Significant progress has been made in circular RNA (circRNA) research in recent years. Increasing evidence suggests that circRNAs play important roles in many cellular processes, and their dysregulation is implicated in the pathogenesis of various diseases. CircRNAs are highly stable and usually expressed in a tissue- or cell type-specific manner. Therefore, they are currently being explored as potential therapeutic targets.

Biogenesis and Properties of CircRNAs

CircRNAs can be categorized as exonic (ecircRNA), exon-intron (EIcircRNA), or intronic (ciRNA) circRNAs. The majority of circRNAs are ecircRNAs, which are predominantly located in the cytoplasm. In contrast, EIcircRNAs and ciRNAs are usually located in the nucleus. There are three proposed models of circRNA biogenesis: direct back-splicing, RNA-binding protein-mediated circularization, and lariat-driven circularization, which are depicted in Fig. 1.

Fig. 1 Biogenesis and functional mechanisms of circular RNAs (circRNAs). (He, A. T. et al., 2021)

Aberrant regulation of circRNA biogenesis may play a role in disease. In general, circRNA biogenesis uses canonical splice sites, and thus back-splicing can compete with linear splicing of mRNA. Under physiological conditions, back-splicing is usually less efficient than linear splicing. However, depleting the activity of core spliceosomal components resulted in increased expression of circRNAs, while expression of their associated linear mRNAs decreased. The balance between back-splicing and linear splicing could also be altered due to RBPs that facilitate circularization. Dysregulation in Quaking and muscleblind splicing factors have been implicated in a wide array of pathological conditions. It is also possible that mutations in the intronic repeats could affect circularization.

Functional Mechanisms of CircRNAs

To date, various circRNA functions have been elucidated. Although microRNA sponging is the most well-known function, circRNAs perform many other functions and exert widespread regulatory effects. A brief overview of circRNA function includes the following:

• Acting as miRNA sponges.
• Regulating transcription and translation.
• Sequestering and translocating proteins.
• Facilitating interactions between proteins.
• Translating to proteins.

Targeting CircRNAs in Disease

The dysregulation of circRNA expression has been implicated in a wide variety of diseases, especially cancers, cardiovascular diseases, and neurological disorders. Many studies have described functional roles for circRNAs in promoting these diseases or exerting protective effects against them.

• Targeting circRNAs in cancer

The expression profile of circRNAs in human cancers is diverse. A recent pan-cancer study performed high-throughput exome capture RNA sequencing on more than 2000 patient samples to provide a global perspective on circRNA expression in cancers.71 Results showed that the expression profile of circRNAs in different cancers are distinct, and the expression of a given circRNA across cancer types is significantly different. These findings, taken together with the stability of circRNAs, suggest that they can be used as potential cancer biomarkers.

• Targeting circRNAs in cardiovascular disease

CircRNAs are abundant in the human heart, and many of them are cardiac specific.90 Numerous studies have elucidated functional roles of circRNAs in aggravating cardiovascular diseases or exerting cardio-protective effects. Currently, studies targeting circRNA in various cardiovascular diseases mainly include: myocardial infarction, cardiac fibrosis, other cardiovascular diseases.

• Neurodegenerative diseases

CircRNAs are also highly abundant in the brain. Studies have evaluated circRNA expression in various brain regions. It was estimated that roughly 30% of genes transcribed in the human brain produce circRNAs. Studies targeting circRNAs for central nervous system (CNS) disorders have focused on neurodegenerative diseases, acute ischemic stroke, neuropathic pain, and other disorders.

Strategies to Target CircRNAs

Several approaches have been developed to study circRNAs and target circRNAs for therapeutic purposes in vivo. These methods include:

• RNA interference-mediated circRNA knockdown.
• CircRNA expression vectors.
• Synthetic circRNAs.
• Nanoparticle delivery of circRNA-based therapeutics.
• Exosome delivery of circRNA-based therapeutics.
• Conditional circRNA knockout or knockdown.
• CRISPR/Cas9-mediated circRNA knockout or knockdown.
• CRISPR/Cas13-mediated circRNA knockdown.

Fig. 2 Strategies used to study circular RNA (circRNA). (He, A. T. et al., 2021)

Challenges in Targeting CircRNAs

To date, circRNA-based therapeutic approaches have only been performed in preclinical studies. There are still many obstacles that need to be overcome in order for the therapeutic potential of these approaches to be achieved.

• Off-target gene silencing

A fundamental concern with RNAi-based strategies is that small molecules like siRNA can potentially induce off-target gene silencing via a miRNA-like effect. Designing siRNA to mitigate off-target effects is an ongoing area of interest for RNAi approaches. The CRISPR/Cas13 system has demonstrated low mismatch tolerance and could knockdown circRNAs with greater specificity than RNAi. However, whether or not this approach will be effective in vivo remains to be investigated.

• Nonspecific tissue or cell type targeting

Although the majority of circRNAs are expressed in a tissue- or cell type-specific manner, some circRNAs are present in more than one tissue or cell type. Common strategies used to target circRNAs may cause adverse effects on off-target tissues or cells. Nanoparticle delivery systems have the potential to improve the targeting of therapeutic agents to specific cells. Alternatively, this challenge could be avoided in cases where it is possible to target circRNAs with highly specific expression patterns.

• Toxicity of gold nanoparticles

Although AuNPs are convenient for delivering circRNA-targeting agents or circRNA plasmids in animal models, it is unclear how safe they are for clinical use. Thus, it is possible that the properties of AuNPs can be fine-tuned to meet safety requirements.

• Mis-spliced products

CircRNA overexpression vectors are usually based on the pairing of intronic complementary sequences. This system can lead to mis-splicing of linear RNAs or circRNAs. The mis-spliced byproducts can cause nonspecific and potentially deleterious effects. Highly purified circRNA molecules synthesized in vitro could potentially be used to overcome the shortcomings of circRNA overexpression vectors.

• Synthetic circRNA immunogenicity

In addition, synthetic circRNAs can induce immune system activation in vivo. It was suggested that foreign circRNAs are distinguished from endogenous circRNAs based on their lack of the m6A modification. Strategies are currently being explored to reduce synthetic circRNA immunogenicity, including introducing chemical modifications and coating them in RBPs.

Perspective

In recent years, many studies have contributed to our increasing understanding of circRNA functions and their important roles in diseases. Due to their stability and tissue- or cell type-specific expression, circRNAs have emerged as promising therapeutic targets. Therefore, it is necessary to develop tools that can effectively target circRNAs.

IntegrateRNA offers a series of premade circular RNA products with high stability and translation efficiency to satisfy your various downstream applications. With our unparalleled expertise and experience in RNA synthesis, we help our customers to expand their understanding of the mechanisms of circRNA function and to further develop specific and effective methods for targeting circRNAs in vivo.