In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA

Nat Biomed Eng. 2021 Feb;5(2):179-189. doi: 10.1038/s41551-020-00671-z. Epub 2021 Jan 25.

Abstract

Base editors are RNA-programmable deaminases that enable precise single-base conversions in genomic DNA. However, off-target activity is a concern in the potential use of base editors to treat genetic diseases. Here, we report unbiased analyses of transcriptome-wide and genome-wide off-target modifications effected by cytidine base editors in the liver of mice with phenylketonuria. The intravenous delivery of intein-split cytidine base editors by dual adeno-associated viruses led to the repair of the disease-causing mutation without generating off-target mutations in the RNA and DNA of the hepatocytes. Moreover, the transient expression of a cytidine base editor mRNA and a relevant single-guide RNA intravenously delivered by lipid nanoparticles led to ~21% on-target editing and to the reversal of the disease phenotype; there were also no detectable transcriptome-wide and genome-wide off-target edits. Our findings support the feasibility of therapeutic cytidine base editing to treat genetic liver diseases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / physiology
  • Animals
  • Cytidine / genetics*
  • DNA / genetics*
  • Gene Editing / methods*
  • Genetic Vectors / physiology
  • HEK293 Cells
  • Hepatocytes / metabolism*
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • RNA / genetics*

Substances

  • Cytidine
  • RNA
  • DNA