RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo

R Alexander Wesselhoeft; Piotr S Kowalski; Frances C Parker-Hale; Yuxuan Huang; Namita Bisaria; Daniel G Anderson

doi:10.1016/j.molcel.2019.02.015

RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo

Mol Cell. 2019 May 2;74(3):508-520.e4. doi: 10.1016/j.molcel.2019.02.015. Epub 2019 Mar 19.

Authors

R Alexander Wesselhoeft¹, Piotr S Kowalski², Frances C Parker-Hale³, Yuxuan Huang⁴, Namita Bisaria⁵, Daniel G Anderson⁶

Affiliations

¹ David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
² David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
³ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Department of Political Science, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
⁴ David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
⁵ Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
⁶ David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Harvard and MIT Division of Health Science and Technology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address: dgander@mit.edu.

Abstract

Circular RNAs (circRNAs) are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Here, we show that unmodified exogenous circRNA is able to bypass cellular RNA sensors and thereby avoid provoking an immune response in RIG-I and Toll-like receptor (TLR) competent cells and in mice. The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses. Unmodified circRNA is less immunogenic than unmodified linear mRNA in vitro, in part due to the evasion of TLR sensing. Finally, we provide the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and we show that circRNA translation is extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs.

Keywords: RIG-I; RNA sensors; circular RNA; immunogenicity; protein expression; synthetic.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
DEAD Box Protein 58 / genetics*
DEAD Box Protein 58 / immunology
Gene Expression Regulation
Gene Regulatory Networks / genetics
Immunity, Innate / genetics
Mice
MicroRNAs / genetics
Protein Biosynthesis*
RNA / genetics*
RNA, Circular
RNA, Messenger / genetics*
Toll-Like Receptors / genetics
Toll-Like Receptors / immunology
Uridine / genetics
Vaccines, Synthetic / genetics

Substances

MicroRNAs
RNA, Circular
RNA, Messenger
Toll-Like Receptors
Vaccines, Synthetic
RNA
Ddx58 protein, mouse
DEAD Box Protein 58
Uridine

Abstract

Publication types

MeSH terms

Substances

Grants and funding