Curing embryos of genetic diseases

The VOICE has on many prior occasions extensively addressed the considerable controversy surrounding “crossing the germline.” This term denotes human interventions into the genome that then will be passed on to future generations. Such interventions are, currently, worldwide restricted by law, regulations and/or ethical considerations.

Some required background

Involving the two genomes every embryo possesses, the nuclear genome (nDNA) representing roughly 99% of total DNA and the mitochondrial genome (mDNA) encompassing only approximately 1% of total DNA, the germline can be crossed in a multitude of ways. Achieving changes in mDNA can have two distinct purposes: One hypothesis proposed for more than a decade, suggests potential fertility benefits from replacing cytoplasm of older women’s eggs with cytoplasm from a young egg donors. As previously discussed in these pages, cytoplasm of eggs contains mitochondria. They are the “batteries” of cells, thus providing the necessary energy to eggs. The hypothesis of cytoplasmic exchange or of mitochondrial exchange suggests that replacing “old” with “young” mitochondria will increase the fertility potential of “older” eggs. So-modified eggs, therefore, would carry 99% of maternal nDNA but instead of 1% of maternal mDNA, they would contain 1% of donor mDNA. If such an egg gets fertilized by sperm, the resulting embryo, therefore, would have 3 genetic parents: the two biological parents would share ca. 99% of nDNA but the offspring’s mDNA would be the donor’s, – therefore the frequently mentioned lay-term in the media, three-parent IVF.

Since every offspring receives his/her mDNA exclusively only from the mother, the donor’s mDNA would be passed on through potential female offspring to all future generations; – i.e. the germline would be crossed.

Distinctively different options of crossing the germline in reference to mitochondria offer themselves, when the purpose of intervention is not exchanging “old” mitochondria but in avoidance of transmission of so called mitochondrial genetic diseases to offspring. As already noted above, mitochondria have their own genome (mDNA), containing only 37 genes (while the nuclear genome is believed to have at least 19,000), which code for 2 rRNAs, 22tRNAs and 13 polypeptides (all subunits of enzyme complexes of the oxidative phosphorylation system). Mitochondrial genetic diseases, therefore, usually affect oxidative phosphorylation, involve multiple organ systems and, ultimately, are terminal at young ages.

Mutations can occur in nDNA and mDNA but are more common in mDNA. Since, as noted before, mDNA always comes from only the mother, diseases caused by an mDNA mutation, therefore, will always be transmitted to offspring only by the mother.

Avoidance of transmission has been a goal for many years. Current clinical practice only allows for either preimplantation genetic diagnosis (PGD), and the discarding of affected embryos, or early pregnancy diagnosis and abortion of affected pregnancies. At least theoretically, affected embryos, however, could be treated in two ways: A first involves the same technique of cytoplasmic or mitochondrial exchange described above. Replacing the total maternal mitochondrial genome (mDNA) with normal donor mDNA, would seemingly remove any disease causing mDNA mutation. Such experiments are in the U.S., however, prohibited, as a recently published “Advisory on Legal Restrictions on the Use of Mitochondrial Replacement Techniques to Introduce Donor Mitochondria into Reproductive Cells Intended for Transfer into a Human Recipient” from the Food and Drug Administration, again reemphasized.

As we previously noted, colleagues in the UK, however, received permission to pursue such trials, and have already published preliminary data, though have not reported yet on transfers of so treated embryos. Such a transfer was, however, as we also extensively discussed in these pages in past months, recently performed on a patient by a New York-based colleague and collaborators resulting in birth of a male infant (Zhang et al., Reprod Biomed Online 2017;34:361-368). This publication has generated considerable controversy, as will be addressed in more detail below.

Assuming that cytoplasmic/mitochondrial exchange works, the mutated mDNA gene would be eliminated. This, however, would occur at the expense of the rest of the maternal mDNA, which in the family forever in future generations would be replaced by the egg donor’s mDNA.

This is why a recent publication by longstanding CHR friend (often mentioned in these pages) Shoukhrat Mitalipov, PhD, from the Center of Embryonic Cell and Gene Therapy at Oregon Health and Science University and collaborators (among those another long-standing CHR friend, Juan Carlos Izpisua Belmonte, PhD, from the Salk Institute for Biological Studies in La Jolla, CA), attracted so much attention from media all around the world with a recent publication in the prestigious science journal Nature, when reporting the first successful use of CRISPR/Cas9 in human embryos in the U.S (Kang et al., Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations. Nature 2017; 540(7632):270-275).

This paper deserved the attention it received because, by injecting CRISPR/Cas9, geared at a single DNA mutation causing a form of hypertrophic cardiomyopathy often resulting in sudden cardiac death in athletes, the investigators were able to eliminate the pathological mutation with greatly improved efficiency over earlier experiments made by Chinese investigators, and with only rare unwanted editing spots at other places of the genome. Though the so obtained embryos were not transferred, this study clearly demonstrates that, in the not too distant future, hopefully avoiding excessive government interventions and prohibitions, CRISPR/Cas9 will find clinical applications in human embryos.

As we previously wrote in these pages, this is a technique that not only lends itself to the pinpoint-targeting of single abnormal DNA mutation but allows for editing of mDNA as well as nDNA mutations. Using this technique mDNA mutations, therefore, could be corrected without need for a third-party oocyte donor who would provide the cytoplasm and, with it, all of her mitochondria. In other words, while British colleagues experiment with the use of plasma/mitochondrial exchange in prevention of transmission of mitochondrial diseases, Kang et al demonstrated already how outdated this technique, likely, soon may be.

Controversies surrounding these treatments of embryos

Crossing the germline has remained a highly controversial issue in professional circles as well as in the lay public and, therefore, presumably in government. The establishment of guidelines and proper conduct of research involving these procedures is, therefore, absolutely essential. CHR recently made this point publically, when criticizing the recent publication by Zhang et al in Reproductive Biomedicine Online (Zhang e al., 2017;34:361-368), in which they reported use of a cytoplasmic/mitochondrial exchange technique in a mother’s embryos, who had previously lost two children to a mitochondrial disease they inherited through her mDNA. Since we discussed this publication in these pages extensively before, we will not be repetitive.

Only so much: As we also previously discussed in the VOICE, their paper was accompanied by an editorial, written by five senior editors of the journal, explaining in detail their internal editorial struggles about whether or not to accept the manuscript for publication (Alikani et al., Reprod Biomed Online 2017;34:333-336). CHR’s writers lauded in their commentary, since published by the journal (Gleicher et al., doi:10.1016/j.rbmo. 2017. 07.006), the openness of the editorial but criticized the editors’ final decision to publish the manuscript for three principal reasons: (i) The Zhang et al manuscript, very obviously, was based on a highly experimental treatment, at least partially illegally administered in the U.S., where such treatments are explicitly prohibited by FDA regulations. (ii) The treatment was, likely, administered without obtaining appropriate informed consent from the parents; and (iii) the treatment was, likely, administered without appropriate Institutional Review Board approval, an absolutely essential step under international publishing guidelines for acceptance of any article for publication in a reputable scientific journal.

The CHR investigators concluded their comments by explaining that they were written “out of concern that opponents of reproductive research will use the irresponsible way in which this research was conducted as example for why all such reproductive research should be prohibited.”

At the time of submission, CHR investigators were, however, unaware that Mina Alikani, PhD, the lead author of the editorial, had a significant undisclosed conflict: Jacques Cohen, PhD, her husband and former Editor-in-Chief of Reproductive Medicine Online, had entered into a business relationship with John Zhang, MD, PhD, in establishing a company (Darwin Life Inc.) that was advertising the use of plasma/ mitochondrial exchange through spindle transfer for older women at the ridiculous price tag of $100,000-120,000 per treatment cycle.

Not knowing this fact at the time, CHR was, therefore, surprised when, with the CHR’s commentary, the journal, published a retort co-signed by Dr. Zhang’s lawyer, James A Boiani, from the law firm of Epstein Becker & Greene PC in Washington, DC, and by Jacques Cohen, PhD, as Emeritus Editor of Reproductive Medicine Online, offering “a point of discussion related to the argument of legality posed by Dr. Gleicher and colleagues.” (Boiani and Cohen, Reprod Biomed Online 2017; doi: 10.1016/j.rbmo.2017. 07.003). They then suggested that “Gleicher and colleague do not provide adequate background information about the definition by the FDA…

On August 4, 2017, the FDA, however, published on its website a cease and desist letter from Mary A. Malarkey, Director, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and Research to John Zhang, PhD, MD, Chief Executive officer, Darwin Life, Inc. and New Hope Fertility Center, fully reaffirming CHR’s interpretation of the current legal status of all plasma/mitochondrial exchange procedures and, likely, all other experimental procedure with potential of crossing the germline.

Where we scientifically, ethically and legally stand today

CHR’s investigators concluded their comments in the Reproductive Biomedicine Online commentary with the following paragraph:

There is, otherwise, only one other lesson to be learned from this case (sic., reported by Zhang et al.), this time relevant to the FDA and other regulatory agencies around the world. What this case demonstrates so well is that if responsible scientists are prohibited from performing responsible research under appropriate guidelines and safeguards, the research will go elsewhere, where there exist neither guidelines, safeguards, nor responsible   researchers.

We fully stand behind this statement and hope that Congress will do so as well. As stated in the letter by the FDA to Dr. Zhang, since December 2015, the FDA has been prohibited by Congress in provisions in annual federal Appropriations Acts from using funds to accept IND submissions for clinical investigations that involve “a human embryo . . . intentionally created or modified to include a heritable genetic modification.”

The letter further stated that “. . . such human subject research cannot legally be performed in the United States. Nor is exportation permitted unless it meets the requirements of an applicable export exemption (sic., in reference to embryo and patient being transported to Mexico for embryo transfer in the reported Zhang et al., case) .  . . . these export exemptions do not apply here because, among other things, export was not to a country described in section 802(b)(1)(A)(i) or (ii).”

As this letter perfectly explains, efforts by Zhang, CHR and others to convince the FDA from reconsidering responsible research of treatments that potentially cross the germline are, a priori, destined to failure because the FDA, under current law, is prohibited by Congress from even considering such discussions with investigators. The impetus for change, therefore, has to come from Congress and/or the Trump administration.

Here is one opportunity for government to do the right thing and quickly, so that responsible investigators in the U.S. know what the guidelines are and, therefore, can follow them in developing potentially revolutionary new treatments to better mankind. If we leave this research to individuals without the necessary moral guidance and personal underpinnings, nothing good will come of it. If Congress, however, reaffirms the FDA’s supervisory power over all responsible medical progress, including processes that may breach the germline, we are convinced that in collaboration with the responsible science community in this country, this kind of research will thrive.

The amazing work of Kang et al in the collaborative effort of the laboratories of Mitalipov and Izpisua-Belmonte are an excellent example of how this kind of research can and should be conducted responsibly. Responsible research in reproductive biology deserves all of our support because it will revolutionize medicine in practically all medical specialty areas, going far beyond the obvious importance this research carries for human fertility.

We are especially pleased that both Prof. Mitalipov and Prof. Izpisua-Belmonte will be speaking at the November Conference in New York City (November 16-19) that CHR is co-sponsoring with The Foundation for Reproductive Medicine. Indeed, Prof. Mitalipov will open the meeting with the “Breaking News Lecture,” dedicated to the recent Nature paper.

And on a side note, if you, a family member or a friend are the known carriers of a specific genetic disease, whether nuclear or mitochondrial, whether you are a female or male, and are interested in contributing your sperm/eggs to responsible approved research, please contact us by calling us at 212-994-4400.

This is a part of the September 2017 CHR VOICE.

Norbert Gleicher, MD, leads CHR’s clinical and research efforts as Medical Director and Chief Scientist. A world-renowned reproductive endocrinologist, Dr. Gleicher has published hundreds of peer-reviewed papers and lectured globally while keeping an active clinical career focused on ovarian aging, immunological issues and other difficult cases of infertility.