Engineering highly efficient backsplicing and translation of synthetic circRNAs
| Autor: | Andrew E. Hale, Katherine E. Simon, William F. Marzluff, Rita M. Meganck, Jeremy E. Wilusz, Jiacheng Liu, Nathaniel J. Moorman, Aravind Asokan, Heather A. Vincent, Marco M. Fanous |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Spliceosome lcsh:RM1-950 Intron Alu element RNA Translation (biology) Computational biology Biology 03 medical and health sciences Internal ribosome entry site Exon 030104 developmental biology 0302 clinical medicine lcsh:Therapeutics. Pharmacology 030220 oncology & carcinogenesis Drug Discovery RNA splicing Molecular Medicine Original Article |
| Zdroj: | Molecular Therapy: Nucleic Acids, Vol 23, Iss, Pp 821-834 (2021) Molecular Therapy. Nucleic Acids |
| ISSN: | 2162-2531 |
| Popis: | Circular RNAs (circRNAs) are highly stable RNA molecules that are attractive templates for expression of therapeutic proteins and non-coding RNAs. In eukaryotes, circRNAs are primarily generated by the spliceosome through backsplicing. Here, we interrogate different molecular elements including intron type and length, Alu repeats, internal ribosome entry sites (IRESs), and exon length essential for circRNA formation and exploit this information to engineer robust backsplicing and circRNA expression. Specifically, we leverage the finding that the downstream intron can tolerate large inserts without affecting splicing to achieve tandem expression of backspliced circRNAs and tRNA intronic circRNAs from the same template. Further, truncation of selected intronic regions markedly increased circRNA formation in different cell types in vitro as well as AAV-mediated circRNA expression in cardiac and skeletal muscle tissue in vivo. We also observed that different IRES elements and exon length influenced circRNA expression and translation, revealing an exonic contribution to splicing, as evidenced by different RNA species produced. Taken together, these data provide new insight into improving the design and expression of synthetic circRNAs. When combined with AAV capsid and promoter technologies, the backsplicing introns and IRES elements constituting this modular platform significantly expand the gene expression toolkit. Graphical Abstract In this study, Asokan and colleagues interrogate a battery of molecular elements including introns, Alu repeats, and internal ribosome entry sites and exploit this information to engineer robust RNA backsplicing. The approach yields a modular circular RNA expression platform for gene therapy applications. |
| Databáze: | OpenAIRE |
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