Efficient Modulation of Exon Skipping via Antisense Circular RNAs

Figures

• 

  Fig. 1.. AS-circRNAs effectively mediate exon skipping in a DMD minigene and endogenous transcripts. (A) Schematic diagram of the DMD minigene and the working principle behind AS-circRNA. The minigene is composed of exons 50, 51, and 52 of the mouse DMD gene. The AS-circRNA binds to exon 51 of the precursor mRNA and impedes the binding of splicing factors, resulting in exon 51 skipping. (B) Detection of exon 51-skipped mRNA by RT-PCR in the AS-circRNA but not AS-RNA (linear) or Ctrl-RNA (random antisense sequence) groups. (C) Sanger sequencing of RT-PCR amplified products in the AS-circRNA group. (D) Quantitative analysis of the exon skipping rate in the different groups; n = 3, mean ± SD. (E) Quantitative analysis of the exon skipping rate at different time points after transfection of HEK293T cells with either an AS-circRNA or an AS-RNA plasmid; n = 3, mean ± SD. (F) The relative expression levels of AS-circRNAs and AS-RNAs at different time points after transfection of HEK293T cells, normalized to GAPDH. Significance was determined by 2-tailed unpaired Student’s t test; n = 3, mean ± SD. (G) Heat map showing the exon skipping rate mediated by different AS-circRNAs and AS-RNAs,…

• 

  Fig. 2.. AS-circRNAs restore dystrophin protein expression in DMD mouse model. (A) Schematic diagram depicting the in vivo experimental design. The AS-circRNA, previously tested in vitro for DMD E51 skipping, was packaged into scAAV. AAV was injected intraperitoneally into 2-week-old DMD mice, and the mice were euthanized for analysis 4 weeks after injection. Different muscles were assessed to determine whether the correct DMD reading frame and dystrophin protein expression were restored. (B) Quantification of exon 51 skipping rate in DMD mouse muscle tissues including heart, quadriceps, gastrocnemius, dorsal muscles, and triceps muscles 4 weeks after AAV injection. n = 3, mean ± SD. (C) Relative expression of AS-circRNA in the heart, quadriceps femoris, gastrocnemius, dorsal muscle, and brachial muscle of AAV-injected mice, normalized to GAPDH; n = 3, mean ± SD. (D) Western blot analysis showing that dystrophin expression was restored in the heart, quadriceps, gastrocnemius, dorsal muscles, and triceps muscles of DMD mice 4 weeks after systemic injection. (E) Percentage of recovered dystrophin protein in the gastrocnemius, quadriceps, triceps, dorsal muscles, and heart. Dystrophin…