Circular RNA as a Drug Platform: Advantages Over Linear mRNA

RNA-based therapeutics have revolutionized modern medicine, with mRNA vaccines serving as a prime example of their clinical potential. However, despite the success of mRNA-based approaches, challenges such as short half-life, susceptibility to exonuclease degradation, and innate immune activation have limited their broader therapeutic applications. Circular RNA (circRNA), a naturally occurring and synthetically engineerable RNA molecule, has emerged as a promising alternative to linear mRNA. By forming a covalently closed-loop structure, circRNA eliminates free 5′ and 3′ ends, rendering it highly resistant to exonuclease-mediated degradation while also enabling prolonged protein expression. These properties make circRNAs an attractive platform for a wide range of applications, including protein replacement therapies, gene editing, cancer immunotherapy, and next-generation RNA vaccines.

Advancements in circRNA engineering, biogenesis control, and optimized translation mechanisms have further enhanced its viability as a drug platform. Unlike linear mRNAs, which rely on 5′ cap-dependent translation, circRNAs can drive protein synthesis via internal ribosome entry sites (IRESs), m6A-driven translation, and rolling-circle translation mechanisms. This not only increases protein yield per transcript but also allows for sustained therapeutic protein production, reducing the need for repeated dosing. Additionally, circRNAs exhibit low immunogenicity, as they lack structural features that trigger pattern recognition receptors (PRRs) such as RIG-I, TLR3, and MDA5, minimizing the risk of inflammatory responses. These properties position circRNA as a next-generation RNA drug platform capable of overcoming the limitations of conventional mRNA-based therapies.

This article provides an in-depth technical analysis of circRNA molecular structure, biogenesis, stability, translational efficiency, and immunogenicity, as well as its key advantages over linear mRNA. Furthermore, we explore the therapeutic applications of circRNA, including protein replacement therapy, gene editing technologies (CRISPR/Cas9 delivery), RNA vaccines, and cancer therapeutics. Finally, we discuss current challenges, regulatory considerations, and future directions for circRNA-based drug development. As research continues to refine synthetic circRNA platforms, these molecules hold immense potential as highly efficient, long-lasting, and safe RNA therapeutics.

Key Points

? Structural Stability: CircRNAs lack 5′ and 3′ ends, making them resistant to exonucleases and ensuring prolonged stability. ? Enhanced Protein Translation: CircRNAs utilize IRES elements, m6A modifications, and rolling-circle translation for cap-independent and sustained protein synthesis. ? Low Immunogenicity: CircRNAs evade immune activation via PRRs (RIG-I, MDA5, TLRs), reducing inflammatory responses. ? Therapeutic Applications: CircRNAs can be used for protein replacement, gene therapy, RNA vaccines, and cancer immunotherapy. ? Challenges and Future Directions: Optimization of in vitro synthesis, translation efficiency, and delivery systems will be critical for clinical success.

Structure and Biogenesis of Circular RNAs

Molecular Structure

CircRNAs are single-stranded RNA molecules that form a closed-loop structure via covalent bonding between the 3′ and 5′ ends. This circular conformation prevents exonuclease-mediated degradation, making circRNAs inherently more stable than their linear counterparts.

Circular RNA (circRNA) is a unique class of non-coding and coding RNA molecules characterized by a covalently closed-loop structure. This architecture differentiates circRNAs from linear RNAs by eliminating free 5′ and 3′ ends, significantly altering their biochemical properties, stability, and function. Below, we delve into the detailed molecular structure of circRNA, focusing on its sequence elements, secondary and tertiary structures, and biochemical stability.

Primary Structure: Nucleotide Composition and Covalent Linkage

Nucleotide Composition

CircRNAs, like all RNA molecules, are composed of ribonucleotides linked via phosphodiester bonds. The nucleotide sequence of circRNAs is determined by the exonic and/or intronic regions from which they are derived. The primary sequence retains:

• Canonical A (Adenine), U (Uracil), G (Guanine), and C (Cytosine) bases
• Post-transcriptional modifications, such as N6-methyladenosine (m6A), pseudouridine (Ψ), and 2′-O-methylation, which influence structure and function.

Covalent Circularization Mechanism

Unlike linear mRNAs, circRNAs lack a 5′ cap and a 3′ poly(A) tail. Instead, the RNA strand undergoes a covalent ligation of its 3′ and 5′ ends, forming a continuous, closed-loop structure. This circularization is typically mediated by:

• Canonical intron-driven back-splicing, forming an exonic circRNA (ecircRNA).
• Intron retention, generating exonic-intronic circRNAs (EIciRNAs).
• Lariat-driven ligation, producing circular intronic RNAs (ciRNAs).

The absence of exposed termini makes circRNAs inherently resistant to exonucleolytic degradation, a feature that contributes to their prolonged stability in cells.

Secondary Structure: Base-Pairing and Motifs

The secondary structure of circRNA plays a crucial role in its function, affecting translation efficiency, RNA-protein interactions, and subcellular localization. Some critical structural elements include:

Stem-Loop Structures

CircRNAs exhibit extensive stem-loop formations, facilitated by complementary base-pairing within their sequence. These structures serve multiple functions:

• Enhancing Stability: Double-stranded regions provide resistance to endonucleases.
• Regulating Translation: Stem-loops can occlude or expose ribosome entry sites (e.g., IRES elements).
• Facilitating RNA-Protein Interactions: Structural motifs act as binding sites for RNA-binding proteins (RBPs), influencing circRNA function.

Internal Ribosome Entry Sites (IRESs) and Pseudoknots

Some synthetic and endogenous circRNAs contain IRES elements, which enable cap-independent translation. These highly structured RNA motifs include:

• Y-type pseudoknots, which stabilize IRES function.
• G-quadruplex structures, which facilitate ribosome recruitment by binding translation initiation factors.

m6A-Modulated RNA Structures

The N6-methyladenosine (m6A) modification creates unique secondary structures that influence translation and degradation. m6A-modified circRNAs can recruit YTHDF proteins, which interact with eIF4G to enhance translation.

Tertiary Structure: Higher-Order Folding and 3D Conformations

CircRNAs adopt specific three-dimensional conformations, which impact their biological roles:

3.1 RNA Tertiary Interactions and Higher-Order Structures

• Long-range intramolecular interactions bring distant nucleotides into proximity, altering accessibility to ribosomes or RBPs.
• Triple helices and kissing-loop complexes stabilize tertiary structures, affecting circRNA turnover.
• Bulged regions and asymmetrical loops influence circRNA-protein binding affinity, affecting its functional specificity.

Effect on Ribosome Binding and Translation

• Unlike linear mRNAs, which require 5′ cap-mediated ribosome recruitment, circRNAs with appropriate tertiary structures can support translation via: IRES-dependent mechanisms Ribosome rolling-circle translation, which allows repeated translation of the same transcript, significantly amplifying protein yield.
• The tertiary structure determines whether a circRNA remains untranslated (non-coding function) or efficiently produces proteins (coding function).

Biochemical Stability and Degradation Resistance

Resistance to Exonucleolytic Degradation

• Absence of 5′ and 3′ termini: Unlike linear mRNAs, circRNAs lack accessible ends, making them impervious to 5′→3′ and 3′→5′ exonucleases such as XRN1 and the exosome complex.
• Resistance to RNA Decay Pathways: The nonsense-mediated decay (NMD) pathway cannot degrade circRNAs due to the absence of premature stop codons in open reading frames (ORFs). CircRNAs evade decapping enzymes and poly(A)-tail-dependent decay pathways, extending their half-life.

Endonuclease Sensitivity and Regulatory RNA Interactions

Although resistant to exonucleases, circRNAs can still be cleaved by endonucleases such as RNase L or Ago2-associated miRNA complexes, regulating their abundance in the cytoplasm.

Functional Consequences of Structural Properties

Prolonged Stability Enables Sustained Protein Expression

CircRNAs remain active for days to weeks in cells, providing a longer window for protein translation than linear mRNAs, which degrade within hours. This makes them advantageous for drug platforms requiring extended protein expression.

Distinct Subcellular Localization

• Certain structural motifs direct circRNAs to the cytoplasm (for translation) or nucleus (for regulatory functions).
• Nuclear circRNAs, particularly EIciRNAs, interact with chromatin and transcriptional regulators, influencing gene expression.

Interaction with MicroRNAs (miRNAs) and RNA-Binding Proteins (RBPs)

• CircRNAs often act as miRNA sponges, sequestering miRNAs that regulate gene expression.
• Certain structural motifs allow circRNAs to bind and modulate RBPs, influencing pathways such as splicing, translation, and signaling.

Engineering CircRNAs for Drug Development

For therapeutic applications, synthetic circRNAs must be designed and optimized for efficiency and stability:

Enhancing Translation via Structural Engineering

• Insertion of optimal IRES elements: Improves translation efficiency.
• Incorporation of stabilizing modifications (m6A, Ψ, or 2′-O-methylation): Reduces immunogenicity and enhances ribosome recruitment.
• Optimizing ORF placement: Ensures ribosome accessibility and minimizes structural hindrance.

Ensuring High Purity and Stability in Manufacturing

• Enzymatic ligation vs. rolling-circle transcription (RCT): Determines yield and integrity.
• Purification of correctly circularized RNA: Eliminates linear byproducts that may trigger immune responses.

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Biogenesis Pathways

Endogenous circRNAs are formed through back-splicing, a process in which a downstream splice donor (5′ splice site) is joined with an upstream splice acceptor (3′ splice site). This can occur through multiple mechanisms, including:

• Lariat-driven circularization: Exon skipping leads to an intron lariat structure, which is subsequently processed into a circRNA.
• Intron pairing-driven circularization: Complementary sequences within flanking introns promote circularization via base-pairing interactions.
• RBP-mediated circularization: RNA-binding proteins (RBPs) facilitate back-splicing by stabilizing splice site interactions.

For therapeutic applications, circRNAs are typically synthesized in vitro using enzymatic ligation or rolling circle transcription (RCT), followed by purification steps to remove linear byproducts.

Biogenesis Pathways of Circular RNA (circRNA)

Circular RNA (circRNA) biogenesis is a highly regulated process that differs significantly from conventional linear mRNA processing. CircRNAs are generated mainly through back-splicing, where a downstream splice donor (5′ splice site) is joined with an upstream splice acceptor (3′ splice site), leading to covalent circularization. The efficiency and specificity of circRNA formation are dictated by cis-regulatory sequences, RNA secondary structures, and trans-acting factors such as RNA-binding proteins (RBPs).

Below, we explore the key biogenesis pathways, their molecular mechanisms, and their implications for RNA-based drug development.

Canonical Splicing vs. Back-Splicing

1.1 Canonical Linear Splicing (Reference for Contrast)

In eukaryotic cells, precursor mRNAs (pre-mRNAs) undergo canonical splicing, where:

• Introns are removed by the spliceosome (a multi-component RNA-protein complex).
• Exons are ligated in a linear order to generate mature mRNA.
• The processed mRNA receives a 5′ cap and 3′ poly(A) tail, ensuring stability and translation efficiency.

Back-Splicing: The Core Mechanism of CircRNA Biogenesis

CircRNA formation deviates from canonical splicing as follows:

• A downstream 5′ splice site (splice donor, SD) is covalently linked to an upstream 3′ splice site (splice acceptor, SA).
• This "back-splicing" reaction generates a closed-loop RNA without a 5′ cap or poly(A) tail.
• The reaction is spliceosome-dependent but can be modulated by cis-elements (intron sequences) and trans-acting factors (RNA-binding proteins).

This unique mechanism is responsible for the three major classes of circRNAs:

1. Exonic circRNAs (ecircRNAs) – Composed exclusively of exons.
2. Exonic-intronic circRNAs (EIciRNAs) – Contain retained intronic sequences.
3. Circular intronic RNAs (ciRNAs) – Derived entirely from introns.

Pathways for circRNA Biogenesis

Lariat-Driven Circularization (Exon Skipping Model)

Mechanism

• This pathway is driven by alternative splicing, particularly exon skipping.
• During splicing, an exon-containing intron lariat intermediate is formed.
• Instead of complete intron degradation, the lariat is processed to retain exonic sequences, resulting in exonic circRNA (ecircRNA).
• The remaining exons are spliced together into linear mRNA.

Key Features

• Requires exon skipping, leading to simultaneous generation of circRNA and an alternative linear mRNA isoform.
• CircRNA formation is influenced by splice site strength and exon length.
• Common in protein-coding genes with long, highly structured exons.

Example Genes

• CDR1as (ciRS-7): A well-known circRNA derived from this mechanism, which functions as a miRNA sponge.

Implications for Therapeutics

• Exon selection can be engineered using specific splice site modifications to favor circRNA production over linear mRNA.
• Synthetic intron lariat scaffolds can be designed for high-efficiency circRNA biogenesis.

Intron-Pairing-Driven Circularization (Direct Back-Splicing)

Mechanism

• CircRNA formation is mediated by complementary intronic sequences flanking the circularized exon(s).
• These sequences form RNA duplexes or stem-loop structures, bringing the donor and acceptor splice sites into proximity.
• The spliceosome catalyzes back-splicing, leading to circRNA formation.

Key Features

• Intronic repeats (e.g., ALU repeats) or short inverted sequences drive circularization.
• This pathway is highly dependent on RNA secondary structure.
• Yields both exonic circRNAs (ecircRNAs) and exonic-intronic circRNAs (EIciRNAs).

Example Genes

• Sry (sex-determining region Y): The first discovered circRNA, generated by this pathway.
• ZNF609: An ecircRNA known to be translated into a functional protein.

Implications for Therapeutics

• RNA engineering strategies can be applied to introduce artificial intronic complementary sequences to enhance circRNA production.
• ALU repeats and other structured motifs can be exploited to optimize synthetic circRNA constructs.

RBP-Mediated Circularization

Mechanism

• RNA-binding proteins (RBPs) such as QKI (Quaking), FUS, and hnRNPs act as stabilizers for back-splicing.
• RBPs bind flanking intronic motifs, facilitating splice site juxtaposition.
• This mechanism often overlaps with intron-pairing-driven circularization but adds an additional regulatory layer.

Key Features

• RBPs can enhance or inhibit circRNA formation.
• Spliceosome-independent or co-transcriptional back-splicing regulation is possible.
• Often occurs in response to cellular signaling or stress conditions.

Example Genes

• QKI-regulated circRNAs: QKI enhances circRNA formation by stabilizing intronic interactions.
• FUS-associated circRNAs: Involved in neurodegenerative disorders.

Implications for Therapeutics

• Synthetic RNA-protein interaction domains could be introduced to artificially modulate circRNA levels.
• Drug-targeted RBP modulation could fine-tune circRNA expression in disease models.

Circular Intronic RNA (ciRNA) Formation

Mechanism

• Unlike exonic circRNAs, ciRNAs arise from intron lariat processing.
• Instead of undergoing full intron degradation, a branched 2′-5′ phosphodiester bond is retained.
• This prevents exosome-mediated degradation, stabilizing the ciRNA in the nucleus.

Key Features

• ciRNAs lack exonic sequences and reside mainly in the nucleus.
• Function as transcriptional regulators, enhancing host gene expression.
• Often contain GU-rich and C-rich motifs, critical for processing.

Example Genes

• ci-ANKRD52: Regulates Pol II transcription elongation.
• ci-METTL3: Implicated in epitranscriptomic regulation.

Implications for Therapeutics

• ciRNAs could be engineered as transcriptional activators for gene therapy applications.
• Stable intron-derived constructs may serve as regulatory RNA therapeutics.

Regulation of CircRNA Biogenesis

3.1 Cis-Elements Regulating circRNA Formation

• Inverted repeat sequences (ALU elements, SINEs, LINEs): Enhance circularization.
• Weak canonical splice sites: Favor back-splicing over linear splicing.
• G-rich and C-rich motifs: Influence intron retention in ciRNAs.

3.2 Trans-Acting Factors

• RBPs: QKI, FUS, hnRNPs, DHX9, ADAR modulate circRNA formation.
• Epigenetic Modifications: m6A modifications influence circRNA biogenesis.

Engineering circRNA Biogenesis for Drug Development

• Synthetic RNA Constructs: Design of optimal intronic motifs for high-yield circRNA production.
• RNA Scaffolds for Translational Efficiency: Optimization of IRES sites and RBP-binding motifs.
• Modulation of Spliceosomal Activity: Small molecules or antisense oligonucleotides (ASOs) targeting back-splicing.

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Engineering Circular RNA (circRNA) Biogenesis for Drug Development

The successful application of circular RNA (circRNA) as a therapeutic drug platform depends on precise engineering of its biogenesis, ensuring high-yield, stable, and translationally efficient constructs. Unlike linear mRNA, circRNAs require intricate design optimizations to maximize stability, translational efficiency, and low immunogenicity while maintaining controlled biogenesis. Below, we discuss the key engineering strategies for circRNA production, including synthetic sequence design, intronic motifs, splice modulation, and delivery optimizations.

Rational Design of circRNA Constructs for High-Efficiency Circularization

Efficient circRNA biogenesis requires careful optimization of back-splicing, which can be achieved through:

Optimization of Back-Splice Sites for Efficient Circularization

• CircRNA formation requires a splice donor (SD) and splice acceptor (SA) brought into close proximity.
• Engineering involves: Strengthening weak splice sites to enhance back-splicing efficiency. Incorporating splice site mutations to prevent linear splicing. Minimizing cryptic splicing sites that may interfere with circularization.
• Example of optimized splice site sequences (based on natural circRNA motifs):

5′-AGGUAAGU [optimized donor site] - (intronic sequence) - [optimized acceptor site] AGG-3′

• Inclusion of strong GT-AG splice signals enhances spliceosome recruitment.
• Incorporation of weak poly-pyrimidine tract sequences minimizes exon skipping.

Synthetic Intron Sequences for Enhanced Circularization

• Natural circRNA formation depends on flanking intronic complementary sequences that facilitate back-splicing.
• Synthetic designs introduce optimized intronic motifs: Alu repeats and other inverted repeats increase back-splicing efficiency. G/C-rich sequences stabilize intron-intron interactions. RNA triplex or G-quadruplex motifs further promote structural integrity.
• Example of complementary inverted repeat sequences to enhance circRNA yield:

Exon1 - (5′-AAAGGUCCGAUCCGGAUUU-3′) - Intron - (3′-UUUCCAGGCUAGGCCUAAA-5′) - Exon2

• These sequences base-pair with each other, forming a stem-loop structure, which promotes circularization.

Enhancing Translation Efficiency of Synthetic circRNAs

While circRNAs naturally lack a 5′ cap and 3′ poly(A) tail, they can still be engineered for efficient protein expression through alternative translation mechanisms.

Incorporation of Internal Ribosome Entry Sites (IRESs)

• CircRNAs must recruit ribosomes cap-independently, making IRES elements essential.
• Optimized IRES elements from viruses (e.g., EMCV, HCV, CrPV) or cellular genes (e.g., c-myc) can be inserted into synthetic circRNA constructs.
• Example of an optimized IRES sequence for circRNA translation:

5′-GCCAGCCACCGGGGTCCGGGTTAACGGAGGCCCGGGTTTTAGCCCTTGAAAGTGAAC-3′

• This sequence recruits eIF4G and eIF3, enabling cap-independent translation.

N6-Methyladenosine (m6A)-Driven Translation Enhancement

• Certain m6A-modified regions can facilitate ribosome recruitment via YTHDF proteins.
• Engineering m6A consensus sites within circRNAs improves translation.
• Example of m6A consensus motifs incorporated into the circRNA sequence:

5′-GGACU[Am6]CAUGG-3′

• The presence of "RRACH" (R = G or A, H = A, C, or U) increases m6A binding affinity.

Ribosome Rolling-Circle Translation for High-Yield Protein Production

• CircRNAs allow continuous translation by enabling rolling-circle ribosome recruitment.
• Design involves: Repeating ORF sequences to amplify protein yield. Optimized Kozak sequences upstream of the start codon.
• Example of rolling-circle ORF design:

5′-AUG (coding sequence) UAG (spacer) AUG (coding sequence) UAG-3′

• The spacer region prevents ribosomal stalling.

Reducing Immunogenicity for Safe Therapeutic Use

A major hurdle in RNA-based drugs is innate immune activation via Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs).

Chemical Modifications to CircRNAs

• Pseudouridine (Ψ) incorporation reduces immune activation.
• 2′-O-methylation at critical sites prevents recognition by PRRs.
• Example of a chemically modified sequence:

5′-ΨAΨGΨCΨUΨAΨG-3′

• Reduces TLR3, TLR7, and TLR8 activation.

Removing Immunogenic dsRNA Structures

• Long double-stranded regions trigger PKR activation, leading to translation inhibition.
• Optimized circRNA constructs minimize long duplexes using short hairpins instead.

Large-Scale Production and Purification of Synthetic circRNAs

Efficient therapeutic application requires high-purity, scalable synthesis.

Rolling Circle Transcription (RCT) for High-Yield Production

• Uses T7 polymerase to transcribe a self-ligating circular template.
• Generates long concatemeric RNA strands, which are then enzymatically circularized.

Enzymatic Circularization Methods

1. T4 RNA Ligase-Based Circularization Uses T4 RNA ligase 1 to join RNA ends. Works best for short circRNAs.
2. Self-Splicing Ribozymes Group I/II self-splicing ribozymes can autonomously circularize transcripts.

Purification Strategies for High-Purity CircRNA

• Size-exclusion chromatography (SEC) to separate circular from linear RNA.
• RNase R digestion: Removes linear RNA while leaving circRNAs intact.

Optimized Delivery Strategies for Synthetic circRNAs

Lipid Nanoparticle (LNP) Encapsulation

• CircRNAs are encapsulated in ionizable lipids, ensuring efficient cellular uptake.
• PEGylated lipids enhance serum stability.

Extracellular Vesicle (EV)-Mediated Delivery

• CircRNAs can be packaged into exosomes, improving biodistribution.

Polymer-Based Nanocarriers

• Cationic polymers like polyethyleneimine (PEI) facilitate efficient RNA transfection.

The engineering of synthetic circRNAs for therapeutic applications involves a multi-faceted approach that optimizes:

1. Efficient back-splicing and biogenesis using intronic motifs and RBPs.
2. High-yield translation via IRES elements, m6A modifications, and rolling-circle translation.
3. Reduced immunogenicity via chemical modifications and structural optimizations.
4. Scalable production methods such as rolling-circle transcription and enzymatic ligation.
5. Effective delivery strategies including lipid nanoparticles, exosomes, and polymeric carriers.

Advances in synthetic RNA biology, ribosome recruitment, and RNA delivery technologies will continue to propel circRNA as the next-generation RNA drug platform for protein replacement therapy, vaccines, and gene therapy.

Key Advantages of Circular RNA over Linear mRNA

Increased Stability and Reduced Degradation

One of the primary limitations of linear mRNA therapeutics is their susceptibility to degradation by exonucleases. Linear mRNAs have free 5′ and 3′ ends, making them highly vulnerable to RNase-mediated degradation. In contrast, circRNAs lack these free ends, rendering them highly resistant to exonucleolytic cleavage.

Studies have shown that circRNAs exhibit a significantly prolonged half-life compared to linear mRNAs, with some synthetic circRNAs maintaining stability for days in mammalian cells. This property is particularly beneficial for drug applications requiring sustained protein expression without the need for repeated dosing.

Enhanced Translational Efficiency

Historically, circRNAs were considered non-coding RNA molecules due to their lack of a canonical cap-dependent translation mechanism. However, recent advances have demonstrated that circRNAs can be engineered to support efficient translation via:

• Internal ribosome entry site (IRES) elements: These RNA motifs enable cap-independent initiation of translation, allowing ribosome recruitment without the need for a 5′ cap structure.
• m6A modification-mediated translation: N6-methyladenosine (m6A) modifications can recruit RNA-binding proteins such as YTHDF3, promoting translation of circRNAs.
• Ribosome rolling-circle translation: CircRNAs allow for continuous translation of peptide repeats, leading to higher protein yields compared to linear mRNA.

The ability of circRNAs to drive prolonged and sustained protein production makes them ideal for applications such as protein replacement therapies and gene therapy.

Reduced Immunogenicity

A major drawback of linear mRNA-based therapies is their inherent immunogenicity, which can trigger innate immune responses via pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), RIG-I, and MDA5. This often necessitates extensive chemical modifications (e.g., pseudouridine incorporation) to mitigate immune activation.

CircRNAs, however, exhibit a much lower immunogenic profile due to their structural properties:

• Lack of exposed 5′ triphosphate groups, which are primary activators of RIG-I.
• Absence of fragmented RNA species that activate TLR3, TLR7, and TLR8.
• Reduced binding to cytoplasmic RNA sensors, leading to lower interferon-stimulated gene (ISG) activation.

This reduced immunogenicity enhances safety and tolerability for therapeutic applications.

Longer-Lasting Protein Expression with Minimal Dosing

Because circRNAs are more stable and less immunogenic than linear mRNAs, they enable longer-lasting protein expression with fewer doses. This has significant implications for diseases requiring sustained protein production, such as enzyme replacement therapies, chronic metabolic disorders, and cancer immunotherapies.

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Key Advantages of Circular RNA Over Linear mRNA

Circular RNA (circRNA) offers several structural and functional advantages over linear messenger RNA (mRNA), making it an attractive platform for therapeutic applications, including vaccines, gene therapy, and protein replacement. The main advantages include increased stability, enhanced translational efficiency, reduced immunogenicity, and prolonged protein expression. Here, we provide an in-depth technical analysis of each of these aspects, supported by the latest molecular insights.

Increased Stability and Resistance to Degradation

One of the most significant advantages of circRNA over linear mRNA is its structural resistance to degradation. The covalently closed-loop structure of circRNA eliminates the 5′ and 3′ ends, making it impervious to exonuclease-mediated degradation.

Resistance to 5′ → 3′ and 3′ → 5′ Exonucleases

• Linear mRNAs are degraded by XRN1 (5′ → 3′ exonuclease) and the RNA exosome complex (3′ → 5′ exonuclease).
• CircRNAs lack these terminal sites, preventing exonucleolytic digestion.
• In vitro studies have demonstrated that synthetic circRNAs can persist 10-20 times longer than linear mRNA in mammalian cells.

Resistance to Nonsense-Mediated Decay (NMD)

• Linear mRNA is subjected to NMD, a surveillance pathway that detects premature termination codons (PTCs) and degrades faulty transcripts.
• CircRNAs lack the necessary 5′ cap and poly(A) tail, making them inherently insensitive to NMD.
• Endogenous circRNAs, such as ciRS-7/CDR1as, have been found to be extremely stable in neuronal cells, persisting for days to weeks.

Protection from Deadenylation and Decapping

• The deadenylation-dependent decay of linear mRNA is mediated by the CCR4-NOT complex, which shortens the poly(A) tail, leading to transcript degradation.
• The DCP1-DCP2 decapping complex removes the 5′ cap of mRNAs, exposing them to XRN1 exonuclease activity.
• CircRNAs, lacking these regulatory elements, evade these degradation pathways, ensuring prolonged transcript availability.

Enhanced Translational Efficiency

Despite lacking a 5′ cap, circRNAs can still support highly efficient protein translation, often exceeding that of linear mRNAs. This is facilitated by:

Internal Ribosome Entry Sites (IRESs) for Cap-Independent Translation

• Unlike linear mRNA, which requires eIF4E-mediated 5′ cap recognition, circRNAs recruit ribosomes via IRES elements.
• Synthetic IRESs derived from viruses (e.g., EMCV, HCV) or cellular genes (e.g., c-Myc, VEGF) allow robust translation.
• Optimized IRES incorporation in synthetic circRNAs enhances translational output up to 30-50% of capped mRNA levels.

m6A-Driven Translation via eIF4G Recruitment

• Recent studies have shown that m6A modifications in circRNAs recruit YTHDF proteins, which in turn interact with eIF4G to initiate translation.
• m6A reader proteins (e.g., YTHDF1/3) enhance translation by recruiting initiation factors independent of the 5′ cap.
• Example: The circRNA FBXW7 harbors an m6A modification that enables efficient cap-independent translation.

Rolling-Circle Translation Enables Continuous Protein Synthesis

• Unlike linear mRNA, which supports single-round translation, circRNAs allow for rolling-circle translation, leading to higher protein yields.
• Mechanism: Ribosomes bind the circRNA and initiate translation at an AUG start codon. Once a stop codon is reached, ribosomes reinitiate translation from the same sequence. This results in circular translation of peptide repeats, significantly increasing protein output.
• Implication for Drug Development: Enzyme replacement therapies (e.g., hemophilia Factor VIII therapy) benefit from prolonged protein production. CircRNA vaccines could elicit sustained antigen presentation due to prolonged translation.

Reduced Immunogenicity and Improved Biocompatibility

Linear mRNAs strongly activate innate immune responses due to the presence of double-stranded RNA (dsRNA) structures and 5′ triphosphate ends, which trigger pattern recognition receptors (PRRs) such as TLR3, TLR7, TLR8, RIG-I, and MDA5.

CircRNA Evades Innate Immune Sensors

• CircRNAs lack a 5′ cap and do not contain a 5′ triphosphate group, preventing RIG-I and MDA5 activation.
• They are less likely to form immunogenic dsRNA structures, reducing TLR3-mediated IFN responses.
• CircRNAs exhibit low activation of interferon-stimulated genes (ISGs), reducing systemic inflammatory responses.

Chemical Modifications Further Minimize Immunogenicity

• Synthetic circRNAs can be modified with pseudouridine (Ψ) or 2′-O-methylation, further reducing immune activation.
• Examples of immune-silent modifications: Ψ modifications prevent TLR7/TLR8 activation in dendritic cells and macrophages. 2′-O-methylation at key residues minimizes RNA-sensing by PKR (protein kinase R), preventing translation shutdown.

Reduced Cytotoxicity Compared to Modified Linear mRNAs

• Lipid nanoparticle (LNP)-delivered mRNAs require extensive pseudouridine modification to reduce immune toxicity.
• CircRNAs naturally avoid activating PKR, which otherwise inhibits translation by phosphorylating eIF2α.

Longer-Lasting Protein Expression and Reduced Dosing Requirements

One of the most critical advantages of circRNA over linear mRNA is its sustained protein expression, which translates to lower dosing frequency in therapeutic applications.

Prolonged Translation Compared to Linear mRNA

• CircRNAs remain translationally active for days to weeks, whereas linear mRNA is degraded within hours.
• In vivo studies show that circRNA-based protein expression can last up to 14 days, compared to ~48 hours for linear mRNA.
• Example: CircRNA-based erythropoietin (EPO) therapy demonstrated long-term protein expression with a single dose, whereas linear mRNA required multiple injections.

Potential for Single-Dose Therapeutics

• The prolonged half-life of circRNAs reduces the need for repeat administration, lowering costs and improving patient compliance.
• Applications in vaccine development: CircRNA vaccines could provide extended antigen presentation, improving immune memory with a single-dose regimen.

The molecular and biochemical advantages of circRNA over linear mRNA position it as a next-generation drug platform. Key benefits include:

1. Increased Stability: CircRNAs resist exonucleolytic degradation, ensuring prolonged cellular retention.
2. Higher Translational Efficiency: Cap-independent mechanisms (IRESs, m6A modifications) enable robust protein synthesis.
3. Reduced Immunogenicity: CircRNAs evade innate immune recognition, improving safety and tolerability.
4. Longer-Lasting Protein Expression: Prolonged translation allows for lower dosing frequency in therapeutic applications.

With continued advancements in circRNA engineering, delivery technologies, and synthetic biology, circRNAs are poised to revolutionize gene therapy, protein replacement therapies, and next-generation vaccines.

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Applications of Circular RNA in Drug Development

Protein Replacement Therapies

CircRNAs can be used to produce therapeutic proteins for conditions such as:

• Hemophilia (e.g., Factor VIII expression)
• Cystic fibrosis (e.g., CFTR protein restoration)
• Lysosomal storage disorders (e.g., enzyme replacement therapy)

Vaccines and Immunotherapies

CircRNA vaccines offer potential advantages over traditional mRNA vaccines:

• Higher antigen stability and prolonged immune stimulation.
• Lower required dosages due to increased protein yield.
• Reduced risk of immune overstimulation, making them safer for repeated administration.

Gene Editing Technologies

CircRNA-based delivery of CRISPR-associated proteins, such as Cas9, enables longer-lasting gene editing activity, making it superior to transient linear mRNA-based delivery.

Cancer Therapeutics

CircRNAs can encode tumor-suppressor proteins or immune-stimulatory cytokines, improving the efficacy of cancer immunotherapies while minimizing off-target effects.

Applications of Circular RNA in Drug Development: A Technical Perspective

Circular RNA (circRNA) has emerged as a highly promising therapeutic RNA platform due to its enhanced stability, efficient protein translation, and reduced immunogenicity. These properties position circRNAs as superior alternatives to linear mRNA for applications such as protein replacement therapies, vaccines, cancer immunotherapies, and gene editing technologies.

In this detailed analysis, we explore the biological mechanisms and molecular optimizations that make circRNAs suitable for various drug development applications.

Protein Replacement Therapies

Sustained Production of Therapeutic Proteins

One of the main challenges in protein replacement therapy is the short half-life of linear mRNA, which necessitates frequent dosing. CircRNAs address this limitation by enabling long-lasting protein expression due to their enhanced stability and resistance to degradation.

Examples of Protein Replacement Therapies Using circRNA

• Hemophilia (Factor VIII & IX Therapy) Hemophilia A and B are caused by deficiencies in coagulation factors VIII (FVIII) and IX (FIX). CircRNA encoding FVIII or FIX can provide long-lasting expression, reducing the need for frequent injections. Rolling-circle translation of FVIII could significantly increase protein yield, making circRNAs an attractive approach.
• Cystic Fibrosis (CFTR Protein Expression) Mutations in the CFTR gene cause defective chloride ion transport in epithelial cells. CircRNA-based delivery of CFTR cDNA could restore functional CFTR protein expression without the risk of rapid degradation. m6A-modified circRNAs enhance translation, ensuring robust protein restoration in lung epithelial cells.
• Lysosomal Storage Disorders (Enzyme Replacement) Diseases such as Gaucher’s, Fabry’s, and Pompe’s disease result from defective lysosomal enzymes. CircRNAs encoding glucocerebrosidase (GBA1), α-galactosidase (GLA), or acid α-glucosidase (GAA) can provide stable, continuous enzyme production, reducing the need for frequent enzyme infusions.

Vaccines and Immunotherapies

CircRNA-Based Vaccines for Infectious Diseases

mRNA vaccines have been highly successful, but rapid mRNA degradation and the need for lipid nanoparticle (LNP) encapsulation remain challenges. CircRNAs, due to their enhanced stability and prolonged antigen expression, offer several advantages.

Mechanisms That Make circRNA Vaccines Superior

1. Rolling-circle translation enhances antigen expression, leading to higher immune response.
2. Reduced innate immune activation (due to lack of a 5′ triphosphate) prevents excessive interferon (IFN) responses.
3. IRES- or m6A-driven translation enables robust, cap-independent protein synthesis.

Example Applications in Vaccines

• COVID-19 and SARS-CoV-2 Variants CircRNA encoding the SARS-CoV-2 spike protein could provide longer-lasting antigen expression than linear mRNA vaccines. CircRNAs avoid PKR-mediated inhibition, ensuring sustained immune activation.
• Influenza and Other Respiratory Viruses CircRNA encoding hemagglutinin (HA) and neuraminidase (NA) proteins could generate more durable protection.
• HIV Vaccines CircRNA encoding HIV envelope (Env) proteins can induce stronger T-cell responses due to prolonged antigen presentation.

Cancer Therapeutics

CircRNA as an Antitumor Agent

Cancer therapy requires sustained protein expression for immune stimulation, apoptosis induction, or tumor suppression. CircRNAs can be engineered to express:

1. Tumor-suppressor proteins
2. Pro-apoptotic factors
3. Immunostimulatory cytokines

Engineering circRNAs for Cancer Therapy

1. CircRNAs encoding tumor suppressors (e.g., p53, PTEN, BRCA1) ensure long-lasting protein production.
2. CircRNA-based immunotherapy can encode cytokines (e.g., IL-12, IFN-γ) to activate immune responses against tumors.
3. CircRNAs delivering chimeric antigen receptors (CARs) could enhance CAR-T cell therapy.

Example Cancer Therapeutic Applications

• CircRNA-based immunotherapy for melanoma CircRNAs encoding IL-2 and IL-12 promote T-cell activation and anti-tumor immunity.
• CircRNA-based apoptosis induction in leukemia CircRNAs encoding Bax, Bak, or TRAIL induce caspase-dependent apoptosis in leukemia cells.
• CircRNA-based personalized cancer vaccines CircRNA encoding neoantigens (tumor-specific antigens) could stimulate cytotoxic T-cell responses.

Gene Editing Technologies

CircRNA-Based Delivery of CRISPR/Cas Systems

CRISPR-based gene editing requires efficient delivery of both:

1. Cas9 (or other CRISPR-associated proteins)
2. Guide RNA (gRNA)

CircRNAs offer an improved platform for delivering gene-editing components due to prolonged expression and reduced immunogenicity.

Engineering circRNAs for CRISPR Therapy

1. CircRNA encoding Cas9 protein with an embedded IRES ensures cap-independent translation.
2. CircRNA-driven expression of base-editing enzymes (e.g., ABE, CBE) enables long-term gene correction.
3. CircRNA encoding gRNA can be designed within an engineered intron, allowing co-expression of Cas9 and gRNA from a single circRNA.

Example Applications in Gene Editing

• CircRNA-driven CRISPR/Cas9 for Duchenne Muscular Dystrophy (DMD) CircRNAs encoding Cas9 and exon-skipping gRNAs restore functional dystrophin expression in muscle cells.
• CircRNA-based gene correction for sickle cell disease CircRNA-mediated expression of adenine base editors (ABEs) could permanently correct the HBB mutation.
• CircRNA for epigenetic editing CircRNAs encoding dCas9 fused to methylation or demethylation enzymes could reprogram gene expression in neurodegenerative diseases.

Delivery Systems for CircRNA Therapeutics

Lipid Nanoparticle (LNP) Encapsulation

• CircRNAs are packaged into ionizable lipid nanoparticles for efficient intracellular delivery.
• PEGylated lipids enhance circulation time.

Extracellular Vesicle (EV)-Mediated Delivery

• CircRNAs can be packaged into exosomes, allowing targeted tissue delivery.
• Exosome-based delivery is particularly promising for brain-targeted therapies (e.g., circRNA for Parkinson’s disease).

Polymer-Based Nanocarriers

• Polyethyleneimine (PEI) nanocarriers enhance cellular uptake of circRNAs.

The unique properties of circRNAs (enhanced stability, sustained translation, and low immunogenicity) make them ideal candidates for a broad range of therapeutic applications, including:

1. Protein replacement therapy for genetic diseases (e.g., hemophilia, cystic fibrosis).
2. Vaccine development for infectious diseases (e.g., COVID-19, influenza, HIV).
3. Cancer immunotherapy through cytokine expression and personalized vaccines.
4. Gene editing technologies using CRISPR/Cas9 and epigenetic modifiers.

With further advancements in circRNA engineering, synthesis, and delivery, circRNAs are poised to become the next-generation RNA-based drug platform, revolutionizing gene therapy, protein therapeutics, and RNA vaccines.

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Challenges and Future Perspectives

Efficient In Vitro Synthesis

Despite its advantages, large-scale production of synthetic circRNA remains a challenge. Current in vitro transcription (IVT) and ligation methods often yield byproducts such as linear RNA fragments, necessitating improved purification strategies.

Translation Optimization

While engineered IRES elements and m6A modifications enhance circRNA translation, further optimizations are needed to achieve consistent and high-efficiency protein expression across different cell types.

Delivery Strategies

The development of effective circRNA delivery systems remains a critical hurdle. Lipid nanoparticles (LNPs), extracellular vesicles, and polymer-based delivery methods are being explored to enhance cellular uptake and bioavailability.

Regulatory and Safety Considerations

Although circRNAs exhibit reduced immunogenicity, long-term safety studies are required to ensure they do not trigger unintended immune responses or interfere with endogenous circRNA functions.

Conclusion

Circular RNAs (circRNAs) represent a paradigm shift in RNA-based therapeutics, offering a robust alternative to linear mRNA with significant advantages in stability, translation efficiency, and immunogenicity. By eliminating the free 5′ and 3′ ends, circRNAs evade exonuclease-mediated degradation, ensuring longer intracellular half-life and sustained protein production. Their cap-independent translation mechanisms, including IRES-mediated translation, m6A-enhanced ribosome recruitment, and rolling-circle translation, enable higher protein yields per transcript, making them particularly advantageous for long-term therapeutic applications. Additionally, circRNAs exhibit reduced innate immune activation, addressing one of the primary challenges faced by traditional mRNA-based drugs.

With continued advancements in circRNA engineering, optimized translation mechanisms, and delivery technologies, the therapeutic potential of circRNAs is rapidly expanding. Applications in protein replacement therapy, RNA vaccines, cancer immunotherapy, and gene editing (CRISPR/Cas9 delivery) highlight the versatility of circRNA-based therapeutics. However, challenges such as scalable in vitro synthesis, efficient purification, and precise translation control must be addressed to enable widespread clinical implementation. Additionally, regulatory frameworks and safety assessments will play a crucial role in determining the long-term viability of circRNA therapeutics in human applications.

As research continues to refine circRNA synthesis, delivery, and translational control, these molecules hold immense promise as the next-generation foundation for RNA-based medicines. Their ability to provide sustained therapeutic protein expression with minimal immune activation makes them a superior alternative to linear mRNA-based therapies. With ongoing preclinical and clinical advancements, circRNAs are poised to revolutionize the fields of gene therapy, protein therapeutics, and vaccine development, ushering in a new era of RNA-based medicine.

Key Takeaways

? Structural and Functional Superiority: CircRNAs provide longer stability, sustained translation, and reduced immunogenicity compared to linear mRNA. ? Versatile Therapeutic Applications: CircRNAs are promising for protein replacement, gene editing (CRISPR), cancer therapy, and next-generation vaccines. ? Challenges to Overcome: Scalable synthesis, efficient delivery, and regulatory approval remain critical hurdles for clinical translation. ? Future Prospects: With continued advancements in RNA engineering and delivery systems, circRNAs have the potential to become the next gold standard in RNA therapeutics.

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