What circular RNA are being developed?

Introduction to Circular RNA

Definition and Basic Concepts
Circular RNAs (circRNAs) are a unique class of RNA molecules that are characterized by a covalently closed loop structure, which distinguishes them from the conventional linear RNAs that have distinct 5′ and 3′ ends. They are generated by a process known as back-splicing where a downstream splice donor site is linked to an upstream splice acceptor site. Initially discovered in plant viroids in 1976 and later observed in mammalian cells in the early 1990s, circRNAs have now been recognized as a prevalent RNA species in eukaryotic cells. Their circular nature grants them high stability and resistance to exonuclease-mediated degradation, providing a significant advantage for both therapeutic and diagnostic applications.

Biological Functions and Characteristics
Biologically, circRNAs are known not only for their structural stability but also for their diverse functions. They can act as microRNA (miRNA) sponges, thereby modulating gene expression by sequestering miRNAs away from their mRNA targets. In certain contexts, circRNAs may function as protein decoys or scaffolds, influencing protein–protein interactions and even serving as templates for protein translation via internal ribosome entry site (IRES)-mediated mechanisms. Their tissue-specific and developmental stage-specific expression profiles add another layer of regulatory potential, making them appealing as both therapeutic tools and diagnostic biomarkers in various diseases including cancer and cardiovascular disorders.

Current Developments in Circular RNA

Therapeutic Applications
Over the past few years, there has been significant progress in the development of circRNA-based therapeutics. These developments reflect the desire to harness the remarkable stability and unique cellular functions of circRNAs for disease treatment.

1. Drug Candidates in Development:
- RXRG-001: This circRNA-based therapeutic, developed by RiboX Therapeutics Ltd., is primarily designed for addressing mouth and tooth diseases, and currently sits in Phase 1/2 clinical development. It targets AQP1 and modulates its function to address disease pathology.
- CircFam53B-219aa DC vaccine: Developed by Sun Yat-Sen Memorial Hospital, this is a circRNA-based dendritic cell (DC) vaccine that represents a promising approach in the immunotherapy space. Being in Phase 1 of clinical trials, it leverages circRNAs to elicit an immune response and potentially treat neoplasms and conditions related to skin and musculoskeletal diseases.
- Circ_0011129-loaded 3D-sEVs: This therapeutic candidate, which is in the preclinical stage and developed by Sun Yat-Sen University, uses a combination of circular RNA and exosome technology. It targets MIR6732 and modulates its function, offering a novel approach particularly in skin and musculoskeletal diseases.
- circCOL-ELNs carried by exosomes: Developed by The Seventh Affiliated Hospital of Sun Yat-sen University, this approach utilizes adipose-derived stem cell exosomes to carry circRNAs. Currently in early Phase 1, it indicates the versatility of using naturally derived carriers for enhancing circRNA delivery.
- HM-2002: This therapeutic candidate under development by Hormos Medical Oy focuses on cardiovascular diseases. It targets VEGF and modulates its activity, taking advantage of the high stability of circRNAs to sustain therapeutic effects.

2. Discovery-Stage Candidates:
Several circRNA molecules are currently in the discovery phase, particularly those developed by Shanghai Huanma Biopharmaceutical Co., Ltd. Examples include:
- CC-2106, CC-2202, CC-2201, CC-2207, and CC-2208: All of these candidates are at the discovery stage and represent the efforts of the company to expand the circular RNA drug portfolio. Their mechanism is based on the inherent properties of circRNAs to modulate specific molecular targets associated with various diseases.

3. Patented Technologies:
Advances in circRNA therapeutics are also being protected and developed through intellectual property filings. For example, engineered circular guide RNAs are being developed for therapeutic targeting of specific genes, potentially allowing for the treatment or prevention of diseases using guide RNAs that harness the unique properties of circularization. Another example is the development of circular RNA compositions that are optimized for high stability and efficient translation, which can be used to produce therapeutic proteins in vivo. Additionally, improved methods of synthesizing circular RNAs with reduced immunogenicity are being patented, reflecting the need to fine-tune the balance between efficacy and safety in RNA therapeutics.

4. Mechanistic Rationale:
The therapeutic promise lies in several intrinsic circRNA characteristics:
- Stability: The closed-loop structure enables circRNAs to resist degradation and maintain steady expression levels in vivo, which is essential for long-term therapeutic applications.
- Translational Capacity: Some circRNAs can be engineered to include internal ribosome entry sites (IRES) or N6-methyladenosine (m6A) modifications, facilitating the translation of encoded proteins. This opens a new avenue for protein replacement therapies and vaccines.
- Modulatory Roles: Their ability to act as miRNA sponges offers a versatile mechanism to indirectly modulate gene expression, which can be harnessed to correct dysregulated pathways in diseases such as cancer, cardiovascular diseases, and even neurodegenerative disorders.

5. Combination Approaches:
Beyond monotherapy, circRNAs are being explored as components of combination therapies. For instance, circRNA-loaded exosomes or nanoparticles—serving as both the delivery vehicle and the therapeutic payload—are designed to achieve targeted delivery, enhanced uptake, and reduced off-target effects. These approaches combine the benefits of high stability and effective delivery systems, which are critical for the success of RNA-based therapeutics.

Diagnostic Applications
Alongside therapeutics, circRNAs are showing tremendous potential as diagnostic biomarkers due to their stability and differential expression in various disease states.

1. Biomarker Potential in Cancer:
In cancers such as hepatocellular carcinoma (HCC) and gastric carcinoma, circRNAs have been extensively studied. For example, circRNA profiles in tissue and body fluids (such as serum/plasma and exosomes) have displayed moderate to high diagnostic accuracy. Several meta-analyses have shown that circRNAs have sensitivity and specificity values ranging from 0.68 to 0.91, with corresponding area under the curve (AUC) values indicating their strong potential as non-invasive biomarkers for early detection and prognosis in cancers.

2. Cardiovascular Disease Diagnosis:
In cardiovascular diseases (CVD), circRNAs have been demonstrated as promising biomarkers, with several studies highlighting their ability to differentiate between CVD patients and healthy subjects. For example, circRNAs detected in serum have shown high sensitivity (around 0.81) and specificity (approximately 0.74), and meta-analyses report a diagnostic odds ratio (DOR) of 12, suggesting their robustness as diagnostic indicators in CVD.

3. Prostate and Female Reproductive System Disorders:
CircRNAs are also being developed for diagnostic purposes in prostate cancer and female reproductive system diseases. In prostate cancer, differential expression of circRNAs correlates with clinical parameters such as Gleason score and tumor stage, while diagnostic meta-analyses yield an overall AUC of 0.81, underlining their clinical utility. Similarly, analyses in female reproductive system diseases have shown a moderate diagnostic value with pooled sensitivity and specificity both around 0.70, demonstrating the versatile diagnostic potential across diverse disease areas.

4. Circulating circRNAs as Liquid Biopsy Markers:
Emerging evidence has supported the use of circulating circRNAs—found in blood, plasma, and even exosomes—as non-invasive biomarkers. This is particularly valuable in the context of diseases such as congenital heart diseases in children, where circRNAs such as hsa_circRNA_004183, hsa_circRNA_079265, and hsa_circRNA_105039 have shown AUC values up to 0.965 when combined, reflecting the high potential for early and precise disease screening.

5. Standardization and Validation Efforts:
Despite these promising results, the clinical translation of circRNA biomarkers requires standardization in detection methods, normalization strategies, and validation in large-scale cohorts. Several studies emphasize the need for improved circRNA databases to consolidate findings and reduce inter-study variability. These developments are critical for moving circRNA diagnostic applications from research laboratories into clinical practice.

Research and Development Methodologies

Techniques for Circular RNA Identification
Accurate identification of circRNAs is crucial for both therapeutic and diagnostic applications.

1. High-Throughput Sequencing and Bioinformatics:
Modern RNA sequencing (RNA-Seq) techniques coupled with specialized computational pipelines have been instrumental in circRNA discovery. rRNA depletion, RNase R treatment, and the use of divergent primers in reverse transcription–PCR are some of the standard techniques employed.
- Bioinformatics Pipelines: Numerous algorithms have been developed specifically for the detection of backsplice junctions characteristic of circRNAs. These tools help distinguish circRNAs from linear transcripts and enable precise quantification. Enhancements in these pipelines have also contributed to the establishment of comprehensive circRNA databases, although challenges remain regarding nomenclature and sequence overlap.

2. Experimental Validation Methods:
Experimental validation, such as RT-PCR followed by Sanger sequencing, northern blotting, and microarray-based detection, further confirms the existence and expression levels of identified circRNAs. These methods not only affirm the circular nature of the RNA but also provide insight into their abundance and tissue specificity.

Methods for Circular RNA Synthesis
Developing circRNA-based therapeutics demands robust and scalable synthesis methods.

1. In Vitro Synthesis and Circularization Strategies:
Two main methodologies are generally used for in vitro synthesis of circRNAs:
- Enzymatic Ligation: This method typically uses T4 RNA ligase 1 to join the 5′ and 3′ ends of an RNA molecule, forming a covalently closed circle. It is advantageous for producing long circRNAs and can be adapted for incorporation of functional elements such as IRES sequences.
- Ribozyme-Mediated Circularization: Group I intron self-splicing or permuted intron-exon (PIE) constructs represent another strategy that allows for high-efficiency circularization. This method can yield circRNAs with minimal immunogenicity if optimized properly, which is essential for clinical applications.

2. Chemical Synthesis Techniques:
Although more challenging for long RNA sequences, chemical synthesis allows the incorporation of nonnatural nucleotides and modifications that enhance stability and reduce immunogenicity. Hybrid approaches combining chemical and enzymatic methods are also emerging to optimize the synthesis of longer, highly modified circRNAs.

3. Scalability and Quality Control:
For practical therapeutic applications, large-scale synthesis must be efficient, cost-effective, and yield high-purity circRNAs. This involves challenges in circularization efficiency, purification from linear RNA precursors, and comprehensive analytical characterization to ensure that the desired circular conformation has been achieved. Patented methods are actively seeking to address these issues by improving both the synthesis and purification processes.

Delivery Systems for Circular RNA
Effective delivery remains one of the critical bottlenecks in the translation of circRNA therapeutics.

1. Nanoparticle-Based Delivery:
Lipid nanoparticles (LNPs) have been widely used to deliver mRNA therapeutics and are now being adapted for circRNAs as well. LNPs can protect circRNAs during systemic circulation and facilitate their uptake by target cells. Recent advancements in LNP chemistry promise improved targeting specificity and reduced toxicity, which is critical given the larger size and unique structure of circRNAs.

2. Exosome-Based Delivery:
Exosomes, being natural carriers of RNA molecules, offer an attractive approach for circRNA delivery. Studies have shown that exosomes derived from adipose-derived stem cells or other sources can serve as efficient vehicles, as seen with the circCOL-ELNs therapeutic candidate. Exosome encapsulation not only protects the circRNA but also enhances cell-specific delivery.

3. Viral and Non-Viral Vectors:
In addition to nanoparticles and exosomes, engineered viral vectors are sometimes employed for gene therapy applications involving circRNAs. However, the safety profile of viral vectors necessitates cautious optimization. Non-viral methods, including physical delivery techniques like electroporation or microinjection, are also being explored particularly for localized treatments, such as cardiac or tumor therapy.

4. Hybrid and Targeted Delivery Systems:
To maximize therapeutic index and minimize off-target effects, next-generation delivery systems may combine improved nanoparticle formulations with targeting ligands (e.g., antibodies, peptides). This dual approach aims to achieve high specificity by directing the circRNA directly to the diseased tissue while bypassing healthy cells.

Challenges and Future Perspectives

Current Challenges in Circular RNA Research
Despite the promising developments in circRNA-based therapeutics and diagnostics, several challenges remain that need to be addressed.

1. Efficiency and Standardization of Synthesis:
Achieving high-yield and reproducible synthesis of circRNAs remains a technical challenge. The efficiency of circularization, subsequent purification of circRNAs away from linear by-products, and maintaining functional integrity are ongoing areas of research. The variability introduced by different circularization methods can affect their immunogenicity and functionality.

2. Delivery and Cellular Uptake:
While delivery systems like LNPs and exosomes are promising, ensuring efficient cellular uptake and site-specific delivery without triggering unintended immune responses is still a major obstacle. The size, residual linear RNA contaminants, and the inherent properties of the circRNA must be carefully balanced to avoid toxicity while ensuring therapeutic efficacy.

3. Immunogenicity Concerns:
Although circRNAs have relatively low immunogenicity compared to linear RNAs, extraneous sequences or structural conformations introduced during synthesis can stimulate immune responses. Understanding and mitigating the immunogenicity caused by by-products or non-native RNA folding remains a critical aspect of circRNA therapeutic design.

4. Regulatory and Analytical Challenges:
The lack of standardized protocols for circRNA detection, quantification, and validation across laboratories leads to data variability. Additionally, the development of comprehensive databases and uniform nomenclature is essential to streamline research and support clinical translation.

5. Mechanistic Uncertainties:
Although many functions of circRNAs have been suggested, including acting as miRNA sponges or translation templates, the full spectrum of their biological roles is not yet completely understood. This gap in knowledge poses a challenge for designing therapies that rely on precise modulation of circRNA function.

Future Directions and Potential
Looking forward, the future of circRNA research is rich with potential both in therapeutic applications and as diagnostic tools.

1. Enhanced Therapeutic Modalities:
The continued exploration of circRNA as a platform for protein replacement therapies, vaccines, and gene regulation presents a multifaceted opportunity. Future work will likely focus on the development of standardized, high-efficiency circRNA synthesis protocols, along with the optimization of delivery systems tailored to specific diseases.
- Emerging strategies might include the combination of circRNA therapeutics with other modalities such as CRISPR/Cas systems, enabling precise gene editing or modulation in a controlled manner.
- The use of engineered circRNAs for the development of next-generation vaccines, especially given their prolonged half-life, could revolutionize the vaccine landscape and address complex diseases such as cancer and infectious diseases.

2. Diagnostic and Prognostic Biomarkers Development:
As circRNAs show distinct expression patterns in various diseases, further research is expected to refine their use as non-invasive biomarkers. Large-scale clinical studies and standardized detection methodologies will be pivotal for translating these discoveries into routine diagnostic assays.
- Integration with liquid biopsy technologies and robust bioinformatics pipelines will further enhance the sensitivity and specificity of circRNA-based diagnostics, making early disease detection more feasible.

3. Integration of Multi-Omics and Systems Biology:
Future research may integrate circRNA studies with genomics, transcriptomics, proteomics, and metabolomics to uncover intricate networks of gene regulation. Such holistic approaches will help in understanding the interplay between circRNAs and other regulatory molecules, and in identifying novel therapeutic targets.

4. Improved Computational Tools and Databases:
With advances in high-throughput sequencing and bioinformatics, the development and refinement of computational methods for circRNA detection will continue to improve. Future databases with more comprehensive and standardized circRNA annotations will greatly facilitate research reproducibility and enable cross-study comparisons.

5. Clinical Translation and Regulatory Approval:
The ultimate goal of circRNA research is the clinical translation of these molecules into safe and effective therapeutics and diagnostics. A concerted effort between academic research, industry, and regulatory agencies will be needed to navigate the challenges of preclinical validation, clinical trials, and eventual market approval.
- Ongoing and future patents and clinical studies will pave the way for establishing circRNA-based drugs, as exemplified by the current pipeline from companies like RiboX Therapeutics, Sun Yat-Sen Memorial Hospital, and Hormos Medical Oy.

Conclusion

In summary, the development of circular RNAs is an expansive and evolving field with applications spanning therapeutic and diagnostic modalities. On a general level, circRNAs are defined by their covalently closed-loop structure, which grants them exceptional stability and resistance to degradation. This structural hallmark is coupled with diverse biological functions that include serving as miRNA sponges, scaffolds for protein interactions, and templates for protein translation. These attributes have fueled their exploration as promising therapeutic agents for a wide range of diseases including mouth and tooth disorders, cancer, and cardiovascular diseases.

When delving into specifics, several circRNA-based therapeutic candidates such as RXRG-001, CircFam53B-219aa DC vaccine, Circ_0011129-loaded 3D-sEVs, and circCOL-ELNs are under development in different stages from discovery to clinical trials. Moreover, additional circRNA candidates for cardiovascular diseases and other conditions are emerging from research and patent portfolios, reflecting a growing interest in harnessing these molecules for therapeutic interventions. Concurrently, circRNAs are being developed as diagnostic biomarkers with encouraging sensitivity and specificity metrics in cancer, cardiovascular diseases, prostate cancer, and even female reproductive system diseases. Studies indicate the potential of circulating circRNAs as non-invasive liquid biopsy markers with further advancements expected as detection and quantification methods continue to be refined.

On the research methodology front, sophisticated identification techniques based on high-throughput RNA sequencing and bioinformatics, combined with robust in vitro synthesis methods, are paving the way for large-scale circRNA production. Advanced delivery systems including lipid nanoparticles, exosomes, and hybrid vectors are being engineered to overcome the challenges of cellular uptake and tissue specificity, which are crucial for the success of circRNA-based therapeutics.

From a general perspective, while considerable progress has been made, challenges remain concerning circRNA synthesis efficiency, standardization of clinical assays, delivery optimization, and comprehensive understanding of circRNA mechanisms. Future research is likely to focus on integrating multi-omics data, refining computational tools, and improving purification and delivery systems. Moreover, enhanced collaboration among academic institutions, biotech companies, and regulatory bodies will be essential for the successful translation of circRNAs from bench to bedside.

In conclusion, the development of circRNAs represents a paradigm shift in the field of RNA therapeutics and diagnostics. Their unique structure and multifaceted biological roles offer significant advantages over traditional linear RNAs, positioning them as next-generation tools for precision medicine. As research continues to address the current challenges and develop innovative synthesis and delivery methods, circRNAs are poised to become a cornerstone in the treatment and early diagnosis of a wide spectrum of diseases, with a future rich in clinical potential and transformative healthcare impacts.