Selective Elimination of Mitochondrial Mutations in the Germline by Genome Editing

Abstract

SUMMARY Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mitochondrial DNA (mtDNA). In most of these patients, mutated mtDNA coexists with wild type mtDNA, a situation known as mtDNA heteroplasmy. Here we report on a strategy towards preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber’s hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP), in mammalian oocytes using mito-TALENs. Altogether, our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders

“…Recent breakthroughs in the field of mitochondrial medicine might yield methods to prevent the transmission of pathogenic mtDNA mutations in the future (Mitalipov and Wolf, 2014;Reddy et al, 2015); but, currently no cures or effective interventional therapies are available for patients who have already inherited the mutations and are subject to such disorders (Pfeffer et al, 2013).…”

Assisted reproductive technologies to prevent human mitochondrial disease transmission

“…At present, there is no clear consensus on the relevance or mechanism of reversion. If it did turn out to be a risk factor in MRT, then methods to avoid carryover of any mutant mtDNA, which could conceivably involve its targeted destruction using mitophagy or genome editing methods 51 , might provide solutions (see below).…”

“…Several findings point to the possibility of eliminating mtDNA from donor samples in MRT. Transcription activator-like effector nucleases (TALENs) have been targeted to mitochondrial genomes of a specified haplotype in heteroplasmic mouse 1-cell embryos 51 , and mitochondrially targeted zinc finger nucleases (mtZFNs) can efficiently discriminate between, and selectively eliminate, mutant mitochondrial genomes in a human cell line in vitro 61 . One report suggests that Cas9 containing a mitochondria-targeting peptide fusion ('mitoCas9') can also effectively edit mtDNA, although it is unclear how the gRNA component enters the mitochondria or to what extent the editing efficiency depends on the targeted mtDNA sequence 62 .…”

Prévenir la transmission des mutations de l’ADN mitochondrial par don de cytoplasme : où en est-on ?

“…De plus, certaines patientes ont très peu de chances de donner naissance à un enfant porteur d'un faible taux d'hétéroplasmie, dans la mesure où elles-mêmes portent un taux élevé de mutation, d'où un risque important d'interruption de grossesse en cas de DPN (50 % dans notre population) ou de non-transfert embryonnaire en cas de DPI. Les nouvelles techniques d'édition du génome comme les enzymes TALEN (transcription activator-like effector nuclease) [5], le système CRISPR(clustered regularly interspaced short palindromic repeat)/Cas 9 [6], voire les ARN anti-réplicatif [7], pourraient être appliquées aux embryons in vitro, dans le but de diminuer leur taux d'hétéroplasmie. Cependant, pour les patientes homoplasmiques (qui sont porteuses uniquement de molécules d'ADNmt mutées), ces options ne sont pas applicables.…”