April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Mitochondrial DNA integration and replication in transgenic mutant ND4 mice
Author Affiliations & Notes
  • John Guy
    Bascom Palmer Eye Institute, University of Miami, Miami, FL
  • Hong Yu
    Bascom Palmer Eye Institute, University of Miami, Miami, FL
  • Arpit Mehta
    Hussman Institute Human Genomics, Miami, FL
  • Alfred S Lewin
    University of Florida, Gainesville, FL
  • Footnotes
    Commercial Relationships John Guy, None; Hong Yu, None; Arpit Mehta, None; Alfred Lewin, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 6209. doi:
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      John Guy, Hong Yu, Arpit Mehta, Alfred S Lewin; Mitochondrial DNA integration and replication in transgenic mutant ND4 mice. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6209.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: To evaluate integration and replication of the mutant human ND4 in mitochondria following injection of a mitochondrially targeted AAV into the blastocyst of mice. Last year we showed that these transgenic mice develop optic neuropathy that is vertically transmitted from mother to offspring.

Methods: Mutant human G11778A ND4 responsible for most cases of LHON was inserted into a mito-targeted AAV containing the COX8 leader sequence inserted into the VP2 capsid then microinjected into the mouse blastocyst to generate transgenic mutant ND4 mice. Next generation sequencing was performed on mitochondrial DNA extracted from the retina, brain and muscles of these transgenic mice and the retinas of mice injected with the MTS-AAV as a positive control. We performed alignments with the human and mouse mitochondrial genomes and also with the virus AAV genome looking for reads which align one part to the virus and other to mouse or human mtDNA and determining the depth at each base level to look for variants. To determine whether the AAV transferred mutant human ND4 replicated, we performed in vitro replication of extracted mtDNA.

Results: In each of the samples we identified 0 to 208 reads that mapped to the MTS-AAV containing human ND4. In retinal samples from transgenic mice and retinas directly injected with the MTS-AAV we identified some retention of the first iTR as well as the iTR at the other end of the vector with muscle and brain samples showing deletion of the iTR. Chimeras of human ND4 and the mouse mitochondrial genome were absent. No insertions of human ND4 or the transferred viral genome were detected in the mouse mitochondrial genome. In vitro replication revealed a ~2kb band of the vector in transgenic mice, absent in normal B6 mice, with hybridization to a human ND4 probe.

Conclusions: Optic neuropathy detected in transgenic mice generated from blastocyst injections appears to be due to mutant human G11778A ND4 and not due to recombination with mouse mtDNA or deletions of host mtDNA. Replication of transferred viral DNA occurred in the absence of inclusion of the CSB regions of the D loop into our AAV construct suggesting that the vector iTR may be driving replication of the episomal mutant human ND4. These findings suggest that MTS-AAV mediated transfer of a normal human ND4 allele to the human embryo may be a yet unexplored option to prevent transmission of blindness in subjects with mutated ND4.

Keywords: 600 mitochondria • 538 gene transfer/gene therapy • 613 neuro-ophthalmology: optic nerve  

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