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Gene Editing Used to Repair Inherited Mutation in Embryos

NEW YORK (GenomeWeb) – For the first time ever, researchers have reported using the gene-editing tool CRISPR/Cas9 to correct a heart disease mutation in viable human embryos.

Researchers from Sun Yat-sen University in China previously reported using the genome-editing tool to modify the gene that causes beta-thalassemia in non-viable human zygotes, while a team at Guangzhou Medical University reported using it to inactivate the CCR5 gene to make embryos HIV-resistant.

However, this work, which was conducted by Oregon State Health and Science University researchers, is the first to use viable embryos and to be done in the US. News of it first circulated last week after a report appeared in MIT Technology Review, and the results have now been published in Nature.

The OHSU-led team used the CRISPR/Cas9 gene-editing system to correct mutations within MYBPC3 in embryos. They targeted an autosomal dominant mutation in MYBPC3 that leads to hypertrophic cardiomyopathy, which affects about one in every 500 people and is a common cause of sudden death in young people.

The researchers said they were able to edit the embryos with high efficiency, while largely avoiding mosaicism and off-target cleavage, two key concerns with the use of the technology, particularly in the clinic.

"The goal of this study was to correct a disease-causing gene mutation in viable human embryos using the CRISPR gene-editing tool," co-senior author and OHSU researcher Paula Amato said during a press briefing. She added that if this embryo gene-correction approach is proven safe, it "can potentially be used to prevent transmission of genetic disease to future generations."

The researchers collected skin, blood, and semen samples from an adult man with familial hypertrophic cardiomyopathy caused by a heterozygous dominant deletion in MYBPC3. They developed two single-guide RNA-Cas9 constructs that targeted the deletion, optimized them in patient-derived induced pluripotent stem cells, and selected the more efficient sgRNA-Cas9 for further use.

They co-injected the combination of sgRNA, Cas9, and template repair DNA into eggs from healthy donors with donor sperm. They used eggs that were in the M phase of their cell cycle.

Of the 58 resulting embryos, 42 had only wild-type MYBPC3, the researchers reported. That is, the mutation was corrected with very high efficiency, higher than what is seen in somatic cells and what was reported in previous human embryo studies, Amato noted.

Surprisingly, the researchers found that the embryos didn't use the template DNA they included to fix the mutation after being cut. Instead, they noted that the sole method of repair was homology-directed repair in which the wild-type maternal copy acted as the repair template. This suggested to the researchers that the double-stranded breaks introduced by the gene-editing machinery might attract the native oocyte homology-directed repair machinery.

In their work, the Sun Yat-sen University researchers had reported that the embryos they developed were mosaic and harbored off-target cleavage sites. But here, the researchers reported only one instance of mosaicism. They attributed this low level to co-injecting the sperm and gene-editing machinery while the oocyte was in the M phase of its cell cycle.

They similarly did not observe any off-target changes when they sequenced the cells, leading them to conclude that the targeting was accurate.

"Overall, the researchers seem to have overcome the main problems encountered in the Chinese experiments — mosaicism and off-target mutations — but it will require considerably more research on many more embryos and with many more genes before confidence in this technique can be established," the University of London's Shirley Hodgson said in a statement.

The researchers' findings also uncovered a new wrinkle — because maternal DNA, rather than template DNA, was used to fix the mutation, it's uncertain whether this approach could be used to fix maternal mutations or homozygous mutations.

During the press briefing, OHSU's Shoukhrat Mitalipov said he suspected that this method should also be able to target maternal mutations. He added, however, that targeting homozygous mutations might be trickier as the researchers would have to find a way to make the embryo use the external template DNA provided.

Still, OHSU's Amato suggested that gene editing could be used — after further safety testing — in conjunction with in vitro fertilization and pre-implantation genetic diagnosis. In the case of an autosomal dominant condition, about half the embryos would be affected, but by folding in this approach, they could boost the likelihood of IVF success by increasing the number of healthy embryos and limiting the number of IVF cycle a woman has to go through, she said.

Mitalipov added that he and his colleagues are interested in targeting other high-penetrance disease genes, like those linked to cystic fibrosis and breast cancer, for editing.

But in a related commentary appearing in Nature, the Karolinska Institute's Nerges Winblad and Fredrik Lanner wrote that more study and optimization of the technology is needed. However, they added that the study shows the promise of CRISPR/Cas9 editing. 

There are also regulatory issues to be addressed, as germline editing is prohibited in the US. A recent report from the National Academy of Sciences, though, gave a cautious go-ahead for germline editing in cases of serious disease.