For what indications are circular RNA being investigated?

Introduction to Circular RNA

Definition and Biological Role
Circular RNAs (circRNAs) are a distinct class of covalently closed-loop RNA molecules that differ fundamentally from their linear counterparts by lacking free 5′ and 3′ ends. This unique structure confers remarkable stability, making circRNAs resistant to degradation by exonucleases, as well as enabling prolonged half-lives both in vitro and in vivo. Beyond being mere by-products of splicing, circRNAs have been found to fulfill a wide variety of biological roles. For instance, they can function as molecular sponges that sequester microRNAs (miRNAs) or RNA-binding proteins (RBPs), thereby modulating gene expression at various regulatory levels. Furthermore, some circRNAs possess the capability to be translated into peptides, thus adding another layer of gene regulation. Their cell-type specificity and tissue-specific expression further suggest that circRNAs play critical roles in both normal physiological processes and pathological conditions across different systems.

Historical Overview of Circular RNA Research
The discovery of circRNAs dates back several decades. Early electron microscopy studies identified circular forms of RNA in plant viroids as early as the 1970s, and subsequent work confirmed similar structures in viral genomes like the Hepatitis delta virus (HDV). In the 1990s, endogenous circRNAs derived from genes such as DCC, SRY, and ETS-1 in humans and other mammals were documented, initially dismissed as splicing errors. It was not until the advent of high-throughput RNA sequencing and sophisticated bioinformatics tools that researchers began to systematically uncover circRNAs throughout the transcriptome. This technological revolution spurred an exponential growth in circRNA research, revealing not only their widespread presence across species but also their involvement in key cellular processes and diseases. As a result, the past decade has seen a shift from regarding circRNAs as mere accidental molecules to recognizing them as vital regulators with significant diagnostic and therapeutic potential.

Current Indications for Circular RNA

CircRNAs are currently being investigated for a broad array of clinical indications. Their intrinsic stability, specificity, and multifaceted roles in gene regulation have made them prime candidates for both biomarker discovery and therapeutic intervention. Here, we discuss the major indications under active investigation.

Cancer Treatment
Cancer remains a central focus in circRNA research. Multiple studies have established that circRNAs are dysregulated in various types of cancer and, owing to their resistance to degradation, they serve as promising biomarkers for early diagnosis, prognostic evaluation, and therapeutic targeting.

1. Biomarkers and Diagnostic Tools:
CircRNAs detected in tissues and bodily fluids are being used as reliable indicators of tumor presence. For example, certain circRNAs have been implicated as diagnostic and prognostic biomarkers in colorectal cancer, liver cancer, gastric cancer, lung cancer, and thyroid cancer. Their unique expression patterns, often independent of host gene expression, facilitate the development of highly specific assays. The detection of circRNA signatures in plasma may also enable real-time monitoring of disease progression and response to therapy.

2. Therapeutic Targets and RNA-Based Therapies:
Multiple preclinical studies are investigating the therapeutic modulation of circRNAs. They can be targeted using antisense oligonucleotides, RNA interference, or CRISPR-Cas systems aimed at restoring or inhibiting circRNA functions. Notably, circRNAs can function as microRNA sponges that modulate oncogenic or tumor-suppressive pathways. For instance, synthetic circRNA molecules are being engineered to alter key regulatory networks in cancer cells, effectively “sponging” miRNAs that promote tumor growth and metastasis.

3. Vaccine Development and Immune Modulation:
The use of circRNA-based vaccines has emerged as an innovative strategy in oncology. Their enhanced stability compared to linear RNAs allows for prolonged antigen expression, potentially eliciting more robust anti-tumor immune responses. Several studies and patents have described circRNA constructs as candidates for cancer vaccines and immunotherapies. These approaches target not only the tumor cells directly but also the surrounding tumor microenvironment by modulating immune responses, a factor crucial in immunotherapy.

4. Combination Therapies and Personalized Medicine Approaches:
Given the heterogeneous nature of cancers, circRNAs are also being explored as tools for personalized cancer therapy. Their tissue-specific expression profiles may help in stratifying patients for targeted therapies, as well as in monitoring treatment responses over time, thus paving the way for more individualized treatment protocols.

Neurological Disorders
The investigation of circRNAs in neurological disorders has gained substantial momentum, driven in part by their high abundance in neural tissues and their involvement in brain-specific regulatory pathways.

1. Neurodegenerative Diseases:
Several studies have identified circRNA dysregulation in neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). For example, a comprehensive study using laser-captured neurons from postmortem brain samples revealed more than 11,000 circRNAs in neurons, some of which were associated with PD and AD pathology.

2. Biomarkers for Early Diagnosis:
The exceptional stability of circRNAs makes them attractive candidates as biomarkers for early detection of neurological disorders. Specific circRNA expression patterns have been correlated with disease progression in PD, where altered levels of circRNAs generated from Parkinson’s-associated genes precede symptom onset. In Alzheimer’s disease, circRNAs may regulate key proteins involved in amyloid precursor processing and tau aggregation.

3. Therapeutic Interventions:
Beyond diagnostics, circRNAs are being targeted for therapeutic intervention. Strategies include the modulation of circRNA expression to restore normal cellular homeostasis in neuronal cells. Loss-of-function studies using antisense oligonucleotides (ASOs) have shown that reducing the levels of pathogenic circRNAs can alleviate neurodegenerative changes. Additionally, their role as miRNA sponges can be harnessed to disrupt detrimental regulatory cascades in diseased neurons, opening potential new avenues for treatment.

4. Other Cognitive Impairments and Brain Injuries:
Emerging evidence suggests that circRNAs may also play roles in traumatic brain injury (TBI), stroke, and various cognitive disorders, where their regulation of neuroinflammation and synaptic function is critical. The capacity of circRNAs to modulate extracellular vesicle-mediated intercellular communication further highlights their therapeutic potential in central nervous system (CNS) injuries.

Cardiovascular Diseases
Cardiovascular diseases (CVDs) represent another major area of circRNA research, owing to the high morbidity and mortality associated with these conditions and the urgent need for novel diagnostic and therapeutic strategies.

1. Biomarkers for Diagnosis and Prognosis:
The highly stable nature of circRNAs, as well as their specific expression in cardiac tissues, makes them ideal candidates for non-invasive biomarkers for various cardiovascular conditions, including acute myocardial infarction and heart failure. CircRNAs have been detected in circulating blood and exosomes, enabling potential development of liquid biopsies for early diagnosis, monitoring disease progression, and predicting outcomes.

2. Therapeutic Targets and Gene Regulation:
CircRNAs modulate key signaling pathways involved in cardiomyocyte hypertrophy, fibrosis, autophagy, and apoptosis. For example, certain circRNAs act as “sponges” for miRNAs that regulate vascular endothelial growth factor (VEGF) and other critical mediators in cardiovascular physiology. Experimental approaches utilizing engineered circRNAs show promise in restoring proper cardiac function and ameliorating pathologic remodeling.

3. RNA-Based Interventions:
Advanced methodologies in RNA therapeutics have paved the way for employing circRNAs as novel agents in cardiovascular treatment. Synthetic circRNAs can be designed to express therapeutic proteins or regulatory molecules in a controlled and sustained manner to counteract dysfunctional signaling pathways in diseased hearts. The efficient delivery systems optimized for mRNA drugs provide a robust framework to translate these approaches into clinical practice.

4. Combination with Regenerative Medicine:
Interdisciplinary approaches involving circRNAs in combination with stem cell therapies or biomaterials are being explored to promote cardiac repair and regeneration. Studies indicate that circRNAs may enhance the survival, differentiation, and integration of transplanted cells into the injured myocardium, thereby improving functional recovery following myocardial infarction.

Mechanisms of Action

The multifaceted roles of circRNAs in various disease states are underpinned by their complex mechanisms of action. Their ability to influence gene expression and regulate cellular pathways occurs primarily through two interrelated mechanisms.

Gene Regulation
CircRNAs can modulate gene expression at both the transcriptional and post-transcriptional levels. They have been implicated in the regulation of parental gene transcription by sequestering regulatory proteins and by influencing the epigenetic landscape of cells. For instance, circRNAs that retain intronic sequences (ciRNAs) or exon–intron circRNAs (EIciRNAs) have been shown to interact with transcriptional complexes within the nucleus, modulating the expression of neighboring genes. Additionally, their capability to function as protein scaffolds can facilitate the assembly of multiprotein complexes that regulate gene transcription, splicing, and mRNA stability. These functions are particularly crucial in diseases like cancer and cardiovascular conditions, where deregulated gene expression is a central pathogenic event.

Interaction with MicroRNAs
One of the best-characterized functions of circRNAs is their role as “sponges” for miRNAs. By binding to miRNAs through complementary sequences, circRNAs can inhibit miRNA activity, thereby releasing suppression on target messenger RNAs (mRNAs). This interaction is crucial in various pathologies. For example, in cancer, circRNAs may sequester tumor-suppressive miRNAs that normally inhibit oncogenic pathways, or conversely, bind to oncogenic miRNAs to restore the expression of anti-tumor genes. Similarly, in neurological diseases, circRNA-mediated miRNA sponging has been linked to the dysregulation of proteins involved in neurodegeneration. In cardiovascular contexts, the regulation of miRNAs by circRNAs affects key processes such as apoptosis, fibrosis, and angiogenesis, thereby influencing cardiac remodeling and disease progression. This miRNA-interaction paradigm is central to many of the therapeutic strategies currently under investigation, as it offers a mechanism to modulate aberrant signaling events in a precise manner.

Research and Clinical Trials

Both preclinical research and early-phase clinical investigations are currently underway to leverage the therapeutic potential of circRNAs across various indications.

Preclinical Studies
Preclinical studies have provided fundamental insights into the function and therapeutic utility of circRNAs. Numerous in vitro and in vivo experiments have established the feasibility of targeting circRNAs to alter disease outcomes. For instance, several drug candidates based on circRNA technology have been reported in early-phase studies, such as RXRG-001 for mouth and tooth diseases, which is a circular RNA targeting AQP1 and is in Phase 1/2 development. Likewise, circRNA constructs like CircFam53B-219aa DC vaccine and circCOL-ELNs carried by exosomes have been evaluated in preclinical tumor models, demonstrating promising immunomodulatory effects and cancer cell targeting. These studies employ gain-of-function and loss-of-function approaches to delineate circRNA functions in pathological processes. Furthermore, engineered circRNAs have been utilized to modulate gene expression in cardiovascular disease models, providing evidence for their efficacy in ameliorating cardiac dysfunction.

Ongoing Clinical Trials
Although the clinical application of circRNAs is still in its infancy compared to established mRNA technologies, several clinical trials are emerging, leveraging the lessons learned from RNA drug development. For example, circRNA-based vaccines and therapeutic constructs are now being optimized for stability and delivery, taking into account issues such as efficient production and immunogenicity. Some circRNA formulations are already in early-phase human trials for cancer immunotherapy, where they function as both diagnostic tools and therapeutic agents. Additionally, circRNA interventions are being designed for neurological and cardiovascular applications, guided by robust preclinical data that supports their safety and efficacy. These clinical investigations are informed by a comprehensive understanding of circRNA biology and are rapidly evolving due to recent advancements in delivery systems and synthesis technologies.

Challenges and Future Prospects

Despite the extensive promise of circRNAs, several challenges remain that must be overcome to fully realize their clinical utility.

Current Challenges in Circular RNA Research
One of the primary challenges in circRNA research pertains to the methodological issues associated with their detection and validation. CircRNAs lack polyadenylated tails, which means traditional RNA isolation methods often fail to capture them, necessitating specialized protocols such as ribosomal RNA depletion and RNase R treatment. Furthermore, the overlapping sequence content between circRNAs and their linear counterparts can complicate bioinformatic analyses, resulting in potential false positives. Researchers are now advocating for the use of multiple algorithms (e.g., find_circ, CIRI2, CIRCexplorer2) to improve accuracy.

Another significant challenge is related to the delivery of circRNA therapeutics. Although circRNAs have notable stability, achieving efficient and targeted delivery to specific tissues remains a hurdle. Advances in lipid nanoparticle (LNP) formulations and viral as well as non-viral delivery systems are actively being pursued to address these issues. Additionally, controlling off-target effects and ensuring the specificity of circRNA-based interventions are critical aspects that need further investigation.

Immunogenicity is also a concern. Even though circRNAs exhibit a favorable immunological profile compared to linear RNAs, there is still the risk of eliciting unwanted immune responses, particularly when synthetic circRNAs are introduced in vivo. Researchers must refine their methods to reduce immunogenicity while maintaining therapeutic efficacy.

Future Directions and Potential Applications
Looking forward, the potential applications of circRNAs extend far beyond current indications. On the diagnostic front, the development of sensitive, high-throughput circRNA detection technologies promises to revolutionize early diagnosis of diseases such as cancer, cardiovascular conditions, and neurodegenerative disorders. As more comprehensive circRNA databases are established, integrated multi-omics approaches will likely enhance our ability to identify disease-specific circRNA signatures.

Therapeutically, the future of circRNA research lies in the development of improved synthesis and purification methods. Innovations in circularization techniques—such as those incorporating group I intron fragments and IRES elements—are expected to enhance production efficiency and scalability, ultimately leading to cost-effective clinical-grade circRNAs. Moreover, the design of synthetic circRNAs that can encode therapeutic proteins or modulate specific signaling pathways offers a promising avenue for innovative treatments. These approaches could be particularly transformative in the context of cancer immunotherapy, where circRNA vaccines may provide durable and robust immune responses.

In the realm of neurological disorders, future research will likely focus on elucidating the specific circRNA–miRNA–mRNA networks that underpin neurodegenerative processes. This could lead to targeted therapies that restore normal neuronal function or delay disease progression in conditions like Parkinson’s and Alzheimer’s diseases. Similarly, in cardiovascular diseases, further investigations into the role of circRNAs in modulating cardiomyocyte function and intercellular communication could result in novel interventions to prevent or reverse heart failure and post-infarction remodeling.

Another promising area is the integration of circRNA research with regenerative medicine. Combining circRNA-based therapies with stem cell technologies has the potential to enhance tissue regeneration and repair, particularly in tissues with limited regenerative capacity such as the heart and brain. This interdisciplinary approach may open up entirely new therapeutic paradigms.

Finally, as circRNA research continues to evolve, overcoming current challenges—such as delivery efficiency, specificity, and immunogenicity—is paramount. Collaborative efforts among academic institutions, biotechnology companies, and regulatory agencies will accelerate the translation of circRNA research from bench to bedside. The lessons learned from mRNA drug development, including successful clinical delivery platforms, can serve as a blueprint to address these challenges and usher in a new era of RNA-based therapies.

Conclusion

In conclusion, circular RNAs are being investigated for a broad range of clinical indications with significant promise in cancer treatment, neurological disorders, and cardiovascular diseases. Their inherent stability, tissue specificity, and diverse mechanisms of action—particularly in gene regulation and miRNA sponging—have made them attractive candidates for both diagnostic and therapeutic applications. Preclinical studies and early-stage clinical trials underscore the potential of circRNAs as innovative tools, ranging from biomarkers for early diagnosis to vehicles for gene-targeted therapies and vaccines.

Nevertheless, critical challenges remain. These include technical hurdles in circRNA detection and validation, efficient delivery to target tissues, control of off-target effects, and minimization of immunogenicity. Addressing these challenges through advanced synthesis methods, refined bioinformatics pipelines, and innovative delivery systems is essential for the successful clinical translation of circRNA-based interventions.

Looking ahead, the future of circRNA research is bright. The development of highly specific circRNA assays will greatly enhance early disease detection and personalized treatment strategies. Additionally, the potential of circRNAs in regenerative medicine and immunotherapy signifies a transformative shift in how chronic and complex diseases are managed. As our understanding deepens and technological advances continue, circRNAs are poised to become integral components of next-generation therapeutics, offering new hope for patients across a wide spectrum of diseases.

In summary, circular RNAs represent a versatile and promising platform that is being explored extensively for cancer, neurological, and cardiovascular indications, among others. Their multifunctional roles in gene regulation and cell signaling, coupled with advances in RNA technology, make them exciting candidates for future therapeutic breakthroughs. Continued research, multidisciplinary collaboration, and clinical innovation will be key to unlocking the full potential of circRNAs in transforming modern medicine.