June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Mitochondrial Gene Therapy for G11778A LHON: Safety of Human ND4 in Nonhuman Primates and Expression in Ex Vivo Human Eyes
Author Affiliations & Notes
  • Rajeshwari Koilkonda
    Ophthalmology, Bascom Palmer Eye Institute, University of Miami-Miller School of Medicine, Miami, FL
  • David Tse
    Ophthalmology, Bascom Palmer Eye Institute, University of Miami-Miller School of Medicine, Miami, FL
  • William Hauswirth
    Department of Ophthalmology, University of Florida, College of Medicine, Gainesville, FL
  • Vince Chiodo
    Department of Ophthalmology, University of Florida, College of Medicine, Gainesville, FL
  • Sanford Boye
    Department of Ophthalmology, University of Florida, College of Medicine, Gainesville, FL
  • Martha Neuringer
    Oregon Health and Science University, Beaverton, OR
  • Tim Stout
    Oregon Health and Science University, Beaverton, OR
  • John Guy
    Ophthalmology, Bascom Palmer Eye Institute, University of Miami-Miller School of Medicine, Miami, FL
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2738. doi:
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      Rajeshwari Koilkonda, David Tse, William Hauswirth, Vince Chiodo, Sanford Boye, Martha Neuringer, Tim Stout, John Guy; Mitochondrial Gene Therapy for G11778A LHON: Safety of Human ND4 in Nonhuman Primates and Expression in Ex Vivo Human Eyes. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2738.

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

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Abstract

Purpose: To demonstrate the safety and expression of allotopic human ND4 to be used for the Leber’s hereditary optic neuropathy gene therapy trial.

Methods: We modified ND4 in the nuclear genetic code for import into mitochondria. The protein was targeted into the organelle by ATPc (P1) sequence. The gene was packaged into self-complementary AAV2 packaged with triple Y-F capsid mutant (Y444F+Y500F+Y730F) VP3 then injected into rodent, nonhuman primate and ex vivo human eyes. Expression and integration into complex I of rodents and ex vivo human eyes was tested by immunohistochemistry and blue native (BN) PAGE. Three rhesus macaques (1m, 2f, ages 3-7 yrs) were injected intravitreally (iv) with scAAV2(Y444,500,703F)-smCBA-P1ND4. The vector titer was 1.23x1011 vg/ml, and a volume of 200 μL was injected for a total dose of 2.46x1010 vg. We next tested for transgene expression in two normal human eyes that were surgically removed for control of periocular spread of cancers. After incising the cornea, eyes were immersed immediately in tissue culture media. One eye received iv injection of 120 μL of scAAV-ND4FLAG (1.0 x 1012 vg/ml) the other 20 µL (4.0 x 1012 vg/ml).

Results: Rodents.2-D BN PAGE with the anti-FLAG antibody reacted against infected retinal mitochondrial extracts from rats 1month post injection (PI) of scAAV-ND4FLAG showed a distinct band of FLAG, absent in uninfected control retinal mitochondria. Antibodies against NDUFA9, NDUFS3, NDUFS4, NDUFB8 or NDUFA6 detected these nuclear encoded complex I subunits. Human Eyes. 3days PI, ND4FLAG co-localized with mitochondrial porin. Quantitative analysis revealed, injection of more than 1011 vg, 84% of RGCs expressed ND4FLAG. Nonhuman Primates. Clinical examinations and retinal fundus photography done at baseline, 5, 8 and 15days PI revealed no clinical signs of inflammation, such as haze or presence of inflammatory cells in the anterior chamber or vitreous and looked similar to the uninjected left eye. One animal developed mild vitritis ~2m after AAV injection. OCT images of the retina and optic nerve head showed no structural changes from baseline in all 3 animals, but cells were present in the vitreous of one animal.

Conclusions: Expression of allotopic ND4 in the ex vivo human eye, safety of the test article in rodent and primate visual systems and the severe irreversible loss of RGCs in LHON support clinical testing.

Keywords: 538 gene transfer/gene therapy • 600 mitochondria • 629 optic nerve  
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