March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Mutant ND4 Gene Delivery to Mitochondria by Targeting Sequence-Modified AAV Induces Visual Loss and Optic Atrophy in Mice
Author Affiliations & Notes
  • John Guy
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida
  • Hong Yu
    Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami, Miami, Florida
  • Vittorio Porciatti
    Bascom Palmer Eye Inst, Univ of Miami Miller Sch Med, Miami, Florida
  • William W. Hauswirth
    Ophthalmology, University of Florida, Gainesville, Florida
  • Vince Chiodo
    Ophthalmology, University of Florida, Gainesville, Florida
  • Sanford L. Boye
    Ophthalmology, University of Florida, Gainesville, Florida
  • Tsung-Han Chou
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida
  • Footnotes
    Commercial Relationships  John Guy, None; Hong Yu, None; Vittorio Porciatti, None; William W. Hauswirth, AGTC (I); Vince Chiodo, None; Sanford L. Boye, None; Tsung-Han Chou, None
  • Footnotes
    Support  R01 EY017141
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 3166. doi:
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      John Guy, Hong Yu, Vittorio Porciatti, William W. Hauswirth, Vince Chiodo, Sanford L. Boye, Tsung-Han Chou; Mutant ND4 Gene Delivery to Mitochondria by Targeting Sequence-Modified AAV Induces Visual Loss and Optic Atrophy in Mice. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3166.

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

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Purpose: : While mutated G11778A ND4 mitochondrial DNA was firmly linked to the blindness of LHON in 1988, an animal model system with mutated mtDNA complex I subunits has never been developed.

Methods: : The mutant human ND4 gene with a G to A transition at position 11778 with an attached FLAG epitope under control of the mitochondrial heavy strand promoter (HSP) was inserted into a modified self-complementary (sc)AAV backbone. The HSP-ND4FLAG was directed towards the mitochondria by addition of the 23 amino acid cytochrome oxidase subunit 8 (COX8) pre-sequence fused in-frame to the N-terminus of GFP into the AAV2 capsid open reading frame. The packaged scAAV-HSP-ND4 was injected into the vitreous cavity of normal mice (OD). Contralateral eyes received scAAV- GFP (OS). Translocation and integration of human ND4 in mouse mitochondria was assessed by PCR, RT-PCR, sequencing, immunoblotting and immunohistochemistry. Visual function was monitored by pattern electroretinography (PERG) and in vivo structure by spectral domain OCT. Animals were euthanized at one year.

Results: : PCR of mitochondrial and nuclear DNA extracted from retina and optic nerve gave the expected 1.4 Kb band. DNA sequencing confirmed PCR products were mutant human ND4 (OD only). RT-PCR revealed the expected 500 bp product. Immunoblotting revealed the 52 Kd band of mutant ND4FLAG (OD only). Mutant ND4FLAG expressed in the RGC layer. PERGs showed a significant decrement in RGC function OD relative to OS at 1 month (24%, p =0.033), 6 months (25%, p= 0.029) and one year (20%, p = 0.039) after AAV injections. Spectral domain OCT showed optic disc edema starting at 1 month and optic nerve head atrophy with marked thinning of the inner retina at one year that was confirmed postmortem. Histopathology of optic nerve cross sections revealed reductions in optic nerve diameters of OD vs OS where transmission electron microscopy revealed significant loss of optic nerve axons in mutant ND4 injected eyes (p = 0. 0025) where mitochondria of remaining axons were swollen with dissolution of cristae and also showing electron dense aggregations characteristic of wallerian degeneration. Normal appearing and degenerating mitochondria were sometimes evident in the same axon.

Conclusions: : Due to the TGA codon at amino acid 16 that encodes for tryptophan in the mitochondria that is a stop codon in the nuclear genetic code, mutant G11778A ND4 can only be translated in the mitochondria (and not on cytoplasmic ribosomes) where its expression led to loss of visual function and optic nerve degeneration characteristic of LHON, thus providing a bona fide animal model for testing potential therapies that common to all mitochondrial disorders is woefully inadequate.

Keywords: neuro-ophthalmology: optic nerve • mitochondria • retinal degenerations: hereditary 

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