Juan Carlos Izpisua Belmonte’s group published a Cell paper today on using gene editing to reverse mutations associated with human mitochondrial disease.

The paper is Reddy, et al. and is entitled, “Selective Elimination of Mitochondrial Mutations in the Germline by Genome Editing”.

The authors report success using TALEN-based gene editing or mitochondrial-direct restriction enzyme (mito-ApaLI) to reduce the burden of mutant mitochondrial DNA (mtDNA).

Their work was done primarily in mice, but also using chimeras made with murine oocytes fused with human cells bearing mitochondrial mutations.

They sum up their work in this way:

“Using mitochondria-targeted nucleases, mtDNA mutations are specifically eliminated in the germline to prevent their transgenerational transmission. This strategy represents a potential therapeutic avenue for treating human mitochondrial disease.”

The graphical abstract is above.

I am of two minds about this paper.

On the one hand, I think technically it is intriguing that they could take a gene editing approach to mitochondrial mutations, but on the other hand the notion that this approach could be safely and effectively used clinically in a human context in the germline as a way to prevent mitochondrial disease is somewhat concerning. This also resonates more strongly because of yesterday’s report out of China of gene editing using CRISPR of human embryos.

There is a growing trend of scientists heading the direction of human genetic modification.

Could it be safe?

What would be the ethical considerations?

Focusing first on the technical side of this new paper, the data look convincing to me after a quick read. One model was heteroplasmic mice (NZB/BALB) that contain two distinct mtDNA mutation types. In this model they were able via gene editing-based targeting one of the two mutation types (BALB) to introduce “heteroplasmic shift” demonstrating reduced abundance of the mutation. They have published this kind of work in the past on somatic cells and others have shown the same so I suppose that makes this work a bit less novel.

They did most of the work with mito-ApaLI targeting a unique cutting site in BALB. The gene edited one-cell embryo was also able to produce healthy mice (Figs. 3-4), but one caveat there is that it wasn’t clear to me from the paper and a quick Google search whether the NZB/BALB heteroplasmic mice normally have any phenotype due to the mitochondrial mutations or not. I suppose at least the gene editing did not seem to affect normal development.

The other model system here (Fig. 6) was the fusion of mouse oocytes with human cells (see Fig. 6B) containing mtDNA mutations. Using mito-TALEN technology they could again observe a heteroplasmic shift in this context as well. Depending on the human mtDNA mutation that was target, the efficiency of the gene editing to remove the mutant form ranged from a modest just over two-fold (NARP) to an impressive near complete elimination (LHOND).

What about potential off-target effects?

They reported that CGH (Figure S3C) and exomic sequencing (I believe this was not shown) did not indicate significant off-target effects. I would have liked to have seen more data on this however. It’s difficult to imagine no off-target effects were created and if true that’d be great, but it seems like they should have shown more data on this.

Shifting now to the bigger picture, what about possible clinical application of this technology in humans?

The authors are fairly gung-ho about the translational potential of their approach in humans, especially relative to so-called “3-person IVF” or “mitochondrial transfer” technology, recently approved for human use in the UK, but not permitted in the US:

“Mitochondrial replacement techniques involve a series of complex technical manipulations of nuclear genome between patient and donor oocytes that will result in the generation of embryos carrying genetic material from three different origins. For these reasons, mitochondrial replacement techniques have raised some biological, medical, and ethical concerns (Hayden, 2013; Reinhardt et al., 2013). Despite their great potential, more studies are still required to show that these techniques are safe in human oocytes. The approach presented here relies on a single injection of mRNA into patient oocytes, which is technically simpler and less traumatic to the oocyte compared to mitochondrial replacement techniques (Craven et al., 2010; Paull et al., 2013; Tachibana et al., 2013; Wang et al., 2014). Importantly, it does not require healthy donor oocytes, thus avoiding ethical issues related to the presence of donor mtDNA.”

The idea that a gene editing approach to mitochondrial disease in humans could be superior to the so-called “3-person IVF” or “mitochondrial transfer” approach to mitochondrial disease is kind of provocative. The Scientist provides a quote suggestive of a possible tension between gene editing and “mitochondrial replacement” approaches to mitochondrial disease prevention:

“While mitochondrial replacement advocate Doug Turnbull of Newcastle University, U.K., praised this latest advance in an e-mail as “elegant and exciting,” he cautioned that the technique “may be of limited value for those women whose oocytes have either large amounts of mutated mitochondrial DNA or all mutated mitochondrial DNA.”

Some dispute the notion that alteration of the mitochondrial genome is a “genetic modification”, but frankly I think that kind of argument is more political that scientific. I don’t buy it. We would be talking about genetically modified human beings whether with 3-person IVF or the gene editing approach described in this new paper.

For many families facing mitochondrial disease and wanting to have a genetically related baby, a better and far safer approach is preimplantation genetic diagnosis (PGD), although admittedly that is not an option for 100% of families.

It is also notable that multiple groups and I myself on this blog have called for restrictions on clinical application of gene editing technology in humans.

Clearly there is a lot more to learn. Great care should be exercised in discussion of potential human clinical applications of human germline modifications from this kind of research.

From Nature News, it seems that despite ethical concerns, researchers want to forge ahead into human embryos and ultimately the clinic with gene editing:

“Nevertheless, Ocampo and Izpisua Belmonte say that they are in the process of acquiring discarded human eggs and embryos from a fertility clinic in California, and waiting for approval from an ethics board. They plan to develop a line of stem cells from these modified cells, but say that they will not implant embryos into mothers or allow them to grow.”

Are we really ready?