Mitochondrial replacement therapies can circumvent mtDNA based disease transmission

Abstract

Mitochondrial DNA diseases are relatively common, sometimes devastating and transmitted exclusively through the egg to children of carrier mothers. The study by Wang et al. (2014) adds the exciting possibility of a new therapy for preventing mitochondrial disease transmission predicated on the use of polar body genomes in mice.

Mitochondrial DNA diseases are relatively common, sometimes devastating and transmitted exclusively through the egg to children of carrier mothers. The study by Wang et al. (2014) adds the exciting possibility of a new therapy for preventing mitochondrial disease transmission predicated on the use of polar body genomesin mice.

Mitochondria are energy generators, present in thousands of copies per cell, and inherited maternally. They contain their own small genome in a circular chromosome that is uniquely susceptible to pathologic mutations. Diagnosis of mitochondrial DNA (mtDNA) disease is often difficult given the potential involvement of multiple tissues, organs, or systems. In extreme cases of homoplastic mutations when all mtDNA is mutated, e.g., Leigh’s Syndrome, catastrophic outcomes can occur in affected children. The pervasive nature of the disease translates into a paucity of specific clinical therapies for the affected patient. However, several approaches to prevent second generation transmission of mitochondrial DNA disease include pronuclear and spindle transfer, both relying on replacement of defective mitochondria with non-pathogenic mtDNA from donor zygotes or oocytes (Wallace and Chalkia, 2013). Spindle transfer involves isolating and transplanting meiotic meta phase chromosomes from a mature oocyte into the cytoplasm of enucleated donor oocyte. During pronuclear transfer, both male and female pronuclei are removed from the one-celled embryo or zygote and transferred to the cytoplasm of donor zygote. Efforts began in the United Kingdom in 2005 with Human Fertilization and Embryology Authority approval of pronuclear transfer experiment ationusing clinically discarded zygotes.

Subsequently, pronuclear transfer was reported with minimal (<2%) co-transfer of mtDNA (Craven et al., 2010). An alternative approach involving the use of spindle transfer has since been championed because it involves manipulation of oocytes, not zygotes, with efficacy and safety demonstrated in monkeys by live births, normal growth curves and maintained low levels of carryover mtDNA from spindle donors (Lee et al., 2012; Tachibana et al., 2009). Additionally, spindle transfer in human oocytes from donors with different mtDNA genotypes supported fertilization, embryo development, and stem cell isolation, all with low mitochondrial carryover (Tachibana et al., 2013).

In a recent Cell paper, Wang et al. directly compare pronuclear and spindle transfer outcomes in mice and demonstrate that the latter is associated with a significant mtDNA carryover (over 20%) in live offspring (2014). The authors further explored whether polar bodies extruded during meiosis could be used in the context of mtDNA replacement (Figure 1A). Wang et al. reasoned that the genetic material present in these polar bodies could be recovered if these structures were harvested in a timely manner and transferred into enucleated oocytes or zygotes. Their initial focus was on the first polar body that was used to replace a sister spindle in metaphase II oocytes (Figure 1B). Polar bodies were transferred into enucleated oocytes from a mouse strain carrying different mtDNA haplotype allowing quantitation of donor versus host mtDNA. In efforts to double the yield of reconstructed oocytes, the authors carried out spindle and polar body transfer from the same oocyte. Manipulated oocytes were fertilized and mtDNA presence was subsequently quantitated in embryos and/or pups. Starting with a relatively low number of mitochondria in the first polar body culminated in near complete mitochondrial replacement in offspring, there by demonstrating the desirable outcome of circumventing co-transfer of potentially pathogenic mtDNA.

Figure 1. Recycling genetic material from polar bodies in mice.

(A)A schematic drawing of first (PB1) and second polar (PB2) body extrusion during meiosis. The fully grown, diploid oocyte undergoes two reductive divisions during meiosis in the process of producing one mature, haploid oocyte. Nuclear DNA is first duplicated and divided into two sister chromatids for each pair of parental homologous chromosomes. Homologous chromosomes pair up and may exchange genetic material by recombination. The first reductive division is accompanied by segregation of two chromosomal complements, one of which is eliminated from the egg in the first polar body during asymmetric division of the cytoplasm. In the second reductive division, meiosis initially arrests at metaphase II (MII) until the oocyte is activated by sperm entrance. During this division, each sister chromatid splits into two with one being extruded into a second polar body. The female haploid complement remaining in the egg and the entered sperm form two pronuclei that migrate centrally and fuse to set the stage for the first mitotic division.

(B) First polar body recovery and transfer into an enucleated MII oocyte.

(C)Second polar body recovery and transfer into a zygote from which the female pronucleus has been removed.

The other approach tested by Wang et al. involved transfer of the haploid chromosomal complement from the second polar body (Figure 1C). In this case, second polar bodies were recovered from fertilized oocytes and fused with recipient zygotes from which the female pronucleus had been removed. Again, the resultant embryos and/or pups were relatively free from carryover of polar body mitochondria. Wang et al. further studied mtDNA segregation in the second generation and reported that F2 offspring from mice produced by first polar body transfer were all homoplasmic with no detectable mtDNA carryover. However, heteroplasmy was observed in the second generation offspring derived by second polar body transfer, spindle transfer, and pronuclear transfer. While the authors acknowledge that first and second polar bodies in the mouse have been used to produce normal offspring, here they demonstrate for the first time that polar body genomes are capable of supporting normal development in the mouse and that their recovery and transfer into enucleated recipient oocytes or zygotes can achieve mitochondrial replacement.

Spindle transfer as employed in the Wang et al. study, and as previously described in monkeys and humans, has gained credit ability for circumventing mtDNA-based disease (Wallace and Chalkia, 2013). Although reported mtDNA carryover levels were slightly higher than those for polar body transfer, they remained below the threshold for clinical significance (Samuels et al., 2013). Mitochondrial replacement approaches as described here in might next be evaluated in mouse models carrying pathogenic mtDNA mutations as a prelude to human studies (Ross et al., 2013). Importantly, first polar body transfer coupled with spindle transfer carries the intriguing possibility of increasing the yield of reconstructed oocytes. In contrast, second polar body transfer is complicated by the need to identify pronuclear gender, which is relatively easy in mice though more complicated in primates.

The ultimate objective of this body of work is to initiate human clinical trials, in carrier women with a prior affected child, in efforts to prevent disease transmission in subsequent children. Since mitochondrial replacement involves germline gene therapy, however, such approaches are currently restricted. Regulatory agencies in the United States and United Kingdom are evaluating safety and efficacy issues based on animal model studies such as Wang et al, 2014 and those described herein (Callaway, 2014); however, it remains to be seen when authorization will be forthcoming and whether polar body transfer will be included as an approved approach.