Correction of a pathogenic gene mutation in human embryos.

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

Germline transmission of donor, maternal and paternal mtDNA in primates.

STUDY QUESTION: What are the long-term developmental, reproductive and genetic consequences of mitochondrial replacement therapy (MRT) in primates? SUMMARY ANSWER: Longitudinal investigation of MRT rhesus macaques (Macaca mulatta) generated with donor mtDNA that is exceedingly distant from the original maternal counterpart suggest that their growth, general health and fertility is unremarkable and similar to controls. WHAT IS KNOWN ALREADY: Mitochondrial gene mutations contribute to a diverse range of incurable human disorders. MRT via spindle transfer in oocytes was developed and proposed to prevent transmission of pathogenic mtDNA mutations from mothers to children. STUDY DESIGN, SIZE, DURATION: The study provides longitudinal studies on general health, fertility as well as transmission and segregation of parental mtDNA haplotypes to various tissues and organs in five adult MRT rhesus macaques and their offspring. PARTICIPANTS/MATERIALS, SETTING, METHODS: MRT was achieved by spindle transfer between metaphase II oocytes from genetically divergent rhesus macaque populations. After fertilization of oocytes with sperm, heteroplasmic zygotes contained an unequal mixture of three parental genomes, i.e. donor (≥97%), maternal (≤3%), and paternal (≤0.1%) mitochondrial (mt)DNA. MRT monkeys were grown to adulthood and their development and general health was regularly monitored. Reproductive fitness of male and female MRT macaques was evaluated by time-mated breeding and production of live offspring. The relative contribution of donor, maternal, and paternal mtDNA was measured by whole mitochondrial genome sequencing in all organs and tissues of MRT animals and their offspring. MAIN RESULTS AND THE ROLE OF CHANCE: Both male and female MRT rhesus macaques containing unequal mixture of three parental genomes, i.e. donor (≥97%), maternal (≤3%), and paternal (≤0.1%) mtDNA reached healthy adulthood, were fertile and most animals stably maintained the initial ratio of parental mtDNA heteroplasmy and donor mtDNA was transmitted from females to offspring. However, in one monkey out of four analyzed, initially negligible maternal mtDNA heteroplasmy levels increased substantially up to 17% in selected internal tissues and organs. In addition, two monkeys showed paternal mtDNA contribution up to 33% in selected internal tissues and organs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Conclusions in this study were made on a relatively low number of MRT monkeys, and on only one F1 (first generation) female. In addition, monkey MRT involved two wildtype mtDNA haplotypes, but not disease-relevant variants. Clinical trials on children born after MRT will be required to fully determine safety and efficacy of MRT for humans. WIDER IMPLICATIONS OF THE FINDINGS: Our data show that MRT is compatible with normal postnatal development including overall health and reproductive fitness in nonhuman primates without any detected adverse effects. 'Mismatched' donor mtDNA in MRT animals even from the genetically distant mtDNA haplotypes did not cause secondary mitochondrial dysfunction. However, carry-over maternal or paternal mtDNA contributions increased substantially in selected internal tissues / organs of some MRT animals implying the possibility of mtDNA mutation recurrence. STUDY FUNDING/COMPETING INTEREST(S): This work has been funded by the grants from the Burroughs Wellcome Fund, the National Institutes of Health (RO1AG062459 and P51 OD011092), National Research Foundation of Korea (2018R1D1A1B07043216) and Oregon Health & Science University institutional funds. The authors declare no competing interests.

Reduced anti-Müllerian hormone action in cumulus-oocyte complexes is beneficial for oocyte maturation without affecting oocyte competence.

Anti-Müllerian hormone (AMH) is a key paracrine/autocrine factor regulating folliculogenesis in the postnatal ovary. As antral follicles mature to the preovulatory stage, AMH production tends to be limited to cumulus cells. Therefore, the present study investigated the role of cumulus cell-derived AMH in supporting maturation and competence of the enclosed oocyte. Cumulus-oocyte complexes (COCs) were isolated from antral follicles of rhesus macaque ovaries for in vitro maturation with or without AMH depletion. Oocyte meiotic status and embryo cleavage after in vitro fertilization were assessed. In vitro maturation with AMH depletion was also performed using COCs from antral follicles of human ovarian tissue. Oocyte maturation and morphology were evaluated. The direct AMH action on mural granulosa cells of the preovulatory follicle was further assessed using human granulosa cells cultured with or without AMH supplementation. More macaque COCs produced metaphase II oocytes with AMH depletion than those of the control culture. However, preimplantation embryonic development after in vitro fertilization was comparable between oocytes derived from COCs cultured with AMH depletion and controls. Oocytes resumed meiosis in human COCs cultured with AMH depletion and exhibited a typical spindle structure. The confluency and cell number decreased in granulosa cells cultured with AMH supplementation relative to the control culture. AMH treatment did not induce cell death in cultured human granulosa cells. Data suggest that reduced AMH action in COCs could be beneficial for oocyte maturation. Cumulus cell-derived AMH is not essential for supporting oocyte competence or mural granulosa cell viability.

Molecular basis for maternal inheritance of human mitochondrial DNA.

Uniparental inheritance of mitochondrial DNA (mtDNA) is an evolutionary trait found in nearly all eukaryotes. In many species, including humans, the sperm mitochondria are introduced to the oocyte during fertilization1,2. The mechanisms hypothesized to prevent paternal mtDNA transmission include ubiquitination of the sperm mitochondria and mitophagy3,4. However, the causative mechanisms of paternal mtDNA elimination have not been defined5,6. We found that mitochondria in human spermatozoa are devoid of intact mtDNA and lack mitochondrial transcription factor A (TFAM)-the major nucleoid protein required to protect, maintain and transcribe mtDNA. During spermatogenesis, sperm cells express an isoform of TFAM, which retains the mitochondrial presequence, ordinarily removed upon mitochondrial import. Phosphorylation of this presequence prevents mitochondrial import and directs TFAM to the spermatozoon nucleus. TFAM relocalization from the mitochondria of spermatogonia to the spermatozoa nucleus directly correlates with the elimination of mtDNA, thereby explaining maternal inheritance in this species.

Limitations of gene editing assessments in human preimplantation embryos.

Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.

Haploidy in somatic cells is induced by mature oocytes in mice.

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.

Horizontal mtDNA transfer between cells is common during mouse development.

Cells transmit their genomes vertically to daughter cells during cell divisions. Here, we demonstrate the occurrence and extent of horizontal mitochondrial (mt)DNA acquisition between cells that are not in a parent-offspring relationship. Extensive single-cell sequencing from various tissues and organs of adult chimeric mice composed of cells carrying distinct mtDNA haplotypes showed that a substantial fraction of individual cardiomyocytes, neurons, glia, intestinal, and spleen cells captured donor mtDNA at high levels. In addition, chimeras composed of cells with wild-type and mutant mtDNA exhibited increased trafficking of wild-type mtDNA to mutant cells, suggesting that horizontal mtDNA transfer may be a compensatory mechanism to restore compromised mitochondrial function. These findings establish the groundwork for further investigations to identify mtDNA donor cells and mechanisms of transfer that could be critical to the development of novel gene therapies.

Germline and somatic mtDNA mutations in mouse aging.

The accumulation of acquired mitochondrial genome (mtDNA) mutations with aging in somatic cells has been implicated in mitochondrial dysfunction and linked to age-onset diseases in humans. Here, we asked if somatic mtDNA mutations are also associated with aging in the mouse. MtDNA integrity in multiple organs and tissues in young and old (2-34 months) wild type (wt) mice was investigated by whole genome sequencing. Remarkably, no acquired somatic mutations were detected in tested tissues. However, we identified several non-synonymous germline mtDNA variants whose heteroplasmy levels (ratio of normal to mutant mtDNA) increased significantly with aging suggesting clonal expansion of inherited mtDNA mutations. Polg mutator mice, a model for premature aging, exhibited both germline and somatic mtDNA mutations whose numbers and heteroplasmy levels increased significantly with age implicating involvement in premature aging. Our results suggest that, in contrast to humans, acquired somatic mtDNA mutations do not accompany the aging process in wt mice.

Age-related changes of NADPH-diaphorase-positive neurons in the rat inferior colliculus and auditory cortex.

Nitric oxide (NO) has been implied in age-related changes of the central nervous system (CNS) and the central auditory pathway. The present study was conducted to investigate whether the number of NO-producing cells and their morphometric characteristics in the inferior colliculus (IC) and the auditory cortex (AC) are changed with the increasing age of the subjects. IC and AC sections of adult and senile Wistar rats were studied using the histochemical detection of NADPH-diaphorase activity (NADPH-d), a marker for neurons containing nitric oxide synthase (NOS). Our results showed a decreased area of the somas of NADPH-d-positive neurons in the dorsal cortex (DC) of the IC and a diffuse loss of NADPH-d-positive neurons in the senile IC and primary cortical auditory area (Te1). However, an increased number of NO-producing cells have been shown by other authors in different parts of the ageing auditory pathway and CNS. It seems that age-related changes in NADPH-d-positive cells may follow a region-specific route. These changes may be related to hearing impairments with increasing age.

Functional Human Oocytes Generated by Transfer of Polar Body Genomes.

Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.

Incompatibility between Nuclear and Mitochondrial Genomes Contributes to an Interspecies Reproductive Barrier.

Vertebrate cells carry two different genomes, nuclear (nDNA) and mitochondrial (mtDNA), both encoding proteins involved in oxidative phosphorylation. Because of the extensive interactions, adaptive coevolution of the two genomes must occur to ensure normal mitochondrial function. To investigate whether incompatibilities between these two genomes could contribute to interspecies reproductive barriers, we performed reciprocal mtDNA replacement (MR) in zygotes between widely divergent Mus m. domesticus (B6) and conplastic Mus m. musculus (PWD) mice. Transfer of MR1 cybrid embryos (B6nDNA-PWDmtDNA) supported normal development of F1 offspring with reduced male fertility but unaffected reproductive fitness in females. Furthermore, donor PWD mtDNA was faithfully transmitted through the germline into F2 and F3 generations. In contrast, reciprocal MR2 (PWDnDNA-B6mtDNA) produced high embryonic loss and stillborn rates, suggesting an association between mitochondrial function and infertility. These results strongly suggest that functional incompatibility between nuclear and mitochondrial genomes contributes to interspecies reproductive isolation in mammals.