Challenges in targeting circRNAs

https://www.nature.com/articles/s41392-021-00569-5

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. Major limitations with these techniques and potential mitigation strategies are outlined in this section.

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.¹⁶⁵ siRNA can target transcripts through partial complementarity, which usually occurs between the 3’UTR of the transcript and seed region of the siRNA.^166,167 In circRNA knockdown experiments, it is usually verified that the corresponding linear mRNA levels are unaffected. However, off-target effects beyond their linear counterparts are less predictable. Designing siRNA to mitigate off-target effects is an ongoing area of interest for RNAi approaches.^127,157 The CRISPR/Cas13 system has demonstrated low mismatch tolerance and could knockdown circRNAs with greater specificity than RNAi.³⁵ 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.²⁵ 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.^31,32,168 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. Previous studies on AuNPs draw inconsistent conclusions about their toxicity.¹⁶⁹ It has been suggested that its toxic effects are dependent on the size of the particles, with smaller AuNPs causing more harmful effects.¹⁷⁰ Thus, it is possible that the properties of AuNPs can be fine-tuned to meet safety requirements. Of note, a LNP-siRNA system has already been approved for the treatment of hereditary transthyretin amyloidosis³⁰ and could potentially be used to deliver siRNA targeting disease-promoting circRNAs.

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. currently, there are still no vectors that can generate target circRNAs without mis-spliced products. Highly purified circRNA molecules synthesized in vitro could potentially be used to overcome the shortcomings of circRNA overexpression vectors. However, inherent problems with large-scale synthesis may limit the therapeutic potential of synthetic circRNAs.

Synthetic circRNA immunogenicity

In addition, synthetic circRNAs can induce immune system activation in vivo.¹⁷¹ It was suggested that foreign circRNAs are distinguished from endogenous circRNAs based on their lack of the m⁶A modification.¹³⁸ Strategies are currently being explored to reduce synthetic circRNA immunogenicity, including introducing chemical modifications and coating them in RBPs.¹³⁹

Other questions

https://www.sciencedirect.com/science/article/abs/pii/S0167779919301775

Will circular RNAs be optimally delivered using lipid-based or exosome-mediated delivery?

ActD treatment is commonly used to evaluate circular RNA stability, but its toxicity to cells limits long exposure studies. Could alternative methods of evaluating RNA stability aid in understanding the limits of circular RNA stability?

How difficult would it be to translate circular mRNA therapeutics in a targeted cell-specific manner? Does this involve the use of multiple input regulatory molecules as well as endogenous regulators?

To date all proteins generated from exogenous circular RNAs have been translated from a single open reading frame. For more complex molecules such as antibodies the best strategy for producing multiprotein complexes is not yet known: will it be a single circular mRNA with multiple open reading frames or multiple independent circles?

A comparative study evaluating the performance of in vitro versus in vivo generated circular RNAs is lacking. For example, what are the challenges in scaling up the production of synthetic circular RNAs?