Targeting circular RNAs as a therapeutic approach: current strategies and challenges

Abstract

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. Gain-of-function and loss-of-function approaches are typically performed using circRNA expression plasmids and RNA interference-based strategies, respectively. These strategies have limitations that can be mitigated using nanoparticle and exosome delivery systems. Furthermore, recent developments show that the cre-lox system can be used to knockdown circRNAs in a cell-specific manner. While still in the early stages of development, the CRISPR/Cas13 system has shown promise in knocking down circRNAs with high specificity and efficiency. In this review, we describe circRNA properties and functions and highlight their significance in disease. We summarize strategies that can be used to overexpress or knockdown circRNAs as a therapeutic approach. Lastly, we discuss major challenges and propose future directions for the development of circRNA-based therapeutics.

Fig. 1

Biogenesis and functional mechanisms of circular RNAs (circRNAs). A Back-splicing driven by the pairing of intronic complementary sequences, RNA-binding protein (RBP), or lariat structure containing skipped exons or introns. B Sponging microRNA (miRNA) to decrease their availability to bind target mRNA. C Sponging RNA-binding protein (RBP) to decrease their availability to bind target mRNA. D Interacting with eukaryotic translation initiation factor 4 G (eIF4G), poly(A)-binding protein (PABP), and cognate mRNA to disrupt the assembly of the translation initiation machinery. E Translocating proteins to the nucleus or sequestering them in the cytosol. F Facilitating interactions between specific proteins. G Translating to protein in a cap-independent manner. H Exon-intron circRNAs (EIcircRNAs) can form a complex with the U1 small nuclear ribonucleoprotein (U1 snRNP) that binds RNA polymerase II (RNA pol II) to enhance transcription of parental genes. Intronic circRNAs (ciRNAs) can interact with elongating RNA pol II complex to enhance transcription

Fig. 2

Strategies used to study circular RNA (circRNA). A CRISPR/Cas9-mediated circRNA knockout via removal of intronic complementary sequence flanking circularized exon involved in circRNA biogenesis. This system has also been used to target the entire gene locus and a transcription factor to knockout and knockdown circRNA, respectively (not shown). B Conditional circRNA knockdown mediated by a cre-dependent short hairpin RNA (shRNA), which is subsequently processed into short interfering RNA (siRNA) to induce circRNA cleavage. C CRISPR/Cas13-mediated circRNA knockdown directly targets the back-splice junction of circRNAs to induce circRNA cleavage. D CircRNA expression plasmid leads to circRNA overexpression. E siRNA/shRNA targeting the back-splice junction of circRNAs induces circRNA cleavage

Fig. 3

Strategies used to target circular RNAs (circRNAs) as a therapeutic approach in vivo. A, B Exosome-mediated delivery of A short interfering RNA (siRNA) targeting the back-splice junction of circRNAs to induce circRNA cleavage and B circRNA expression plasmid to overexpress circRNAs. C–E Gold nanoparticle-mediated delivery of C siRNA targeting the back-splice junction of circRNAs, D circRNA expression plasmid, and E antisense oligonucleotide (AON) blocking protein interaction site on circRNAs