Towards germline gene therapy of inherited mitochondrial diseases

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Abstract

Mutations in mitochondrial DNA (mtDNA) are associated with severe human diseases and are maternally inherited through the egg’s cytoplasm. Here we investigated the feasibility of mtDNA replacement in human oocytes by spindle transfer (ST; also called spindle–chromosomal complex transfer). Of 106 human oocytes donated for research, 65 were subjected to reciprocal ST and 33 served as controls. Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA. The mtDNA can be efficiently replaced in human oocytes. Although some ST oocytes displayed abnormal fertilization, remaining embryos were capable of developing to blastocysts and producing embryonic stem cells similar to controls.

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Figure 1: Experimental design and main outcomes after ST with human oocytes.
Figure 2: Abnormal pronuclear formation and spindle morphology in human ST zygotes.
Figure 3: Genetic analysis of ESCs derived from human ST embryos.

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Acknowledgements

The authors would like to acknowledge the Oregon Health & Science University (OHSU) Embryonic Stem Cell Research Oversight Committee and the Institutional Review Board for providing oversight and guidance. We thank oocyte and sperm donors and staff at the Women’s Health Research Unit at the Center for Women’s Health, University Fertility Consultants and Reproductive Endocrinology & Infertility Division at the Department of Obstetrics & Gynecology of Oregon Health & Science University for their support and procurement of human gametes. The Division of Animal Resources, Surgery Team, Assisted Reproductive Technology & Embryonic Stem Cell Core, Endocrine Technology Core, Imaging & Morphology Core, Flow Cytometry Core and Molecular & Cellular Biology Core at the Oregon National Primate Research Center provided expertise and services for the nonhuman primate research. Hamilton Thorne Inc., donated an XYClone laser system for this study. We are grateful to W. Sanger and D. Zaleski for karyotyping services, C. Penedo for microsatellite analysis and J. Hennebold for consulting on metabolic assays. We are also indebted to A. Steele, R. Cervera Juanes and E. Wolff for their technical support. The human oocyte/embryo research was supported by grants from the OHSU Center for Women’s Health Circle of Giving and other OHSU institutional funds, as well as the Leducq Foundation. The nonhuman primate study was supported by grants from the National Institutes of Health HD063276, HD057121, HD059946, EY021214 and 8P51OD011092.

Author information

M.T., P.A., J.J. and S.M. conceived the study, designed experiments and wrote institutional review board protocols. P.A., M.S. and N.M.G. coordinated recruitment of participants. P.A., K.M., D.B., D.L., D.W. and P.P. performed ovarian stimulation and oocyte recovery. M.T. conducted ST micromanipulations. M.S., K.M. and S.M. performed ICSI. M.T., M.S., J.W., D.M.S., N.M.G., R.T.-H. and E.K. conducted ESC derivation and characterization. S.G. analysed teratoma tumours. M.T., H.M. and D.M.S. performed DNA/RNA isolations, metabolic and mtDNA analyses. C.R., M.T., M.S.,H.-S.L., R.S. and S.M. conducted monkey studies. M.T., R.S., J.J., P.P. and S.M. analysed data and wrote the paper.

Correspondence to Shoukhrat Mitalipov.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-10, Supplementary Tables 1-7, Supplementary Methods and Supplementary References. (PDF 1329 kb)

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Tachibana, M., Amato, P., Sparman, M. et al. Towards germline gene therapy of inherited mitochondrial diseases. Nature 493, 627–631 (2013) doi:10.1038/nature11647

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