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Study verifies gene repair breakthrough

Research published in Nature confirms discovery of DNA repair mechanism
Germline editing
This sequence of images shows the development of embryos after co-injection of a gene-correcting enzyme and sperm from a donor with a genetic mutation known to cause hypertrophic cardiomyopathy. New research published in Nature verifies the findings in a groundbreaking study that used genome editing to repair a deadly genetic mutation in human embryos. (OHSU)

New research published today in the journal Nature verifies the findings in a groundbreaking study that used genome editing to repair a deadly genetic mutation in human embryos. Much of the research was conducted at OHSU in Portland, Oregon.

The findings came in response to a critique raised by a group of stem cell scientists and geneticists who questioned the conclusions of the landmark Nature paper published Aug. 2, 2017. The journal published the verification today following a period of rigorous peer review, along with a pair of critiques of the original study. One of the critiques was initially posted Aug. 28, 2017, to the bioRxiv preprint server.

An international team of researchers collaborated on the response. Led by senior author Shoukhrat Mitalipov, Ph.D., director of the Center for Embryonic Cell and Gene Therapy at OHSU, researchers re-tested embryonic samples generated in the initial study and cited additional research that bolsters the study’s central finding: that a precisely targeted break in the mutant DNA is repaired by copying the genetic code of the normal gene from the second parent as a template. By repairing the mutation at the embryonic stage, the technique would not only remove it from the developing embryo but also prevent it from being inherited by succeeding generations. 

Shoukhrat Mitalipov, Ph.D.
Shoukhrat Mitalipov, Ph.D., principal investigator for the Center for Embryonic Cell and Gene Therapy. (OHSU/Kristyna Wentz-Graff)

“The additional retesting of the samples confirmed our original conclusion that this repair occurred as we described,” Mitalipov said.

Mitalipov noted that the study’s co-authors and OHSU welcome scientific debate over the findings, and the new paper concludes by noting that the mechanism of DNA repair should be further explored by the scientific community. In fact, new research published this year by a team at the Massachusetts Institute of Technology confirmed the paper’s key finding by documenting the same method of DNA self-repair in mice.

“Independent replication of our work provides additional evidence that our discovery may lead to the prevention of inherited disease,” Mitalipov said.

The study provided new insight about the workings of DNA, the foundational building block of life. Using molecular scissors known as CRISPR, Mitalipov and colleagues discovered that by snipping a specific target sequence on a mutated gene – in this case, a mutation carried by a male study participant that causes hypertrophic cardiomyopathy, a common genetic heart disease that can cause sudden cardiac death and heart failure – human embryos effectively repaired the break using the normal copy of the gene from a second parent as a blueprint.

This stunning biological finding, if confirmed through continued testing and eventual clinical trials, could apply to more than 10,000 monogenic inherited disorders that currently affect an estimated 600 million people worldwide with limited treatment options. These disorders are caused by a mutation in a single gene.  

Juan Carlos Izpisua Belmonte
Juan Carlos Izpisua Belmonte, Ph.D.

“For an issue with as much potential to impact human health as gene editing, it is critical that scientists engage in robust debate at every step of the research process, always keeping in mind both safety and ethical considerations,” said co-author Juan Carlos Izpisua Belmonte, Ph.D., professor and Roger Guillemin chair in the Gene Expression Laboratory of the Salk Institute in La Jolla, California. “We are pleased that this new work verifies our previous results.”

Mitalipov said the repair mechanism discovered in one-cell human embryos may be at work in other cells within the body. If so, the researchers may have uncovered a fundamental principle in how DNA repair works, which ultimately could be useful in treating many forms of genetic disease.

“This could have huge implications for DNA repair with aging and in cancer,” Mitalipov said.

It also could improve the success of infertility treatments by increasing the number of embryos potentially available for transfer.

Paula Amato, M.D.
Paula Amato, M.D.

"If proven safe, gene editing can increase the efficiency of preimplantation genetic diagnosis and reduce the burden of in vitro fertilization on couples at risk of transmitting inherited disease," said co-author Paula Amato, M.D., associate professor of obstetrics and gynecology in the OHSU School of Medicine.

CRISPR, which stands for clustered regularly interspaced short palindromic repeats, offers promise as a readily available, precise and relatively inexpensive gene-editing tool. Scientists are exploring avenues to use it to treat genetic diseases. Through his work in embryonic cells, Mitalipov is focused on correcting mutations in the human genome to prevent inherited disease.

Mitalipov and colleagues conducted additional research to address questions raised by the original study.

Critics suggested that the authors may have been misinterpreting their results and concluding that they had corrected the mutation but instead may have lost altogether the mutant paternal gene in most embryos due to large deletions. However, subsequent re-testing of the embryonic cells found no such deletions. Further, Mitalipov and colleagues noted that the CRISPR enzyme and guide was meticulously designed and pre-tested in skin cells, with minimal deletions, before it was applied to embryos.

The research team also ruled out another possibility raised by critics, that the early-stage embryos developed without a genetic contribution from sperm – a development known as parthenogenesis. The investigators excluded the possibility of parthenogenesis and confirmed that the embryos contained both maternal and paternal genes.

Finally, the research team cited other recent research – in tomatoes, mice and human zygotes – suggesting the DNA repair mechanism described by Mitalipov and colleagues appears to be conserved across many species.

“Mounting evidence suggests that the two parental homologs provide more than a genetic diversity contributed by parents,” the researchers wrote. “Recent developments in custom-designed nucleases allowing selective targeting of one of the two parental alleles have provided evidence for inter-chromosomal pairing, interaction and contribution to DNA repair across plant and animal species.”

The work at OHSU marked the first time scientists successfully tested the method on embryos generated from donated clinical-quality eggs and sperm.

Research adhered to guidelines established by OHSU’s Institutional Review Board and additional ad-hoc committees established for scientific and ethical review. Further, the work is consistent with recommendations issued in February of 2017 by the National Academy of Sciences and the National Academy of Medicine joint panel on human genome editing.

The work involved human oocytes donated by healthy volunteers and sperm carrying a genetic mutation that causes cardiomyopathy. Embryos created in the study were used to answer pre-clinical questions about safety and effectiveness.

In addition to the OHSU Center for Embryonic and Gene Therapy, OHSU Knight Cardiovascular Institute and Division of Reproductive Endocrinology and Infertility, co-authors on the response represented the Center for Genome Engineering, within the Institute for Basic Science, and the Department of Chemistry at Seoul National University in South Korea; the Salk Institute for Biological Studies in La Jolla, California; and BGI-Shenzhen, BGI-Qingdao, the China National GeneBank and Shenzhen Engineering Laboratory for Innovative Molecular Diagnostics in China.

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