Emerging Technologies: Genetic Interventions in the Human Germ Line: Mitochondrial Replacement and Gene Editing

Mitochondrial replacement is a heritable genetic change that can prevent mitochondrial disease but is not involve an alteration of the actual DNA sequence and is thus distinct from gene editing.
Gene editing may provide a path to prevent the transmission of heritable mutations in the nuclear genome.
Novel powerful tools to edit genes continue to emerge, but most have not been tested extensively in human embryos and may result in outcomes other than the intended correction.

Introduction

The genomes received at conception determine much of our health starting at birth and continuing through to adulthood. The brief interval from oocyte retrieval, in vitro fertilization, and culture to the blastocyst stage on day 6 of development, provides—in principle—an extraordinary window for therapeutic intervention. Prevention of disease at the earliest stage possible is meaningful before cell divisions amplify the genetic defect to billions of cells. Genetic defects can result in irreversible damage to the embryo and developing fetus that drug or gene therapies in utero or after birth may not be fully able to correct. Even if interventions in the soma do have a therapeutic benefit, they would not solve the issue of transmission of a mutation to the next generation through the germ line. Preventing transmission of disease-causing mutations is, in principle, like removing errors before a book goes to print, avoiding the need of sending a corrigendum or addendum to all buyers. However, while methods to select embryos based on genotype and discard those with mutations have been available for decades, technologies to correct the genome are still new and emerging, and their consequences are not well understood. More broadly, the consequences of introducing an entirely novel tool into medicine that can be repurposed must be considered. Of particular concern is the potential for nontherapeutic use, or enhancement, which is—by design—aligned to a specific interest depending on the circumstances. This is particularly problematic as the genetic makeup of the child will be decided by, and thus will be aligned with the interests of someone other that the child. Human reproduction’s sole and unbiased agenda is the propagation and survival of the species, which includes the introduction of genetic diversity. The inability to intervene thus has a function: it ensures autonomy of the child, and through the stochastic nature of genetic inheritance, ensures a tremendous degree of diversity, as well as surprise and serendipity in human reproduction. But it also comes with a high cost in some: genetic disease. How governments can pass regulations that can enable genetic intervention in the embryo to prevent disease while ensuring that other important functions of reproduction remain uncompromised is not currently known, and thus there are biological, technical, and regulatory questions that must be addressed before novel genetic technologies can be applied in human reproduction. The medical risks, as well as the risks of insufficient or ineffective regulation and enforcement, need to be weighed against other approaches, including preimplantation genetic testing, embryo selection, and somatic gene therapies.

Mitochondrial Replacement

Inheritance of Mitochondrial Disease

Human mitochondria are organelles that produce adenosine triphosphate (ATP) through oxidative respiration. ATP provides energy that cells require to drive enzymatic reactions, build nucleic acids and proteins, and transduce signals in brain cells and signals that allow all cells to respond to specific cues. Mitochondria have their own genome of only 16,569 base pairs, including 14 protein-coding genes and 24 noncoding RNA genes required for mitochondrial protein synthesis. Natural mutations have been documented in 13 of the 14 protein-coding genes, and all noncoding RNA genes. Organs that are most affected include those with high energy needs, in particular the brain and heart. Symptoms of mutations to mitochondrial genes can range widely, from a lethal disorder to a relatively mild impairment and exercise intolerance. Mitochondrial disorders are difficult to treat and have no cure. This is because there is no substitute for the ATP a cell requires, which is made autonomously in each cell of the body. Mitochondrial diseases exemplify why amplification of a genetic defect during development results in an intractable therapeutic problem after birth. This is why effective prevention of mitochondrial disease is a priority ( ).

Mitochondria are located in the cytoplasm, and thus their inheritance from one cell to another also occurs through the cytoplasm. The cytoplasm is where there is one of the greatest asymmetries in human reproduction: the oocyte, a cell of more than 100 micrometers in diameter, is mostly composed of cytoplasm and contains hundreds of thousands of mitochondrial DNA copies. The sperm is small, tailored to transport the paternal genome to the oocyte, and has been stripped from essentially all cytoplasm during maturation. Only a tiny number of mitochondria are left to help drive sperm tail movement, but these mitochondria are lost at, or just after, fertilization ( ). Even when fertilization is performed through intracytoplasmic sperm injection, sperm mitochondria are lost or degraded ( ). The inheritance of mitochondria from one generation to the next is thus maternal, with a few notable exceptions, which show a mixture of maternal and paternal contribution ( ). This is why fathers with mitochondrial disease do not transmit them to their offspring. When mothers with mitochondrial DNA mutations have children, they are at risk of having a child with the disease. Because there are many copies of mitochondrial genomes, usually mutant and normal copies co-exist as a mixture. This mixture can change in composition from one cell division to the next. Major shifts in the relative proportion of mutant and normal mitochondrial genomes are observed, especially during germ cell development, such that oocytes can have little or no mutant mitochondrial DNA copies, or very high proportions of them. This form of inheritance is fundamentally different from Mendelian inheritance of nuclear genes. Of our >20,000 genes, most reside in the nucleus and are present in two copies: one maternal and one paternal. Their inheritance is according to predictable patterns governed by meiotic recombination and segregation and allelic ratios almost always remain constant after fertilization. The non-Mendelian inheritance of mitochondria brings unique challenges to disease prevention, and a limited predictive value of genotyping, as the proportion of mutant mitochondrial DNA can change in different tissues, and the tissue used for genotyping does not necessarily reflect another. Despite these challenges, when embryos with very low levels of mutant mitochondrial DNA are selected for implantation, a child with a low mutation load compatible with normal health can be the outcome ( ). Through selection, most of the embryos are lost. In this particular case report, only 2 in 19 embryos met the criteria of less than 18% mutant mitochondrial genomes in total. For some couples, there will be no viable embryos that can be transferred. These are the cases where genetic interventions are being considered to help the couple have a healthy genetically related child.

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