Circular RNA Therapeutics: Boosting Next-Gen Treatments

Researchers are exploring circular RNA (circRNA) as a promising tool for next-generation therapeutics. Unlike traditional linear mRNA-based treatments, circRNAs offer enhanced stability and resistance to degradation, making them attractive for drug development and gene therapy. Their potential spans from protein expression to regulatory functions in disease pathways.

Advancements in synthetic biology and RNA engineering have accelerated efforts to harness circRNA for medical use. However, optimizing its design, production, and delivery remains a challenge.

Structure And Formation

Circular RNA (circRNA) is distinguished by its covalently closed-loop structure, which lacks the free 5′ and 3′ ends of linear RNA. This conformation arises from back-splicing, where a downstream splice donor joins an upstream splice acceptor, forming a continuous loop. The absence of exposed termini makes circRNA highly resistant to exonuclease-mediated degradation, significantly extending its half-life compared to linear counterparts.

The formation of circRNA is regulated by cis-acting elements and trans-acting factors that influence back-splicing efficiency. Intronic repeat sequences, such as Alu elements in primates, facilitate circularization through complementary base pairing. RNA-binding proteins (RBPs) like Quaking (QKI) and Muscleblind-like (MBNL) stabilize the looping structure or enhance splice site recognition. These mechanisms dictate circRNA abundance and functional diversity, as different sequence configurations influence interactions with cellular machinery.

Beyond structural integrity, circRNA adopts secondary and tertiary conformations that impact its biological roles. Stem-loop structures serve as binding platforms for microRNAs (miRNAs) or RBPs, modulating their availability for other cellular processes. Some circRNAs contain internal ribosome entry sites (IRES) or N6-methyladenosine (m6A) modifications, enabling cap-independent translation into functional peptides. These features expand circRNA’s therapeutic potential, allowing it to function as both a regulatory RNA and a protein-coding entity.

Biogenesis Mechanisms

Circular RNA is generated through a non-canonical splicing process called back-splicing, where a downstream 5′ splice donor covalently links to an upstream 3′ splice acceptor, forming a looped structure. This process is influenced by cis-acting elements and trans-acting factors that coordinate splice site proximity, exon selection, and regulatory control. The efficiency of circRNA formation varies across cell types and tissues, underscoring the complexity of its biogenesis.

Cis-regulatory elements facilitate back-splicing by bringing distant splice sites into proximity. Intronic repeat sequences, particularly Alu elements in primates, mediate this process through base-pairing interactions that form RNA secondary structures conducive to circularization. Other species utilize different intronic motifs, such as inverted repeat sequences or GC-rich regions, to drive circRNA production. The presence and arrangement of these elements dictate circularization efficiency and exon selection.

Trans-acting factors further modulate circRNA biogenesis by influencing splice site recognition and RNA folding. RBPs such as QKI, MBNL, and FUS enhance or suppress circRNA formation depending on their binding patterns. QKI stabilizes RNA duplexes by binding to flanking intronic regions, promoting back-splicing in neuronal and muscle cells. Conversely, splicing factors such as heterogeneous nuclear ribonucleoproteins (hnRNPs) can inhibit circularization by competing for splice site binding, favoring linear mRNA production.

In some cases, circRNA biogenesis is coupled with transcriptional activity, linking RNA polymerase II elongation dynamics to back-splicing likelihood. Slow transcriptional elongation rates promote circularization by extending the time window for splice site recognition, whereas rapid elongation favors canonical splicing. Chromatin modifications, including histone methylation and acetylation, also influence circRNA production by altering splicing machinery accessibility to nascent transcripts.

Key Differences From Linear Transcripts

Unlike linear mRNAs, which have distinct 5′ and 3′ termini, circRNAs form covalently closed loops. This structural difference enhances their stability, as the absence of free termini makes them highly resistant to exonuclease-mediated degradation. Studies show that circRNAs persist for over 48 hours in mammalian cells, whereas most linear mRNAs degrade within hours. This prolonged stability enhances their potential as therapeutic molecules, particularly for sustained gene expression.

CircRNAs also differ from linear transcripts in translation and regulation. While linear mRNAs rely on 5′ cap-dependent translation initiation, circRNAs employ alternative mechanisms such as IRES or m6A-mediated initiation. These pathways allow circRNAs to produce functional peptides independently of the 5′ cap, a feature useful for therapeutic protein production. Additionally, circRNAs often act as molecular sponges, sequestering miRNAs and RBPs that regulate linear mRNA expression, thereby altering post-transcriptional gene regulation.

Another distinction is intracellular localization. While linear mRNAs predominantly localize to the cytoplasm for translation, circRNAs exhibit varied distribution patterns. Many accumulate in the nucleus, where they participate in transcriptional regulation, while others reside in the cytoplasm, modulating gene expression post-transcriptionally. This functional versatility expands their potential applications, from gene modulation to protein replacement strategies.

Strategies To Enhance Translational Efficiency

Optimizing the translational efficiency of circRNA is essential for therapeutic applications, as its closed-loop structure lacks traditional cap-dependent translation mechanisms. One strategy involves incorporating IRES elements, which recruit ribosomes independently of the 5′ cap. IRES sequences from viruses such as hepatitis C virus (HCV) or encephalomyocarditis virus (EMCV) have shown varying degrees of efficiency in initiating translation. Synthetic IRES elements have also been engineered to enhance ribosome recruitment.

Beyond IRES elements, m6A modifications facilitate translation by creating binding sites for RBPs like YTHDF3, which interact with the translation initiation machinery. m6A-enriched circRNAs exhibit significantly higher translation rates, making methylation a viable strategy for boosting protein expression. The density and positioning of m6A sites within the circRNA sequence influence translational efficiency, underscoring the need for optimization in therapeutic applications.

Approaches For Large Scale Production

Scaling up circRNA production for therapeutics presents challenges due to its distinct biogenesis and structural properties. Unlike linear mRNA, which can be synthesized using conventional in vitro transcription (IVT) methods, circRNA requires precise enzymatic or cellular processing for efficient circularization.

One approach involves enzymatic ligation, where linear RNA precursors are synthesized via IVT and circularized using RNA ligases such as T4 RNA ligase or autocatalytic ribozymes. While specific, these methods often suffer from low efficiency and require extensive purification to remove unligated linear RNA. Advances in ribozyme-based circularization, particularly self-splicing intron sequences from Tetrahymena or permuted tRNA introns, have improved yield and reproducibility. Another strategy leverages cellular splicing machinery by transfecting cells with plasmids encoding circRNA precursors, allowing endogenous spliceosomal components to drive circularization.

Ensuring scalability requires optimizing purification and quality control measures. Chromatography techniques such as size-exclusion or affinity purification separate fully circularized RNA from linear intermediates. High-performance liquid chromatography (HPLC) and gel electrophoresis verify circularization efficiency and RNA integrity. Advances in nanopore sequencing and mass spectrometry provide tools for characterizing circRNA purity and structural fidelity. As demand for circRNA-based therapeutics grows, integrating automated, high-throughput production pipelines with stringent quality control protocols will be essential for clinical-grade manufacturing.

Intracellular Localization Patterns

The subcellular distribution of circRNA influences its function, determining whether it acts as a regulatory molecule, a translation template, or a structural component. Unlike linear mRNA, which primarily resides in the cytoplasm for protein synthesis, circRNA exhibits diverse localization patterns, accumulating in either the nucleus or cytoplasm depending on sequence composition and binding proteins.

Nuclear-retained circRNAs often regulate gene expression. Some associate with RNA polymerase II, influencing alternative splicing or transcription elongation, while others interact with chromatin-modifying enzymes to alter epigenetic landscapes. Nuclear localization signals within circRNA sequences and interactions with nuclear transport proteins contribute to retention.

Cytoplasmic circRNAs primarily function in post-transcriptional regulation and protein synthesis. Many act as miRNA sponges, sequestering miRNAs to prevent them from downregulating target mRNAs, thereby altering gene expression networks. Certain circRNAs harbor IRES or m6A modifications, enabling them to serve as templates for protein translation. Understanding these localization patterns provides insights into how circRNAs can be engineered for therapeutic applications, from gene modulation to stable protein-coding entities.