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Reduction of mtDNA heteroplasmy in mitochondrial replacement therapy by inducing forced mitophagy

Abstract

Mitochondrial replacement therapy (MRT) has been used to prevent maternal transmission of disease-causing mutations in mitochondrial DNA (mtDNA). However, because MRT requires nuclear transfer, it carries the risk of mtDNA carryover and hence of the reversion of mtDNA to pathogenic levels owing to selective replication and genetic drift. Here we show in HeLa cells, mouse embryos and human embryos that mtDNA heteroplasmy can be reduced by pre-labelling the mitochondrial outer membrane of a donor zygote via microinjection with an mRNA coding for a transmembrane peptide fused to an autophagy receptor, to induce the degradation of the labelled mitochondria via forced mitophagy. Forced mitophagy reduced mtDNA carryover in newly reconstructed embryos after MRT, and had negligible effects on the growth curve, reproduction, exercise capacity and other behavioural characteristics of the offspring mice. The induction of forced mitophagy to degrade undesired donor mtDNA may increase the clinical feasibility of MRT and could be extended to other nuclear transfer techniques.

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Fig. 1: Design of forced mitophagy.
Fig. 2: CISD1-Binding induces forced mitophagy in HeLa cells.
Fig. 3: Forced mitophagy degrades C57/6j mtDNA in BALB/c embryos.
Fig. 4: Forced mitophagy is efficient for clearing mtDNA carryover in pronuclear transfer for MRT in mice.
Fig. 5: CISD1-Binding-mediated MRT has little effect on the reproduction, growth curves and behaviour of offspring.
Fig. 6: Forced mitophagy is efficient and safe for the elimination of external mtDNA in human embryos.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. Source data for the figures are provided with this paper. Other raw data generated during the study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank those who donated gametes for this study, and R. Chen (South China Normal University, Guangzhou, China) and H. Schatten (University of Missouri-Columbia) for editing and proofreading the manuscript. This study was supported by the National Key Research and Development Program of China (2018YFC1004800), the National Natural Science Foundation of China (82071714, 81971357) and the Key-Area Research and Development Program of Guangdong Province (2019B030335001).

Author information

Authors and Affiliations

Authors

Contributions

S.-M.L. and X.-Y.F. conceived the idea. S.-M.L., X.-H.O., Q.-Y.S., X.-Y.F., L.G., L.-N.C., S.Y., C.H., L.Z., J.-Y.M., S.L., T.J., M.-X.J., W.S., Z.-J.G., Z.-B.W. and M.C. designed experiments and interpreted the results. X.-Y.F., S.-M.L., L.G., J.W., X.-H.S., F.W., C.-F.Z. and X.-H.W. carried out the experiments. S.-M.L., S.Y., X.-Y.F., C.H. and Q.-Y.S. wrote the manuscript.

Corresponding authors

Correspondence to Qing-Yuan Sun, Xiang-Hong Ou or Shi-Ming Luo.

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Nature Biomedical Engineering thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 CISD1-RFP-SQSTM1 induces forced mitophagy and reduces the mitochondrial number.

a. HeLa cells were stably transfected with GFP-LC3 (green) and stained with MitoTracker Deep Red FM (grey) to label mitochondria. CISD1-RFP-SQSTM1 (red) was transiently transfected into the HeLa cells. Images were obtained 60 hours after CISD1-RFP-SQSTM1 transfection. The insets indicate that CISD1-RFP-SQSTM1-labeled mitochondria co-localizes with GFP-LC3. b. CISD1-RFP-SQSTM1-labelled mitochondria fuse with lysosomes. HeLa cells were stably transfected with pmEmerald-Mito (grey) to label mitochondria, transiently transfected with CISD1-RFP-SQSTM1 (red), and stained with LysoTracker Blue DND-22 (green) to label lysosomes. Images were obtained 48 hours after CISD1-RFP-SQSTM1 transfection. The insets indicate that CISD1-RFP-SQSTM1-labeled mitochondria fuse with lysosomes. c. The CISD1-RFP-SQSTM1-expressed cells have fewer mitochondria than the unexpressed cells. The stably pmEmerald-Mito (green)-transfected HeLa cells were transiently transfected with CISD1-RFP-SQSTM1 (red) and stained with Hoechst 33342. Images were obtained24 and 48 hours after CISD1-RFP-SQSTM1 transfection. White arrowheads indicate that the CISD1-RFP-SQSTM1-expression cells decrease the pmEmerald-Mito fluorescence signals. In ac, scale bars, 30 μm, and three independently repeated experiments with similar results. d. The fluorescence analysis of mitochondria in the CISD1-RFP-SQSTM1-expressed and control cells at different time points. Data were analyzed by two-tailed unpaired t-test and expressed as average ± SEM, and each point represents one cell from 3 biological replicates. For the 24 hours group, n = 106, the control value is 945 ± 84.97, and the CISD1-RFP-SQSTM1 value is 950.8 ± 90.19. For the 48 hours group, n = 67, the control value is 815.9 ± 51.32, and the CISD1-RFP-SQSTM1 value is 455.7 ± 39.86. e. The mtDNA number decreased after CISD1-RFP-SQSTM1 expression. Forty-eight hours after CISD1-RFP-SQSTM1 transfection, a single cell with or without CISD1-RFP-SQSTM1 expression was picked up, and the mtDNA quantity was analyzed by qPCR. Data were analyzed by two-tailed unpaired t-test and expressed as average ± SEM. n = 27 for control, and n = 30 for CISD1-RFP-Binding from 3 biological replicates. The value of control is 2357 ± 104.3, and the value of CISD1-RFP-Binding is 1274 ± 79.14.

Source data

Extended Data Fig. 2 CISD1-Binding expresses immediately and localizes to mitochondria after its mRNA microinjection in mouse oocytes and Zygotes.

The mRNA encoding CISD1-Binding was microinjected into mouse GV oocytes, MII oocytes, and early pronuclear embryos. Images were obtained 1.5 hours after microinjection. Red indicates CISD1-Binding proteins and Green indicates MitoTracker Deep Red FM. Pearson’s correlation coefficient assessed co-localization between the CISD1-Binding and Mitotracker. Data were analyzed from three independently repeated experiments, and each point represents one cell. n = 10 for GV oocytes; n = 11 for MII oocytes and n = 12 for zygotes. The mean ± SEM values are 0.899 ± 0.01286 (GV), 0.9491 ± 0.00667 (MII) and 0.9467 ± 0.0076 (Zygote). Bar = 30 μm.

Source data

Extended Data Fig. 3

CISD1-Binding recruits GFP-LC3 at different mouse-embryo stages. Mouse zygotes were microinjected with the CISD1-Binding (red) and GFP-LC3 (green) mRNA and cultured to 2-cell, 4-cell and 8-cell stages. The insets indicate that CISD1-Binding recruits GFP-LC3. Bar = 30 μm. Also, see Fig. 3a and c. Four independently repeated biological experiments with similar results.

Extended Data Fig. 4 CISD1-Binding-labelled mitochondria begin to fuse with lysosomes after 4-cell stages.

Mouse embryos were microinjected with the CISD1-Binding mRNA at the zygote stage and stained with Blue DND-22 to label lysosomes at 1-cell, 2-cell and 4-cell stages before imaging. The insets indicate that CISD1-Binding-labeled mitochondria fuse with lysosomes (orange only). Bar = 30 μm. Also, see Fig. 3b and e. Three independently repeated biological experiments with similar results.

Extended Data Fig. 5 CISD1-RFP-Binding(mutation)-labelled mitochondria do not recruit LC3 proteins and fuse with lysosomes in mouse embryos.

a. Mouse zygotes were microinjected with the mRNA encoding CISD1-RFP-Binding(mutation) and GFP-LC3. The insets indicate that the CISD1-RFP-Binding(mutation)-labelled mitochondria do not recruit GFP-LC3 in mouse embryos. b. Mouse zygotes were microinjected with the mRNA encoding CISD1-RFP-Binding(mutation) and stained with Blue DND-22 to label lysosomes before imaging. The insets indicate no fusion between the CISD1-RFP-Binding(mutation)-labelled mitochondria and lysosomes in mouse embryos. In a and b, scale bars, 30 μm, and experiments with similar results were biologically repeated twice.

Extended Data Fig. 6 The capacity of early mouse embryos to degrade CISD1-Binding-labelled mitochondria.

a. Mouse embryos were injected with or without (control) CISD1-Binding mRNA at the zygote stage, cultured to different stages, and then the whole embryos were subjected to absolute quantification of the mtDNA. Data were analyzed by two-tailed unpaired t-test and expressed as average ± SEM. Each point represents one embryo from three biological replicates. For Zygote, n = 14, the values are 2.44 ± 0.1618 (control) and 2.485 ± 0.1586 (CISD1-Binding). For 2-Cell, n = 13, the values are 2.543 ± 0.1016 (control) and 2.642 ± 0.1891 (CISD1-Binding). For 4-Cell, n = 12 (control) and n = 11 (CISD1-Binding), the values are 2.529 ± 0.07611 (control) and 2.405 ± 0.1418 (CISD1-Binding). For 8-Cell, n = 11, the values are 3.109 ± 0.1873 (control) and 2.703 ± 0.1292 (CISD1-Binding). For Morula, n = 12, the values are 3.457 ± 0.2498 (control) and 1.466 ± 0.2157 (CISD1-Binding). For Blastocyst group, n = 12, the values are 2.659 ± 0.1224 (control) and 2.143 ± 0.1093 (CISD1-Binding). b. Morula embryos expressed with or without CISD1-Bindingprotein (red) were stained with Sybgreen I (green) for observing mtDNA. The insets indicate that the embryo with CISD1-Binding protein expression has less mtDNA than in the control embryo. Bar = 40 μm. Three biologically repeated experiments with similar results.

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Fan, XY., Guo, L., Chen, LN. et al. Reduction of mtDNA heteroplasmy in mitochondrial replacement therapy by inducing forced mitophagy. Nat. Biomed. Eng 6, 339–350 (2022). https://doi.org/10.1038/s41551-022-00881-7

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