Synthetic circRNAs employ IRES activity for translation in cells and in cell-free translation systems

This study systematically characterizes the ability of various viral and cellular internal ribosomal entry sites (IRESes) to drive translation in synthetic circular RNAs across human cells and improved cell-free systems, providing critical insights for optimizing circRNA-based therapeutics.

The Gist

Imagine your body is a bustling city, and the instructions for building everything in that city (proteins) are written on long, fragile scrolls called mRNA. Usually, these scrolls have a "cap" at the start and a "tail" at the end. But these ends are weak points; they get chewed up by the city's cleanup crew (enzymes) very quickly. This is great for vaccines that need to work fast and then disappear, but it's bad if you want to keep a factory running for years to replace a missing protein.

Enter the Circular RNA (circRNA). Think of this as taking that fragile scroll, cutting off the ends, and gluing them together to make a tire. A tire has no beginning or end, so the cleanup crew can't chew it up. It lasts much longer, providing a steady, long-term supply of instructions.

However, there's a problem. The city's construction workers (ribosomes) are trained to grab the "cap" at the start of a scroll to begin building. Since a tire has no cap, the workers don't know where to start. To fix this, scientists attach a special "on-ramp" to the tire. This on-ramp is called an IRES (Internal Ribosome Entry Site). It's like a secret highway entrance that lets the construction workers jump straight onto the tire without needing the front door.

The Big Question:
Scientists have many different designs for these "on-ramps." Some are borrowed from viruses (which are very good at hijacking cells), and some are taken from our own human genes. The big mystery was: Which on-ramp works best on a tire? And, do these on-ramps accidentally trigger the city's security alarms (the immune system)?

What the Scientists Did:
The researchers in this paper acted like engineers testing different car parts. They built synthetic tires (circRNAs) and attached various viral and human on-ramps (IRESes) to them. They tested these in two ways:

1. Inside the City (Cells): Putting the tires into human cells to see if they actually started building proteins.
2. In a Test Tube (Cell-Free Systems): Using a simplified, controlled environment to see how the tires behaved without the complexity of a whole living cell.

Key Findings:

• Not All On-Ramps Are Created Equal:
  They found that the viral on-ramps (like those from Hepatitis C or Coxsackievirus) were like high-performance race car ramps. They worked incredibly well, getting the construction workers to start building immediately and efficiently.
  The human on-ramps were more like quiet, local neighborhood entrances. Some worked well, but many were sluggish or didn't work at all when put on a synthetic tire. This suggests that human on-ramps might need extra "keys" or helpers (proteins) that are only present when the tire is made inside a cell's nucleus, not when it's made in a test tube.

• The "Clean Tire" Rule:
  One of the most important discoveries was about purity. When making these tires in a lab, sometimes you accidentally leave behind scraps of the original straight scrolls (linear RNA). These scraps look like "foreign invaders" to the cell's immune system and trigger a massive alarm (inflammation).
  The team realized that if they cleaned the tires extremely well (using a special gel filter), the immune system stayed calm. It didn't matter which on-ramp was on the tire; the alarm was triggered by the dirty scraps, not the on-ramp itself. This means that for medical use, cleaning the product is more important than choosing a specific on-ramp to avoid side effects.

• The Perfect Test Tube:
  They also tested a new, high-tech "test tube" system that mimics human cells much better than old-fashioned ones. This new system was able to tell the difference between a good on-ramp and a bad one, just like a real cell does. This is a huge step forward because it means scientists can now test and design better tires in the lab before ever putting them into a human.

• Tweaking the Engine:
  Finally, they showed that they could take a viral on-ramp and make small "tweaks" to its design (like changing a few letters in the code) to make it work weaker or stronger. This proves that we can engineer these tires to release just the right amount of medicine, no more and no less.

Why This Matters:
This research is like a blueprint for building better long-lasting medicines. If we want to cure diseases where a patient is missing a protein for life (like certain genetic disorders), we need a delivery system that lasts. By figuring out which "on-ramps" work best on "tires" and how to keep them clean from immune alarms, this paper paves the way for a new generation of RNA therapies that are safer, more effective, and can work for years instead of just days.

In a Nutshell:
Scientists built circular "tires" of genetic code to deliver medicine. They tested different "on-ramps" to get the cell's machinery to start working. They found that viral ramps are powerful, human ramps are tricky, and the most important thing is to make sure the tires are perfectly clean so the body doesn't panic. This brings us one step closer to long-term, life-saving RNA medicines.

Technical Summary

1. Problem Statement

Circular RNAs (circRNAs) are emerging as promising therapeutics due to their stability and resistance to exonucleases, offering sustained protein expression compared to linear mRNA. However, a major bottleneck in developing circRNA-based medicines is the lack of systematic characterization of Internal Ribosomal Entry Sites (IRESes) within synthetic circRNA contexts.

• Knowledge Gap: While viral IRESes (e.g., HCV, CVB3) are known to drive translation, the functionality of diverse cellular IRESes in synthetic circRNAs is poorly understood.
• Technical Challenges:
  • Synthetic circRNAs often contain contaminants (linear RNA, dsRNA fragments) from in vitro transcription (IVT) that trigger innate immune sensors (RIG-I, MDA5), confounding translation assays.
  • Existing plasmid-based circRNA systems (relying on nuclear back-splicing or ribozyme ligation) may introduce artifacts, such as "nuclear experience" (RNA processing history) or trans-splicing events, which do not reflect the behavior of purely synthetic, exogenous circRNAs.
  • Standard cell-free translation systems (Rabbit Reticulocyte Lysate, Wheat Germ Lysate) often fail to recapitulate the complex regulation of IRES-dependent translation seen in human cells.

2. Methodology

The authors developed a robust pipeline to generate, purify, and test synthetic circRNAs containing various IRES elements.

• Synthesis & Design:

  • Generated synthetic circRNAs in vitro using T7 transcription.
  • Utilized permuted split group I introns (from phage T4 thymidylate synthase) as self-splicing ribozymes to circularize the RNA.
  • Reporter Constructs: Encoded either EGFP or NanoLuciferase (Nluc) upstream of the IRES element. The IRES was placed downstream of the ORF to ensure consistent ribozyme processing and prevent residual linear RNA from contributing to signal.
  • IRES Selection: Tested 3 viral (HCV, CVB3, EMCV) and 6 cellular (Hoxa9, Chrdl1, Dlx1, c-Myc, Cofilin, Hoxa5) IRESes, alongside negative controls (hHBB) and inverse sequence controls.

• Purification Strategy (Critical Innovation):

  • To eliminate immune-stimulatory contaminants, the authors employed a two-step purification: RNase R digestion (to degrade linear RNA) followed by 4% urea-PAGE gel extraction. This ensured the removal of dsRNA side products and linear IVT byproducts that typically activate immune sensors.

• Assay Systems:

  1. In Cellulo: Transfection of purified circRNAs into HEK293T cells. Measured via FACS (EGFP) and Dual-Luciferase assays (Nluc/Fluc ratio).
  2. Plasmid Comparison: Compared synthetic circRNAs against two plasmid-based systems:
     • ZKSCAN1 back-splicing system: Nuclear pre-mRNA circularization.
     • Tornado system: Cytoplasmic ribozyme cleavage and ligation.
  3. In Vitro Translation: Tested in three systems:
     • Rabbit Reticulocyte Lysate (RRL).
     • Wheat Germ Lysate (WGL).
     • Tripartite Human HEK293T Cell-Free System: A reconstituted system separating ribosomes, cytoplasm, and mRNAs to better mimic human physiology.

• Immunogenicity Assessment:

  • Measured mRNA induction of innate immune sensors (RIG-I, MDA5, OAS1, OASL, PKR) and IFN-β via RT-qPCR to determine if specific IRES sequences trigger immune responses.

• Engineering:

  • Introduced specific mutations in the HCV IRES (deletion of domain II and GGG-to-CCC mutation in domain III) to test if the synthetic system could recapitulate known mechanistic defects.

3. Key Results

• Purification is Paramount:

  • RNase R treatment alone was insufficient to remove all immunostimulatory contaminants.
  • Urea-PAGE purification was essential to obtain "immune-silent" circRNAs. Only with high-purity circRNAs could specific IRES activity be accurately distinguished from background noise caused by contaminants.

• IRES Activity in Synthetic circRNAs (In Cells):

  • Viral IRESes: HCV and CVB3 drove robust, high-level translation (CVB3 showed ~1500-fold activity over empty controls).
  • Cellular IRESes: Activity was variable. Hoxa9 and Chrdl1 showed moderate activity (2.8-fold and 1.5-fold over controls, respectively). Dlx1 and Hoxa5 were largely inactive in synthetic circRNAs, despite being active in plasmid-based back-splicing systems.
  • Inverse Controls: Inverse sequences of active IRESes (CVB3, Hoxa9, Chrdl1, Dlx1) lost activity, confirming specificity.
  • Discrepancy: The "nuclear experience" (splicing history) present in plasmid-derived circRNAs appears necessary for the full activity of certain cellular IRESes (like Dlx1), which are inactive in purely synthetic, exogenous circRNAs.

• Immunogenicity:

  • Purity > Sequence: The level of immune activation (sensor induction) was primarily determined by the purity of the preparation (presence of IVT contaminants) rather than the identity of the IRES (viral vs. cellular) or the translation state.
  • Highly purified circRNAs (PAGE + RNase R) showed minimal immune activation, regardless of the IRES used.

• In Vitro Translation Systems:

  • RRL and WGL: Failed to distinguish between IRESes; translation was generally unregulated and high across all constructs, likely due to the lack of specific human ITAFs (IRES Trans-Acting Factors) or regulatory mechanisms.
  • Human HEK293T Tripartite System: Successfully recapitulated IRES-dependent regulation. It showed strong HCV/CVB3 activity and correctly identified the inactivity of Hoxa9/Chrdl1 (unlike in cells, suggesting specific ITAFs are missing in the extract).
  • Efficiency: IRES-mediated initiation on circRNAs in the human extract was more efficient than cap-dependent initiation on linear mRNAs, scaling exponentially with RNA input.

• Engineering Validation:

  • Mutations in the HCV IRES (ΔdII and dIIId GGG-to-CCC) successfully reduced translation in both transfected cells and the cell-free system, validating the synthetic platform for mechanistic studies.

4. Key Contributions

1. Standardized Synthetic Platform: Established a rigorous protocol for generating, purifying (via PAGE), and testing synthetic circRNAs, separating true IRES biology from artifacts of plasmid-based systems or impure preparations.
2. IRES Characterization: Systematically mapped the activity of viral and cellular IRESes in a synthetic context, revealing that many cellular IRESes require specific cellular processing or ITAFs not present in exogenous circRNAs.
3. Immunogenicity Insight: Demonstrated that circRNA immunogenicity is driven by preparation purity (contaminants) rather than the specific IRES sequence, providing a clear path to "immune-silent" therapeutics.
4. Advanced Cell-Free System: Validated a human tripartite cell-free translation system as a superior tool for studying IRES regulation compared to traditional RRL/WGL, enabling precise engineering of translation kinetics.

5. Significance

• Therapeutic Development: The findings provide a critical guide for selecting IRES elements for circRNA-based medicines. While viral IRESes offer high potency, cellular IRESes (like Hoxa9) may be preferred for sustained, lower-level expression with reduced immunogenicity, provided the specific IRES is active in the target cell type.
• Quality Control: Emphasizes that purification quality is the single most critical factor in circRNA research and therapy, as contaminants can mask true biological activity and trigger false immune responses.
• Mechanistic Biology: Offers a tool to dissect cap-independent translation mechanisms, distinguishing between RNA structure-driven IRES activity and modification-driven (e.g., m6A) initiation.
• Future Applications: The ability to engineer IRES activity in synthetic circRNAs opens avenues for tissue-specific therapies and long-term protein replacement treatments that outperform current linear mRNA technologies.