Generation of mitochondrial replacement monkeys by female pronucleus transfer-Chinese Academy of Sciences

INTRODUCTION

This study successfully generated mitochondrial replacement cynomolgus macaques using female pronucleus transfer (FPNT), a technique that reduces cytoplasmic carryover. The monkeys, born healthy and surviving for over two years, exhibit a unique genetic lineage from three parental genomes and minimal maternal mtDNA carryover (3.8%–6.7%). This research provides a valuable non-human primate model for evaluating mitochondrial replacement therapy (MRT), offering insights into improving its efficiency and safety for human applications.

MATERIALS AND METHODS

Monkey FPNT

In the FPNT procedure, oocytes from two different monkeys were divided into two groups. MII oocytes from a donor monkey were placed in manipulation drops containing HEPES-buffered Tyrode’s lactate medium (TH3) with 5 μg/mL cytochalasin B (CB, Absin, China) in a glass bottom dish to rapidly perform spindle-chromosome complex (SCC) removal using a piezo-driven pipette under polarized light imaging (Oosight Imaging System, Cri, Hamilton Thorne, USA). Sperm were then injected into the enucleated oocytes 1 h later. Concurrently, oocytes from another donor monkey underwent parthenogenetic activation in 5 μmol/L ionomycin (Sigma, USA) for 5 min and were then transferred to 7.5 μg/mL cycloheximide (Sigma, USA) medium for 5–6 h (I/CHX). After this, successful oocyte activation in each group was confirmed by the presence of a single pronucleus. 5–10 I/CHX-activated embryos were then transferred to manipulation drops containing TH3 with 5 μg/mL CB and 5 μg/mL nocodazole (MedChemExpress, USA). A laser objective (Hamilton Thorne, USA) was used to create a hole in the zona pellucida of the embryos, allowing for the precise extraction of the female pronucleus and surrounding cytoplasm using a pipette with a 15–18 μm outer diameter (Supplementary Movie S1). After brief incubation with hemagglutinating virus of Japan envelope (HVJ-E) medium (Cosmo Bio, Japan) at 37°C for 10 s, the female pronucleus were introduced into the zona pellucida space of sperm-activated embryos (Supplementary Movie S2). This process resulted in the successful creation of a reconstructed embryo harboring genetic material from three distinct genomes. All embryos were cultured in HECM-9 (H9) medium at 37°C under 5% CO₂. 

RESULTS

Isolated single pronucleus carried less mitochondria
This study explored a single pronucleus transfer strategy to reduce cytoplasmic carryover in mitochondrial replacement therapy (MRT). Mitochondrial distribution in parthenogenetically activated monkey embryos showed uniform cytoplasmic distribution of mitochondria. Digital PCR analysis revealed that the mtDNA copy number in a single pronucleus was significantly lower than in double pronuclei or whole oocytes. These results suggest that single pronucleus transfer could reduce mitochondrial carryover compared to traditional biparental pronucleus transfer, making it a promising approach for MRT.

Generation of mitochondrial replacement monkeys by FPNT

In this study, MII oocytes were divided into two groups: one for cytoplasm donor oocytes and the other for female pronucleus (FPN) donor oocytes, sourced from different macaques. In the cytoplasm donor group, SCC was removed from oocytes, and sperm was injected into the enucleated oocytes. In the FPN donor group, oocytes were activated using I/CHX. Five hours post-activation, embryos from both groups had a single pronucleus: male pronucleus (MPN) in the cytoplasm donor group and FPN in the FPN donor group. The FPN was then extracted and transferred into the cytoplasm donor embryo after fusion with HVJ-E, resulting in successful FPNT embryo reconstruction after 1 hour.

Using the described approach, 35 reconstructed FPNT embryos were obtained from 125 MII mature oocytes and transferred into 15 macaque surrogates. Pregnancy was confirmed in three surrogates after about four weeks, resulting in a pregnancy rate of 20%, with one surrogate carrying twins. This is similar to the pregnancy rate seen with normal ICSI embryos (28.95%). Four healthy infants were delivered via caesarean section at full-term, and all have survived for over two years.

Genetic origin of FPNT monkeys

To determine the genetic lineage of the FPNT-derived monkeys, genomic DNA was extracted from blood cells, and mtDNA and nuclear DNA origins were identified using STR and SNP analyses. STR analysis of 27 loci confirmed that all monkeys inherited their maternal genome from the FPN donor and paternal genome from the sperm donor. SNP analysis of the mitochondrial D-loop regions and ND5 gene showed that the mtDNA of the FPNT-derived monkeys predominantly came from the cytoplasm donor monkeys.

Postnatal development of FPNT-derived monkeys

Although PNT and early PNT (ePNT) have been performed in human embryos, concerns remain about potential damage that could lead to later developmental disorders. To address this, a two-year follow-up was conducted on four FPNT-produced monkeys. Their health was closely monitored with physical exams every two months to track body weight, head circumference, and body length. Compared to age-matched monkeys conceived via ICSI, the FPNT-derived monkeys showed normal postnatal growth. Viral testing for colony-excluded viruses was negative for all four monkeys, and none showed any serious health issues during development.

MtDNA heteroplasmy dynamics in FPNT-derived monkeys

Similar to the detection of mtDNA copies in pronuclei, a small but measurable fraction of cytoplasm from the FPN donor oocytes is inevitably transferred along with the FPN, resulting in residual FPN donor mtDNA in the FPNT monkeys. Pyrosequencing analysis of blood cells from the four FPNT macaque infants at 3 months post-birth revealed that while the monkeys predominantly carried mtDNA from the cytoplasm donor, a small proportion (3.8%–6.7%) of mtDNA from the FPN donor was also present.

In addition to monitoring health and growth, a two-year follow-up was conducted to assess mtDNA carryover dynamics. Pyrosequencing at 3, 6, 18, and 24 months showed that mtDNA carryover from the FPN donor remained relatively stable during development. Among the four macaques, only FPNT-1 showed an increase in mtDNA carryover as it matured, while the other three monkeys exhibited a decline in initially elevated mtDNA levels.

DISCUSSION
This study is the first to successfully generate healthy mitochondrial replacement non-human primates (NHPs) using female pronucleus transfer (FPNT) into androgenetic embryos. While PNT has been used in human embryos and mouse models, ethical and safety concerns have limited its application in humans to generating ESC lines rather than for embryo transfer. Mouse models produced by traditional PNT show high mtDNA carryover levels (23.7%±11.1% or 5%–44%), which can undermine the effectiveness of preventing mitochondrial disease transmission. In contrast, the FPNT strategy used in this study resulted in lower mtDNA carryover and eliminated the ambiguity of differentiating between male and female pronuclei.

Using FPNT, the researchers successfully produced four healthy mitochondrial replacement cynomolgus monkeys from 35 reconstructed embryos, resulting in a birth rate of 11.43%. These monkeys survived beyond two years and exhibited stable mtDNA carryover levels during postnatal development.

Link:Generation of mitochondrial replacement monkeys by female pronucleus transfer