An Update on Using TALENS to Edit Mitochondrial DNA

Mitochondria are the power plants of the cell, a host of organelles evolved from symbiotic bacteria. They each carry a small amount of DNA, and this accumulates damage with age. Some sorts of damage can spread rapidly within a cell's mitochondria, causing all of them to become dysfunctional. The cell itself also malfunctions as a result, exporting damaging reactive molecules into surrounding tissues. A small but significant portion of all the cells in the body suffer this fate by the time old age rolls around, and their presence contributes to degenerative aging.

Any comprehensive rejuvenation toolkit developed in the near future must include some way to deal with this issue. One possibility is a form of gene therapy for all cells in the body, delivering fixed and fully functional mitochondrial DNA, coupled with removal of the damaged strains to prevent them from spreading once more. The use of TALENs is showing some promise here, but at this point the research community is focused on inherited mitochondrial diseases rather than aging:

Mutations in mitochondrial DNA (mtDNA) can be specifically targeted and removed by transcription activator-like effector nucleases (TALENs) in murine oocytes, single-celled mouse embryos, and fused human-mouse hybrid cells, providing proof of principle for a method that could one day be used to treat certain hereditary mitochondrial disorders in people.

Between 1,000 and 100,000 mitochondria power each human cell. Often, mitochondria in the same cell have different genomes, or haplotypes, a condition known as heteroplasmy. Certain haplotypes include mutations that impact mitochondrial function and cause disease, particularly in energy-hungry organs such as the brain and heart. Because mitochondria segregate randomly as cells divide, it is impossible to determine early in embryonic development how a mix of wild-type and mutated mitochondria inherited from the mother will affect an organism.

To rid mitochondria of these harmful mutations, researchers have used restriction enzymes as well as zinc-finger nucleases (ZFNs) and TALENs, which can be designed to recognize any DNA sequence, to cut and eliminate mutated mitochondrial genomes from heteroplasmic cells. "Because the cell likes keeping the number of mtDNA molecules constant, after elimination of the faulty ones, the wild-type copy will repopulate the cell."

Now, an international team has used mitochondria-targeting restriction enzymes and TALENs in the mammalian germline and early-stage mouse embryos for the first time. Injecting mRNAs encoding each enzyme into mouse cells with two different wild-type mtDNA haplotypes selectively removed the targeted genome variant, and the edited embryos grew into normal mice. The team did not observe any off-target effects. To determine whether the enzymes could be used to edit human mtDNA, the researchers fused mouse oocytes with fibroblast cells from patients with one of two mitochondrial disorders - Leber's hereditary optic neuropathy or neurogenic muscle weakness, ataxia, and retinitis pigmentosa. Unlike in the mouse experiment, mutant mtDNA was still detectable, albeit at lower levels, after TALEN mRNA injection. Mutated mtDNA usually only causes disease if more than 60 percent to 75 percent of a cell's mitochondria harbor the error, so "the reduction that we observed was more than enough for the phenotype to disappear."



It looks like the delivery problem has been solved too:

I think (as a layman) that reducing the mutant mitochondrial DNA is probably an easier approach than expressing replacement genes in the cell nucleus.

I wonder if anyone is thinking of using TALENs/mRNA-based lentiviral delivery in mice? It would be interesting to see the effect on a mouse periodically dosed with TALENs directed at all 13 protein encoding mitochondrial genes. It this had a healthspan/lifespan effect it could put to bed once and for all the argument about whether mitochondrial mutations cause systemic damage and aging.

Posted by: Jim at April 27th, 2015 9:06 AM

Unless I'm missing something, that Nature paper only speaks to cell culture. What we need is highly efficient transduction in adult animals.

Posted by: Gary at April 27th, 2015 11:50 AM

@Gary: Yes, it's really much more aimed at germline manipulation for ensuring that parental mutations are not passed on. It's a whole other set of additional technology and techniques to make this work with adult organisms with sufficient cell coverage.

Posted by: Reason at April 27th, 2015 2:22 PM

@Gary - Yes they didn't test in vivo, but their use of modified lentiviral vectors was done with in vivo tests in mind in the future.

"Combining the high specificity of TALENs with efficient lentiviral gene delivery should advance genome editing in vitro and potentially in vivo".

Posted by: Jim at April 27th, 2015 5:51 PM

Thanks, Jim & Reason for your elaboration. I've been looking but not finding much information on general in vivo transduction efficiency (and preferably as a function of individual tissue/organ type) for the lentivirus vector. Any insights on your part?

Posted by: Gary at April 27th, 2015 10:09 PM

I'm just a layperson on the Internet. Most gene therapy in the clinic right now is ex vivo on removed blood cells (T cells, blood stem cells). I think the closest in vivo clinical study is the lentiviral vector study for cystic fibrosis in 2017 planed by the UK cystic fibrosis gene therapy consortium.

Posted by: jim at April 28th, 2015 12:05 AM

A gene therapy for Beckers MD has just finished up at Nationwide Childrens Hospital that has shown very promising results. It has potential for application in other pathologies and even as a potential aging therapy to combat muscular wastage.

There are a number of very promising gene therapies using AAV (and CRISPR will improve these) but the FDA has been very slow to embrace them.

This gene therapy is apparently the same as the Biotech BioViva is planning to deploy in-vivo this year as an aging therapy. They are intending to combine that with hTERT according to an article I read on H+

Posted by: Steve H at April 29th, 2015 1:11 AM

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