April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Mutant Human T8993G ATP6 Expression by a Mitochondrial Targeting Sequence Modification of AAV Capsid VP2 in Transgenic Mice
Author Affiliations & Notes
  • Huijun Yuan
    Bascom Palmer Eye Institute, Miami, FL
  • John Guy
    Bascom Palmer Eye Institute, Miami, FL
  • Footnotes
    Commercial Relationships Huijun Yuan, None; John Guy, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3311. doi:
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      Huijun Yuan, John Guy; Mutant Human T8993G ATP6 Expression by a Mitochondrial Targeting Sequence Modification of AAV Capsid VP2 in Transgenic Mice. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3311.

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

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Purpose: To create a mouse model of mutant human ATP6, at T8993G of mitochondrial DNA by a mitochondrial targeting seqeunce modifcation of AAV capsid VP2. The ATP6 mutation causes two mitochondrial diseases, neuropathy, ataxia, retinitis pigmentosa (NARP) and maternally inherited Leigh's syndrome (MILS).

Methods: Sc-HSP-ATP6mut fused with FLAG and a second gene mitochondrial mCherry was packaged with mito-targeted (COX8) VP2 capsid into AAV2. ATP6mut (A6M) transgenic mice were created with injections of AAV2-cox8ATP6mutFlag-mcherry into fertilized eggs. The Heidelberg image was used to examine mCherry expression of the A6M mice at two months. The expression of ATP6mutFLAG-cherry was detected by immunohistochemistry and blue native PAGE gel with 2D western blotting. The brain degeneration of ATP6 mutant mice were checked by MRI and H&E staining. The pattern electroretinogram (PERG) and flash ERG examined visual function.

Results: Using Heidelberg images, mCherry was detected by red channels in 80% of the A6M transgenic mice. Two A6M mice died at one month and three others died between 2 to 5 months . MCherry and Flag, tagged human ATP6 mutant gene, exhibited perinucleus distribution and colocalization of the mitochondrial marker porin in the brain, spinal cord, lung, heart and muscles. A blue native PAGE and 2D SDS-PAGE western blotting showed human mutant ATP6 expression was detected in complex V by anti-flag. MRI revealed significant, symmetrical lesions observed in brain. Histopathology of dead mouse brains displayed demyelination in the cerebellum. At 4 months, PERG data showed amplitudes decreased in A6M mice to 10uV compared to 31uV in wild type mice (P=0.0001).

Conclusions: A modified AAV VP2 with a mitochondrial targeting sequencing cox8 can successfully deliver a human ATP6mutant gene to mouse mitochondria, similar to human mitochondrial diseases, NARP and MILS. This mouse model can be used to test therapies for these blinding and fatal disorders.

Keywords: 538 gene transfer/gene therapy  

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