Validation of a mitochondrial RNA therapeutic strategy using fibroblasts from a Leigh syndrome patient with a mutation in the mitochondrial ND3 gene

Abstract

We report on the validation of a mitochondrial gene therapeutic strategy using fibroblasts from a Leigh syndrome patient by the mitochondrial delivery of therapeutic mRNA. The treatment involves delivering normal ND3 protein-encoding mRNA as a therapeutic RNA to mitochondria of the fibroblasts from a patient with a T10158C mutation in the mtDNA coding the ND3 protein, a component of the mitochondrial respiratory chain complex I. The treatment involved the use of a liposome-based carrier (a MITO-Porter) for delivering therapeutic RNA to mitochondria via membrane fusion. The results confirmed that the mitochondrial transfection of therapeutic RNA by the MITO-Porter system resulted in a decrease in the levels of mutant RNA in mitochondria of diseased cells based on reverse transcription quantitative PCR. An evaluation of mitochondrial respiratory activity by respirometry also showed that transfection using the MITO-Porter resulted in an increase in maximal mitochondrial respiratory activity in the diseased cells.

Evolution of drug delivery system from viewpoint of controlled intracellular trafficking and selective tissue targeting toward future nanomedicine

“…Through a series of studies, we succeeded in delivering the therapeutic mRNA (ND3) to mitochondria in the of LS ND3 cells and reducing the content ratio of mutant mRNA (ND3) from 80% to 10%. We also confirmed that the rate of mitochondrial oxygen consumption (mitochondrial function) increased with increasing ratio of mutant mRNA (ND3) [ 95 ]. These findings suggest that the mitochondrial RNA delivery therapeutic strategy using the MITO-Porter is a potentially useful innovative therapeutic method for the treatment of mitochondrial diseases.…”

“…To date, we succeeded in efficiently packaging nanoparticles of therapeutic nucleic acids with polycations in the MITO-Porter. These cargoes include circular DNA [ 89 , 90 ], an antisense RNA oligonucleotide (ASO) [ 91 , 92 ], tRNA [ 93 ] and mRNA [ 94 , 95 ]. In the following, we summarize our efforts in mitochondrial gene therapy based on the MITO-Porter technology.…”

“…With the goal of achieving gene therapy targeting mitochondria, we have mainly focused on research based on the use of a MITO-Porter system, including “Functional molecule delivery aimed at mtDNA [ 96 , 97 ]“, “Construction of mitochondrial gene expression DNA [ 89 , 98 , 99 ]“and “Nucleic acid therapy targeting mitochondria [ 10 ][ 91 , 93 , 95 ]”. As our first trial to validate the nucleic acid delivery targeting mitochondria, we attempted mitochondrial RNA knockdown.…”

Therapeutic Approaches to Treat Mitochondrial Diseases: “One-Size-Fits-All” and “Precision Medicine” Strategies

“…As proof of principle, this method was further exploited to deliver wild-type mitochondrial pre-tRNA Phe to decrease the mutation rate of tRNA Phe in mitochondria of the patient’s cell with a G625A heteroplasmic mutation in the tRNA Phe of mtDNA, with a significant correction of the mutation rate [ 355 ]. Similarly, a therapeutic correction of ND3 mutant fibroblasts’ mitochondrial respiration was obtained by reintroducing the wt mRNA of ND3 via Mito-Porter [ 356 ].…”

An effective in vivo mitochondria-targeting nanocarrier combined with a π-extended porphyrin-type photosensitizer

“…The nanoparticles that make up the system have a highly fusogenic lipid composition that is combined with an arginine-rich cellpenetrating peptide. This system has been used to carry a wide variety of cargoes, ranging from small molecules to genetic materials, into mitochondria to achieve specific targeting goals [19][20][21] . Moreover, we recently utilized the MITO-Porter system to carry an anthracycline antineoplastic agent into the mitochondria of resistant human renal carcinoma cells, both in vitro and in vivo, resulting in a promising level of inhibition of tumor growth 22,23 .…”