Journal Article

mitoTev‐TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels

Zekonyte, Ugne, Edgell, David R, Moraes, Carlos T, Bacman, Sandra R, Arguello, Tania, Pereira, Claudia V, Williams, Sion L

2018

Overview

Pathogenic mitochondrial DNA (mtDNA) mutations often co‐exist with wild‐type molecules (mtDNA heteroplasmy). Phenotypes manifest when the percentage of mutant mtDNA is high (70–90%). Previously, our laboratory showed that mitochondria‐targeted transcription activator‐like effector nucleases (mitoTALENs) can eliminate mutant mtDNA from heteroplasmic cells. However, mitoTALENs are dimeric and relatively large, making it difficult to package their coding genes into viral vectors, limiting their clinical application. The smaller monomeric GIY‐YIG homing nuclease from T4 phage (I‐TevI) provides a potential alternative. We tested whether molecular hybrids (mitoTev‐TALEs) could specifically bind and cleave mtDNA of patient‐derived cybrids harboring different levels of the m.8344A>G mtDNA point mutation, associated with myoclonic epilepsy with ragged‐red fibers (MERRF). We tested two mitoTev‐TALE designs, one of which robustly shifted the mtDNA ratio toward the wild type. When this mitoTev‐TALE was tested in a clone with high levels of the MERRF mutation (91% mutant), the shift in heteroplasmy resulted in an improvement of oxidative phosphorylation function. mitoTev‐TALE provides an effective architecture for mtDNA editing that could facilitate therapeutic delivery of mtDNA editing enzymes to affected tissues. Synopsis This work describes the development of a mitochondrial‐targeted DNA editing enzyme that can specifically cleave the MERRF m.8344A>G mtDNA mutation. The novel feature of this enzyme is that it is monomeric, in contrast to mitoTALEN and mitoZFN, which are heterodimeric. The homing endonuclease I‐TevI was fused to the N‐terminus of a TALE motif that binds specifically to the mtDNA MERRF m.8344A>G site. A mitochondrial targeting sequence and a FLAG tag were also added to the N‐terminus. When MERRF cells harboring heteroplasmic mutant mtDNA were transfected with mitoTev‐TALE there was a reduction in mutant mtDNA by approximately 20%. The monomeric nature of this reagent should facilitate packaging into AAV vectors. Graphical Abstract This work describes the development of a mitochondrial‐targeted DNA editing enzyme that can specifically cleave the MERRF m.8344A>G mtDNA mutation. The novel feature of this enzyme is that it is monomeric, in contrast to mitoTALEN and mitoZFN, which are heterodimeric.

• Publisher: Nature Publishing Group UK,EMBO Press,John Wiley and Sons Inc,Springer Nature
• Subject: Cells, Cultured / Cybrids / Deoxyribonucleic acid / DNA / DNA Repair / DNA, Mitochondrial - metabolism / Editing / EMBO16 / Endonuclease / Endonucleases - genetics / Endonucleases - metabolism / Enzymes / Epilepsy / Heteroplasmy / Homing endonuclease / Humans / Hybrids / Hydrolysis / I‐TevI / MERRF Syndrome / MERRF Syndrome - drug therapy / Mitochondrial DNA / mitoTev‐TALE / Molecular Targeted Therapy - methods / monomeric / Mutation / Nuclease / Oxidative phosphorylation / Phages / Phenotypes / Phosphorylation / Point mutation / Protein Binding / Recombinant Proteins - genetics / Recombinant Proteins - metabolism / Transcription / Transcription Activator-Like Effector Nucleases - genetics / Transcription Activator-Like Effector Nucleases - metabolism / Viral Proteins - genetics / Viral Proteins - metabolism