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. 2013 Jan 31;493(7434):627-31.
doi: 10.1038/nature11647. Epub 2012 Oct 24.

Towards germline gene therapy of inherited mitochondrial diseases

Masahito Tachibana  1 , Paula Amato, Michelle Sparman, Joy Woodward, Dario Melguizo Sanchis, Hong Ma, Nuria Marti Gutierrez, Rebecca Tippner-Hedges, Eunju Kang, Hyo-Sang Lee, Cathy Ramsey, Keith Masterson, David Battaglia, David Lee, Diana Wu, Jeffrey Jensen, Phillip Patton, Sumita Gokhale, Richard Stouffer, Shoukhrat Mitalipov
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Towards germline gene therapy of inherited mitochondrial diseases

Masahito Tachibana et al. Nature. .
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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.

Figures

Figure 1. Experimental design and main outcomes after ST with human oocytes
a, Oocytes were retrieved from two unrelated donors and spindle-chromosomal complexes were reciprocally exchanged. Reconstructed oocytes were fertilized by ICSI and monitored for in vitro development to blastocysts and ESCs. Images in boxes depict a human mature MII oocyte with the spindle visualized under polarized microscope (left), isolated cytoplast and karyoplast (middle) and intact spindles inside recipient cytoplasts after transfers (right). Original magnifications: left and middle, x200; right, x100. b, Experimental outcomes after ST in human oocytes. The top and bottom graphs represent fertilization, blastocyst and ESC isolation rates for intact control and ST embryos, respectively. No statistical differences were found between ST and controls in survival after ICSI, fertilization, blastocyst development and ESC derivation rates (P>0.05).
Figure 2. Abnormal pronuclear formation and spindle morphology in human ST zygotes
a, Proportion of normally fertilized zygotes with 2 pronuclei and 2 polar bodies (2PN/2PB) vs. abnormal zygotes (3PN/1PB; 3PN/2PB; 1PN; multiple PN and 2-cell) after ST. b, Integrity of meiotic spindles in human ST oocytes depicting normal metaphase II (top), and premature progression to the anaphase II (bottom).
Figure 3. Genetic analysis of ESCs derived from human ST embryos
a, Nuclear DNA origin of hESO-ST2, -ST3 and -ST4 determined by microsatellite parentage analysis. The microsatellite markers for D7S513 and D6S1691 loci demonstrate that the nuclear DNA in these ESC lines was from the egg donor 1 (the spindle donor). b, mtDNA genotyping by direct sequencing show that the mtDNA in hESO-ST2, -ST3 and -ST4 is originated from the egg donor 2.
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Comment in

  • Chromosome biology: mixing it up.
    Jones B. Jones B. Nat Rev Mol Cell Biol. 2012 Dec;13(12):750. doi: 10.1038/nrm3476. Epub 2012 Nov 15. Nat Rev Mol Cell Biol. 2012. PMID: 23151661 No abstract available.

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