Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo

Ugne Zekonyte,Jeff Smith,Derek Jantz,Ginger Tomberlin,James Stewart,Carlos T Moraes,Sandra R Bacman,Claudia V Pereira,Wendy Shoop

doi:10.1038/s41467-021-23561-7

Abstract

Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. In recent years, DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. Mitochondrial-targeted restriction endonucleases, ZFNs, and TALENs have been successful in shifting mtDNA heteroplasmy, but they all have drawbacks as gene therapy reagents, including: large size, heterodimeric nature, inability to distinguish single base changes, or low flexibility and effectiveness. Here we report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS®. These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. We show the development of a mitoARCUS specific for the mouse m.5024C>T mutation in the mt-tRNAAla gene and its delivery to mice intravenously using AAV9 as a vector. Liver and skeletal muscle show robust elimination of mutant mtDNA with concomitant restoration of mt-tRNAAla levels. We conclude that mitoARCUS is a potential powerful tool for the elimination of mutant mtDNA.

Highlights

Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure
Analysis of the designer nuclease using a green fluorescent protein (GFP)-based double-stranded break (DSB) recombination assay in Chinese Hamster Ovary (CHO) cells showed that the specific ARCUS nuclease yielded ~80% GFP+ cells when tested against the intended target sites but
Two variants were designed, CF and CSF, which differed by the mitochondrial localization sequence (MLS)

Summary

Introduction

Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. We report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS® These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. Specific threshold levels of mutant mtDNA need to be reached, to compromise cell function and for disease to manifest[2]. This is an important aspect in mtDNA gene therapies, as not all mutant mtDNA need to be eliminated to see improvements in symptoms. The ARCUS gene-editing platform, developed by scientists at Precision BioSciences, is based on the homing endonuclease

Methods

Results

Conclusion