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J. Guy, X. Qi; Efficiency and Safety of Mitochondrial Gene Replacement in the Vertebrae Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3239.
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© ARVO (1962-2015); The Authors (2016-present)
1. To evaluate the efficiency and safety of AAV mediated gene delivery of a normal human complex I subunit (ND4) in the vertebrate visual system. 2. To test rescue of RGC degeneration induced by the mutant human ND4 (R340H) responsible for most cases of Leber Hereditary Optic Neuropathy (LHON).
A normal human ND4 subunit made compatible with the universal genetic code to which a mitochondrial targeting sequence (ATPc) was appended to the N terminus and an epitope for immunodetection (FLAG) added to the C terminus was inserted into a bicistronic AAV vector that also contained GFP. This construct was injected into the vitreous of the right eyes of DBA/1J mice. The vector without the ND4 gene, but containing GFP was injected into the left eyes. One month later, ND4 gene expression was assessed in excised flat mounted retina and the optic nerve immunostained for FLAG by fluorescence, light and electron microscopy and by RT-PCR. Incorporation of the human ND4 into the murine holo-complex was assessed by complex I immunoprecipitation followed by immunobloting for ND4FLAG.Quantitative analysis of ND4FLAG positive RGCs were assessed relative to GFP and Thy1.2 positive RGCs. RGC loss was assessed by Thy1.2 positive cell counts in experimental relative to control eyes. To test rescue, one month after injection of the normal ND4, we injected AAV containing the ND4 gene with a His substitution at amino acid 340 into both eyes. One month later we assessed apoptosis of RGCs in TUNEL stained retinal flat mounts.
RT-PCR of ND4 infected murine retina and optic nerve yielded the expected 242 bp band. Sequencing confirmed it to be the human ND4. Immunoprecipitated murine complex I gave the expected 52 kDa of human ND4FLAG. Fluorescence microscopy revealed a punctate and perinuclear expression of FLAG characteristic of mitochondrial localization in the retina and optic nerve of experimental eyes. In contrast GFP expression was cytoplasmic and nuclear. Relative to the mean number of Thy1.2-positive RGCs quantification of FLAG positive RGCs was 38% and GFP-positive RGCs was 65%. Thy1.2 positive-RGC counts with a mean of 3176 cells/mm2 in ND4 injected eyes and 3316 cells /mm2 in control eyes injected with GFP were similar. The normal ND4 protected against RGC loss, with 33% less apoptotic RGCs in eyes that receiving the rescue normal ND4 and disease inducing mutant ND4 relative to control eyes that received GFP and the mutant ND4 (p <0.05).
Although approximately two-thirds the efficiency of GFP, expression of normal human ND4 in murine mitochondria did not induce loss of RGCs and it rescued a mouse model of LHON suggesting it may safely be used for treatment of patients.
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