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Salk Scientists Develop Method to Edit Mutations in Germline Mitochondrial DNA


NEW YORK (GenomeWeb) – An international team of scientists led by the Salk Institute has used genome editing technologies to remove mutations in mitochondrial DNA from germline cells. The method, demonstrated in mouse embryos and mouse oocytes infused with human mitochondrial DNA, could be applied to human germline cells to edit away heritable mitochondrial diseases, they said.

Led by Pradeep Reddy and Alejandro Ocampo of the Salk Institute for Biological Studies, the scientists used restriction endonucleases and transcription activator-like effector nucleases (TALENs) to demonstrate proof of concept that gene editing technology could reduce mutated in mitochondrial DNA in mice, then showed that this reduction could prevent the transmission to successive generations. They also used TALENs to eliminate mutated mtDNA known to contribute to heritable mitochondrial diseases in humans. The scientists published their results today in Cell.

Their success working in mice indicates that the method is ready to test in human cells, Ocampo and Reddy told GenomeWeb, which could prove the ability to edit mitochondrial inherited diseases. But with high profile calls to halt research into human germline gene editing, uncertainty abounds as they make their next move. Yesterday, a study published in Protein & Cell that used CRISPR/Cas9 to edit the nuclear genome in zygotes highlighted the difficulties of using that technology to safely edit the human germline. But Ocampo and Reddy pointed to several reasons why germline editing of the mitochondrial genome might warrant separate considerations than the nuclear genome.

Mitochondria are organelles that supply energy to the cell, among other functions, and are thought to have originated as bacteria. Mitochondria contain their own DNA (mtDNA), a circular loop that encodes several proteins for their function. Mutations in this mtDNA can lead to dysfunction in cellular respiration and affect tissues with large energy needs, including the brain and heart. Human germline cells, such as eggs, can contain hundreds of thousands of copies of mtDNA, hundreds of times more than somatic cells which might have several thousand. In comparison, human germ line cells only have one copy of the nuclear genome, while somatic cells would have two.

The mtDNA is inherited exclusively from the mother in a non-Mendelian fashion; likewise, inherited mitochondrial diseases do not follow Mendelian patterns of inheritance. "Mitochondrial DNA is present in multiple copies per mitochondrion and, with each cell division, mutated and normal mitochondrial DNA can be non-randomly distributed in daughter cells," Asha Kallianpur, a doctor and researcher at the Genomic Medicine Institute of the Cleveland Clinic's Lerner Research Institute, told GenomeWeb. Thus, many cells are heteroplasmic, meaning they contain both normal and mutated copies of mtDNA, especially in cases of disease.

Most mitochondrial diseases are heteroplasmic, with mutated mtDNA needing to comprise a certain threshold of total mtDNA. "If the proportion of [mutated] mitochondrial DNA is above a certain threshold, disease will be expressed because mitochondrial function will be compromised," Kallianpur said.

Reddy and Ocampo were able to demonstrate that gene editing could reduce mtDNA mutations to levels below the typical threshold necessary for disease, which is typically above 60 percent of mtDNA. In a mouse oocyte fused with a human cell, they were able to eliminate a mutation responsible for the mitochondrial disease Leber's hereditary optic neuropathy (LHON), bringing the percentage of mutated mtDNA down from about 80 percent to below 15 percent in two of the three trials (in one trial the LHON mutation remained at 67 percent).

"Our aim is to get to zero percent," Ocampo said, "but the truth is as long as we reduce the mutation below the clinical threshold, we will generate an embryo that will not have disease."

Having shown that TALENs can shift the balance of human mtDNA mutations in a mammalian germline cell, Reddy and Ocampo said they're looking to move on to human germline editing. Reddy said that they've contacted in vitro fertilization clinics in the US but have yet to submit a study to an institutional review board.

Given the intense scrutiny given to gene editing in the human germline recently, it could be a challenge to convince an IRB and the scientific community at large that such a study would be a good idea, but there are several key differences between nuclear and mitochondrial genome editing.

First, the mechanics of editing are different, Reddy said. "In our case we are not trying to correct the mutations. We are trying to identify the DNA and cut them," he said. Ocampo added that merely cutting the mutation was enough to remove the entire molecule of mtDNA. There's also the fact that there are hundreds of thousands of mitochondrial genomes to target, instead of just the one nuclear genome, and not all of the mistakes need to be edited, just enough to tip the balance.

Additionally, the use of TALENs seems to avoid the problem of off-target effects seen with CRISPR/Cas9. The study authors used whole-exome sequencing to look for off-target effects, but found none, Reddy said. He noted that the method they used steered clear of the nuclear genome and only targeted mtDNA. Kallianpur, the Cleveland Clinic doctor, confirmed that this appeared to be the case.

Another consideration is the nature of mitochondrial diseases, which are "fundamentally different from disease transmitted by the nuclear genome," Kallianpur said. Mitochondrial DNA is transmitted to offspring exclusively through the mother. If a mother carries a serious mtDNA mutation then all of her children will inherit the mutation and be at risk for the disease, even if the father has completely normal mtDNA, she added. 

During in vitro fertilization, pre-implantation diagnosis can attempt to identify any embryos harboring mtDNA mutations, but the irregular dispersal of mutations means that even if several samples are taken, they may not be able to predict a disease.

Recently, the UK Parliament passed a bill permitting the also contentious procedure of mitochondrial transfer to help prevent these diseases. In mitochondrial transfer, mtDNA from a third individual are inserted during in vitro fertilization, essentially giving the embryo genetic material from three individuals. In addition to ethical concerns, there are technical limitations to the technique. "It requires a series of steps that not many laboratories in the world can actually perform," Ocampo said. He said the method only requires the injection of mRNA, which could be injected at the same time as the sperm. "What we are doing could be implemented in any in vitro fertilization clinic in the world, using the same instrumentation used to inject sperm."

"This is a potential treatment option that could be used to prevent transmission of mitochondrial diseases," Kallianpur said, which occur with a frequency of about one in every 5,000 people. "The diseases are quite devastating when they occur, because they involve degeneration of tissues with high metabolic needs, like brain and muscle." They can affect people at any age and patients often develop neurodegenerative symptoms. "For family members it's not only heart rending, but disconcerting to relatives at reproductive age. They don't know if they may transmit this disease to their children," she said.

This story has been updated from a previous version to clarify quotes from a source.