concern with LNP-based systems, which accumulate significantly in liver and spleen. The renal accumulation is perhaps owing to renal clearance of such a relatively smaller molecular weight payload?
They also ran Cy5-labelled study—6 hours post-injection revealed that cy5-labelled Apt-circRNA preferentially accumulated in dendritic cells compared to B cells and macrophages. Apt-circRNA was efficiently internalized by DCs at the injection site!
This contrasts sharply with LNP-circRNA, which primarily remained as intact nanoparticles near the injection site before uptake by both B cells and DCs within lymph nodes. This is consistent with the expectation that aptamer-mediated recognition enables direct DC internalization and lymph node trafficking.
The study also looked at immuno-stimulatory responses. Worth a read.
Surprisingly, Luminex assays revealed that Apt-circRNA elicited lower systemic levels of reactogenicity-associated chemokines and cytokines than LNP-circRNA except IL-12. Apt-circRNA also demonstrated reduced cytotoxicity in BMDCs (murine bone-marrow derived DCs) compared to LNP-circRNA. I would have expected the opposite—recall that native RNA could invoke innate response from pathways that sniff out cytosolic RNA.
First-in-Human Clinical Trial
The authors initiated a Phase I clinical trial at Zhejiang Xiaoshan Hospital with remarkable speed, testing Apt-circRNA-KR2 in healthy volunteers. Here KR2 refers to the RNA payload expressing the mutations (G12D and G12V) most common in KRAS gene. They want to elicit a T-cell response in KRAS-mutant cancer.
G12D and G12V are two of the most common single amino acid substitutions at codon 12 of the KRAS gene. The G12D indicates a substitution of the normal amino acid Glycine (G) with Aspartic acid (D) at position 12, while G12V indicates a substitution with Valine (V).
The trial enrolled 12 healthy volunteers total. Though a small cohort, it’s very encouraging!
- Single-dose escalation cohort: 9 volunteers received 50, 100, or 250 μg doses (n=3/group)
- Multi-dose cohort: 3 HLA-A*02:01 or HLA-A*11:01-positive volunteers received 250 μg on days 1, 7, and 13
- Only 1 of 12 participants experienced any adverse event—transient flu-like symptoms resolving within 12 hours
- Zero injection-site reactions (0/12)
- Zero grade ≥2 adverse events (0/12)
- All hematologic parameters, immune cell subsets, and cytokines remained within normal ranges through 180 days
The safety profile is quite surprising and compares very favorably to LNP-mRNA vaccines, which commonly cause injection-site reactions, fever and systemic symptoms.
High notes
Unlike previous ‘naked mRNA’ approaches that lack stability and targeting, it’s a clever idea to encode aptamer within the RNA sequence itself. However, I am not convinced this is necessary and it will prohibit chemical modifications that can stabilize the aptamer, increase affinity and half-life. One could conjugate the aptamer to the circular RNA and modularize the system further.
The dual aptamer strategy is definitely very interesting. The combination of waz (TfR-targeting) and min2 (DEC-205-targeting) creates a bispecific design that enhances both binding affinity and functional outcomes. Are these serving different purposes in directly the payload to endocytic compartments?
Manufacturing could be quite scalable, as is! The in vitro transcription and ribozyme-mediated circularization can be performed at scale without the complex formulation processes required for LNPs. The >80% circularization efficiency is commercially viable. Also, perhaps this advantage is a counter-point to the first point I made about chemically-modified aptamers, for which the aptamer would have to separately synthesized and somehow conjugated to the RNA at specific sites that does not disrupt expression. Moreover, with multiple aptamers to decorate the RNA, it’s messy. Efficiency and purity of product would be a challenge.
Cellular uptake was still a bit low, and I suspect this is because the aptamers were not really optimized. They took existing aptamer sequences and slapped it on. Flow cytometry data showed that even in draining lymph nodes only a fraction of DCs take up Apt-circRNA. This may necessitate higher doses or more frequent administration compared to LNP-formulated mRNA, partially offsetting the manufacturing advantages. But I strongly believe this is a solvable engineering problem.
Also, the naked RNA formulation and size leads to rapid renal clearance. One could incorporate sugar / lipid modifications to the construct but then the modules need to be separate—the circularized RNA and the aptamer (chemically modified and say, with added ligands like PEG attached, or albumin binding aptamers). Totally doable!
The bigger picture
For decades, the field has focused on engineering increasingly sophisticated nanocarriers—optimizing lipid chemistry, surface modifications etc. What is thought provoking here is, Why engineer a carrier when you can engineer the RNA itself?
It’s a tantalizing possibility—Nucleic acid is all you need.
References
- https://www.biorxiv.org/content/10.1101/2025.09.28.679023v1 ↩︎
- https://patents.google.com/patent/US9439973B2/en ↩︎
- https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(17)30047-1 ↩︎
- https://www.nature.com/articles/s41467-018-04691-x ↩︎
- https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(16)30728-6 ↩︎
- https://www.nature.com/articles/s41467-018-05096-6
↩︎ - https://www.nature.com/articles/s41587-022-01393-0
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