Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner

Abstract

Mutations in mitochondrial DNA are an important source of severe and incurable human diseases. The vast majority of these mutations are heteroplasmic, meaning that mutant and wild-type genomes are present simultaneously in the same cell. Only a very high proportion of mutant mitochondrial DNA (heteroplasmy level) leads to pathological consequences. We previously demonstrated that mitochondrial targeting of small RNAs designed to anneal with mutant mtDNA can decrease the heteroplasmy level by specific inhibition of mutant mtDNA replication, thus representing a potential therapy. We have also shown that 5S ribosomal RNA, partially imported into human mitochondria, can be used as a vector to deliver anti-replicative oligoribonucleotides into human mitochondria. So far, the efficiency of cellular expression of recombinant 5S rRNA molecules bearing therapeutic insertions remained very low. In the present study, we designed new versions of anti-replicative recombinant 5S rRNA targeting a large deletion in mitochondrial DNA which causes the KSS syndrome, analyzed their specific annealing to KSS mitochondrial DNA and demonstrated their import into mitochondria of cultured human cells. To obtain an increased level of the recombinant 5S rRNA stable expression, we created transmitochondrial cybrid cell line bearing a site for Flp-recombinase and used this system for the recombinase-mediated integration of genes coding for the anti-replicative recombinant 5S rRNAs into nuclear genome. We demonstrated that stable expression of anti-replicative 5S rRNA versions in human transmitochondrial cybrid cells can induce a shift in heteroplasmy level of KSS mutation in mtDNA. This shift was directly dependent on the level of the recombinant 5S rRNA expression and the sequence of the anti-replicative insertion. Quantification of mtDNA copy number in transfected cells revealed the absence of a non-specific effect on wild type mtDNA replication, indicating that the decreased proportion between mutant and wild type mtDNA molecules is not a consequence of a random repopulation of depleted pool of mtDNA genomes. The heteroplasmy change could be also modulated by cell growth conditions, namely increased by cells culturing in a carbohydrate-free medium, thus forcing them to use oxidative phosphorylation and providing a selective advantage for cells with improved respiration capacities. We discuss the advantages and limitations of this approach and propose further development of the anti-replicative strategy based on the RNA import into human mitochondria.

Can Mitochondrial DNA be CRISPRized: Pro and Contra

“…To counter the problem of low efficiency, new RNA molecules can be created with greater mitochondrial import capacities, and our previous reports indicate that it is possible. As a matter of fact, our recent studies with 5S rRNA based RNA vectors clearly are supporting this strategy, as increasing expression and import of recombinant RNA also produce a more pronounced anti‐replication effect on mtDNA . All in all, we believe that the current study provides matter to be moderately optimistic about the possibility of CRISPRization of human mitochondrial DNA.…”

“…C ,D, both transient and induced expression of MTS COX8A ‐hCas9 do not significantly influence mtDNA copy number. Finally, it is worth to mention that our previous assays in the frame of the anti‐replicative strategy to address heteroplasmic mtDNA mutations proved to be more effective when imported recombinant RNAs with higher melting temperatures were applied , which can indirectly indicate that the temperature issue is to be seriously taken into account also for CRIPRization.…”

Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

“…Our results show an enhanced delivery of gRNA with RP-loop. A recent study has shown that guide RNA with short hairpin structures can promote mitochondrial import and specific cleavage of mtDNA albeit at low level due to limited import into mitochondrial matrix (41,42). This study further strengthens our hypothesis that specific hairpin structures that are involved in the delivery of nuclear encoded RNA can serve as potential adapter for delivery and PNPase may serve as conduit for channeling this recombinant RNA into mitochondrial matrix.…”

Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

“…Our results show an enhanced delivery of sgRNA with RP loop into the mitochondria. A recent study has shown that guide RNA with short hairpin structures can promote mitochondrial import and specific cleavage of mtDNA albeit at a low level due to limited import into the mitochondrial matrix ( Loutre et al, 2018a , b ). This study further strengthens our hypothesis that specific hairpin structures that are involved in the delivery of nuclear-encoded RNA can serve as potential adapter for delivery, and PNPase may serve as a conduit for channeling this recombinant RNA into the mitochondrial matrix.…”