Gene Editing Approach May Reduce Risks, Experts Say

Ricki Lewis, PhD

August 04, 2017

As reported earlier, researchers in the United States have used CRISPR-Cas9 editing of human embryos to correct an inherited form of hypertrophic cardiomyopathy (HCM). The details of the work, published online August 2 in Nature, show the new methods avoid some of the pitfalls seen in prior attempts and would likely be safer, experts say.

Mutations in any of several genes cause HCM. Although autosomal dominant, these mutations face little selective pressure that might reduce their frequency because heart failure does not typically begin until after reproduction age. The condition, however, is a common cause of sudden death among young athletes.

To prevent the transmission of an HCM, Hong Ma, PhD, from the Center for Embryonic Cell and Gene Therapy at the Oregon Health & Science University and colleagues, intervened in the inheritance of mutations in the MYBPC3 gene. Mutations in MYBPC3 account for 40% of genetic HCM. The gene encodes the thick filament-associated cardiac myosin-binding protein C, which regulates structure of the sarcomeres in cardiac muscle.

At this time, families with this form of HCM can use preimplantation genetic diagnosis (PGD) after in vitro fertilization to select from the half of their embryos that do not inherit a parent's mutation. If available, gene editing, which creates double-strand breaks in the DNA that permit replacement of the mutant allele with a normal (wild-type) one, would expand the pool of embryos from which to select.

However, previous attempts, which introduced the gene editing (Cas9 enzyme and guide RNA) at fertilization, led to mosaic 8-celled embryos, in which some cells had the correction and some did not. Mosaic embryos would make PGD impossible and reproduction more, rather than less, risky for these couples.

In the current work, Dr Ma and colleagues used a technique to avoid mosaicism. They created fully corrected human embryos by shifting the timetable, injecting oocytes on the brink of fertilization, at metaphase of the second meiotic division, with sperm and the CRISPR components. The sperm were from a man with a MYBPC3 4-base deletion mutation in one of the two copies of the gene (heterozygote); 12 healthy young women donated the oocytes, which had normal genes.

Also, unlike previous editing attempts that introduced a standardized corrected version of the gene, Dr Ma and colleagues used a system that copied the wild-type gene from the oocyte. The oocytes provide the template for a natural form of DNA correction (homology-directed repair) that removes the mutant gene from the male genome and replaces it with a copy of the maternal normal gene. The DNA repair system, which is in the cytoplasm, comes from the oocyte because mature sperm have so little cytoplasm, explained senior investigator Shoukhrat Mitalipov, PhD, at a news conference held to announce the work.

Of 58 treated oocytes, 42 (72.4%) divided to yield nonmosaic embryos, some of which progressed to the blastocyst stage, indicating the cells are capable of developing and differentiating into an embryo.

The other 16 embryos had activated a second DNA repair pathway (nonhomologous end-joining) that causes insertion-deletion mutations that lead to mosaic embryos. (The experiments included a marked synthetic gene copy so that the researchers could trace whether embryos copied the maternal allele or inserted the marked template.)

"The introduction of CRISPR at the time of sperm injection eliminated the issue of mosaicism. We assessed off-target effects using whole genome sequencing, and we did not observe any," said team member Paula Amato, MD, from Oregon Health & Science University in Portland.

A News and Views  article from Nerges Winblad and Fredrik Lanner, PhD, from the Karolinska Institute, Stockholm, Sweden, also published online August 2 in Nature, cautions that the approach for an autosomal dominant condition does not apply to an autosomal recessive condition, which is the mode of inheritance most common in young children with genetic diseases. Because both copies of the targeted gene from an affected parent are mutant, an exogenous copy would need to be introduced, and that might not be safe.

"Adapting the technology to correct the two mutant alleles of an autosomal recessive condition could be exposing human gametes or embryos to small molecule inhibitors that could have deleterious effects on embryonic development," Fernando Scaglia, MD, professor of medical and human genetics at Baylor College of Medicine, told Medscape Medical News.

That said, the germline correction of heterozygous fertilized ova for the autosomal dominant condition described in the Nature paper could lower the risk for PGD, especially in older women, Dr Amato said. She described a recent patient who after three rounds of IVF/PGD still had no embryos free of the MYBPC3 mutation and with normal chromosomes.

"Fewer [in vitro fertilization] cycles would be necessary to generate the normal chromosome complement in embryos for transfer, minimizing the risk to a woman undergoing ovarian stimulation. This is especially important for older women who have a higher aneuploidy rate and fewer eggs. Aneuploidy can reach 70% to 90% in older women, and then 50% are excluded because of the disease, so you have to do a lot of cycles to get normal embryos," Dr Amato said.

The technique would work for any heterozygous autosomal dominant condition, the researchers said. "With the MYBPC3 mutation as the groundwork, we're closer to clinical applications. We're also interested in a myosin mutation, MYH7, that causes 40% of hypertrophic cardiomyopathy cases, and we'd like to explore the BRCA cancer genes in the same way. The BRCA mutations have a high prevalence in some populations and a single copy can cause cancer," Dr Mitalipov said.

Dr Scaglia summed up the situation. "The results are very encouraging and promising, but germline-correction genome editing must be improved and optimized before clinical applications are possible."

One coauthor is a cofounder and shareholder of ToolGen, Inc, and another is cofounder and shareholder of Mitogenome Therapeutics, Inc, and Health and Science Center "M1" Inc, and have disclosed competing financial interests. The other authors have disclosed no relevant financial relationships.

Nature. Published online August 2, 2017. Article full text, News and Views full text

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