Dataset Information

Validation of Gene Therapy for Mutant Mitochondria by Delivering Mitochondrial RNA Using a MITO-Porter.

ABSTRACT: Here, we report on validating a mitochondrial gene therapy by delivering nucleic acids to mitochondria of diseased cells by a MITO-Porter, a liposome-based carrier for mitochondrial delivery. We used cells derived from a patient with a mitochondrial disease with a G625A heteroplasmic mutation in the tRNAPhe of the mitochondrial DNA (mtDNA). It has been reported that some mitochondrial gene diseases are caused by heteroplasmic mutations, in which both mutated and wild-type (WT) genes are present, and the accumulation of pathological mutations leads to serious, intractable, multi-organ diseases. Therefore, the decrease of the mutated gene rate is considered to be a useful gene therapy strategy. To accomplish this, wild-type mitochondrial pre-tRNAPhe (pre-WT-tRNAPhe), prepared by in vitro transcription, was encapsulated in the MITO-Porter. The pre-WT-tRNAPhe encapsulated in the MITO-Porter was transfected into diseased mitochondrial cells, and the resulting mutant levels were examined by an amplification refractory mutation system (ARMS)-quantitative PCR. The mutation rate of tRNAPhe was decreased, and this therapeutic effect was sustained even on the 8th day after transfection. Furthermore, mitochondrial respiratory activity of the disease cells was increased after the transfection of therapeutic pre-WT-tRNAPhe. These results support the conclusion that the mitochondrial delivery of therapeutic nucleic acids represents a viable strategy for mitochondrial gene therapy.

SUBMITTER: Kawamura E

PROVIDER: S-EPMC7210581 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Validation of Gene Therapy for Mutant Mitochondria by Delivering Mitochondrial RNA Using a MITO-Porter.

Kawamura Eriko E Maruyama Minako M Abe Jiro J Sudo Akira A Takeda Atsuhito A Takada Shingo S Yokota Takashi T Kinugawa Shintaro S Harashima Hideyoshi H Yamada Yuma Y

Molecular therapy. Nucleic acids 20200419

Here, we report on validating a mitochondrial gene therapy by delivering nucleic acids to mitochondria of diseased cells by a MITO-Porter, a liposome-based carrier for mitochondrial delivery. We used cells derived from a patient with a mitochondrial disease with a G625A heteroplasmic mutation in the tRNA<sup>Phe</sup> of the mitochondrial DNA (mtDNA). It has been reported that some mitochondrial gene diseases are caused by heteroplasmic mutations, in which both mutated and wild-type (WT) genes a ...[more]

PMID: 32388194

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Project description:BackgroundBacterial cancer therapy was first trialled in patients at the end of the nineteenth century. More recently, tumour-targeting bacteria have been harnessed to deliver plasmid-expressed therapeutic interfering RNA to a range of solid tumours. A major limitation to clinical translation of this is the short-term nature of RNA interference in vivo due to plasmid instability. To overcome this, we sought to develop tumour-targeting attenuated bacteria that stably express shRNA by virtue of integration of an expression cassette within the bacterial chromosome and demonstrate therapeutic efficacy in vitro and in vivo.ResultsThe attenuated tumour targeting Salmonella typhimurium SL7207 strain was modified to carry chromosomally integrated shRNA expression cassettes at the xylA locus. The colorectal cancer cell lines SW480, HCT116 and breast cancer cell line MCF7 were used to demonstrate the ability of these modified strains to perform intracellular infection and deliver effective RNA and protein knockdown of the target gene c-Myc. In vivo therapeutic efficacy was demonstrated using the Lgr5creERT2Apcflx/flx and BlgCreBrca2flx/flp53flx/flx orthotopic immunocompetent mouse models of colorectal and breast cancer, respectively. In vitro co-cultures of breast and colorectal cancer cell lines with modified SL7207 demonstrated a significant 50-95% (P < 0.01) reduction in RNA and protein expression with SL7207/c-Myc targeted strains. In vivo, following establishment of tumour tissue, a single intra-peritoneal administration of 1 × 106 CFU of SL7207/c-Myc was sufficient to permit tumour colonisation and significantly extend survival with no overt toxicity in control animals.ConclusionsIn summary we have demonstrated that tumour tropic bacteria can be modified to safely deliver therapeutic levels of gene knockdown. This technology has the potential to specifically target primary and secondary solid tumours with personalised therapeutic payloads, providing new multi-cancer detection and treatment options with minimal off-target effects. Further understanding of the tropism mechanisms and impact on host immunity and microbiome is required to progress to clinical translation.

Project description:Mitochondrial DNA is primarily maternally inherited in most animals and evolves about 10 times faster than biparentally inherited nuclear DNA. Mitochondrial dysfunction (mt-dys) arises when interactions between the co-evolving mitochondrial and nuclear genomes are perturbed in essential processes like oxidative phosphorylation (OXPHOS). Over time mt-dys can lead to mitochondrial diseases (mt-diseases), which are surprisingly prevalent and include common diseases such as Alzheimer's, Parkinson's, and diabetes. Unfortunately, the strong impact that intraspecific mitochondrial and nuclear genetic variation has on mt-disease complicates its study and the development of effective treatments. Animal models have advanced our understanding of mt-disease but their relevance to human conditions is often limited by their relatively low nuclear genetic diversity. Many traditional laboratory models also typically have a single mitochondrial haplotype (mitotype), in stark contrast to over 5,000 mitotypes in humans worldwide. The threespine stickleback fish has an evolutionary history that has made it a favorable evolutionary mutant model (EMM) for studying mito-nuclear interactions and possibly mt-diseases. EMMs are species with naturally evolved states that mimic maladaptive human diseases. In threespine stickleback, a period of isolation followed by introgression of the mitochondrial genome from a sister species resulted in the maintenance of two distinct mitochondrial haplotypes which continue to segregate within many populations of wild stickleback. The existence of two mitogenomes segregating in numerous genetically diverse populations provides a unique system for exploring complex mito-nuclear dynamics. Here we provide the first complete coding region analysis of the two threespine stickleback mitotypes, whose mitogenomic divergence exceeds that of other mammalian models for mitochondrial disease and even that between ancient and modern humans. We find that divergence is not uniform across the mitogenome, but primarily impacts protein coding genes, and significantly impacts proteins in Complex I of OXPHOS. The full characterization of these highly divergent intraspecific mitotypes provides a foundation for the development of threespine stickleback as an EMM for mito-nuclear interactions.

Dataset Information

Validation of Gene Therapy for Mutant Mitochondria by Delivering Mitochondrial RNA Using a MITO-Porter.

Publications

Validation of Gene Therapy for Mutant Mitochondria by Delivering Mitochondrial RNA Using a MITO-Porter.

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