Tackling inherited diseases with CRISPR

Researchers reportedly achieve first safe repairs of single-gene mutations in human embryos

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LA JOLLA, Calif.—As gene-editing technologies have advanced, especially in recent years with CRISPR techniques, the debate over human germline gene modification has heated up. The potential to create “custom human beings” by editing the genome before they are even born, rather than using gene-editing techniques to correct an existing medical condition in a fully developed human, has largely been considered unsafe and unethical and is actually prohibited by law in more than three dozen nations.
As the Center for Genetics and Society puts it on their website, “This controversy marks a new chapter of a profoundly consequential debate about the future of gene editing in humans. If perfected, somatic gene editing (or ‘gene therapy’) holds promise for helping people who are sick, affecting only an individual consenting patient.
“But editing the genes of human embryos in order to create genetically modified people is very different, and raises grave safety, social and ethical concerns. These range from the prospect of irreversible harms to the health of future children and generations, to concerns about opening the door to new forms of social inequality, discrimination and conflict.”
Still, controversy over whether something should be done does not necessarily deter research into whether or how it can be done, and such is the case with recent work coming out of a collaboration between the Salk Institute, Oregon Health and Science University (OHSU) and Korea’s Institute for Basic Science. In that work, researchers have, for the first time, corrected a disease-causing mutation in early-stage human embryos with gene editing using the CRISPR/Cas9 system. In this work, they corrected the mutation for a heart condition at the earliest stage of embryonic development so that the defect would not be passed on to future generations.
The research was described in Nature on August 2, 2017, and the collaborators see it as a potential way to improve in-vitro fertilization (IVF) outcomes as well as to eventually provide cures for some of the thousands of diseases caused by mutations in single genes.
“Thanks to advances in stem cell technologies and gene editing, we are finally starting to address disease-causing mutations that impact potentially millions of people,” says Juan Carlos Izpisua Belmonte, a professor in Salk’s Gene Expression Laboratory and a corresponding author of the paper. “Gene editing is still in its infancy so even though this preliminary effort was found to be safe and effective, it is crucial that we continue to proceed with the utmost caution, paying the highest attention to ethical considerations.”
As Salk notes in a news release about the research, “Though gene-editing tools have the power to potentially cure a number of diseases, scientists have proceeded cautiously, in part to avoid introducing unintended mutations into the germ line (cells that become eggs or sperm). Izpisua Belmonte is uniquely qualified to speak to the ethics of genome editing in part because, as a member of the committee on human gene editing of the National Academies of Sciences, Engineering and Medicine, he helped author the 2016 roadmap ‘Human Genome Editing: Science, Ethics, and Governance.’ The research in the current study is fully compliant with recommendations made in that document, and adheres closely to guidelines established by OHSU’s Institutional Review Board and additional ad-hoc committees set up for scientific and ethical review.”
Still, because the work required the creation and destruction of human embryos, it was barred from receiving U.S. government funding. Also, it was no short walk to get the research started, as embryologist Shoukhrat Mitalipov of OSHU first brought the idea to his university three years ago and many of the institutional review board members were reportedly reluctant to sign off on the research.
Part of the reason for this reluctance was that three other published experiments involving gene editing of human embryo—all from Chinese researchers who utilized small numbers of embryos—have noted that the CRISPR technique can cut unintended DNA targets as well as produce mosaic embryos (that is, portions of those embryos’ cells contained the healthy gene and others kept the mutated one). So, aside from the ethical considerations, there was concern that the technique might be inefficient enough that it wouldn’t represent any improvement over current IVF procedures that work to prevent implantation of fertilized eggs that might produce genetic disease-carrying embryos.
In the work led by Mitalipov for the Salk, OHSU and Institute for Basic Science collaboration, the target was hypertrophic cardiomyopathy (HCM), which affects approximately one in 500 people overall. It is caused by a dominant mutation in the MYBPC3 gene, but often goes undetected until it is too late. Since people with a mutant copy of the MYBPC3 gene have a 50-percent chance of passing it on to their own children, being able to correct the mutation in embryos would prevent the disease not only in affected children, but also in their descendants, Salk notes.
The researchers generated induced pluripotent stem cells from a skin biopsy donated by a male with HCM and developed a gene-editing strategy based on CRISPR/Cas9 that would specifically target the mutated copy of the MYBPC3 gene for repair. As Salk explains, the targeted mutated MYBPC3 gene was cut by the Cas9 enzyme, allowing the donor’s cells’ own DNA-repair mechanisms to fix the mutation during the next round of cell division by using either a synthetic DNA sequence or the non-mutated copy of MYBPC3 gene as a template.
Using IVF techniques, the researchers injected the best-performing gene-editing components into healthy donor eggs newly fertilized with the donor’s sperm. Then they analyzed all the cells in the early embryos at single-cell resolution to see how effectively the mutation was repaired.
Reportedly, the scientists were surprised by just how safe and efficient the method was—in addition to seeing a high percentage of embryonic cells being repaired, there were no detectable off-target mutations or genome instability.
“Even though the success rate in patient cells cultured in a dish was low, we saw that the gene correction seems to be very robust in embryos of which one copy of the MYBPC3 gene is mutated,” remarks Jun Wu, a Salk staff scientist and one of the paper’s first authors. This was in part because, after CRISPR/Cas9-mediated enzymatic cutting of the mutated gene copy, the embryo initiated its own repairs. Instead of using the provided synthetic DNA template, the team found, surprisingly, that the embryo preferentially used the available healthy copy of the gene to repair the mutated part. “Our technology successfully repairs the disease-causing gene mutation by taking advantage of a DNA repair response unique to early embryos."
Izpisua Belmonte and Wu emphasize that, although promising, these are very preliminary results and more research will need to be done to ensure no unintended effects occur.
“Our results demonstrate the great potential of embryonic gene editing, but we must continue to realistically assess the risks as well as the benefits,” adds Izpisua Belmonte.
Future work will continue to assess the safety and effectiveness of the procedure and efficacy of the technique with other mutations.

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