LHON gene therapy vector prevents visual loss and optic neuropathy induced by G11778A mutant mitochondrial DNA: biodistribution and toxicology profile

Abstract

Purpose: To demonstrate safety and efficacy of allotopic human ND4 for treatment of a Leber's hereditary optic neuropathy (LHON) mouse model harboring the G11778A mitochondrial mutation.

Methods: We induced LHON in mice by intravitreal injection of mutant (G11778A) human ND4 DNA, responsible for most cases of LHON, that was directed to mitochondria using an AAV2 vector to which we appended a mitochondrial targeting sequence to the VP2 capsid. We then attempted rescue of visual loss using our test article (ScAAV2-P1ND4v2) containing a synthetic nuclear encoded G11778G ND4 gene that was allotopically expressed. Control mice either were uninjected or received AAV2-GFP or AAV2-mCherry. We performed RT-PCR and confocal microscopy at 2 weeks post injection. Pattern electroretinograms (PERGs), spectral-domain optical coherence tomography (SD-OCT), histology, and transmission electron microscopy (TEM) were performed. For toxicology and biodistribution studies, the test article was administered intravitreally to rats and rhesus macaques at different doses.

Results: Mutant and wild-type ND4 were efficiently expressed in the mitochondria of retinal ganglion cells (RGCs). Visual function assessed by serial PERGs and retinal structure by serial SD-OCT showed a significant rescue by the test article. Histology and ultrastructural analysis confirmed that loss of RGCs and demise of axons was prevented by ScAAV2-P1ND4v2. Rat and nonhuman primate biodistribution studies showed that vector spread outside the injected eye into spleen and lymph nodes was minimal. Histopathology of tissues and organs including the eyes was comparable to that of uninfected and saline-injected eyes.

Conclusions: Allotopically expressed wild-type ND4 prevents the phenotype induced by G11778A mitochondrial DNA with a toxicology profile acceptable for testing in a phase I clinical trial.

Keywords: LHON; gene therapy; mitochondria.

Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Figures

Figure 1

Plasmid maps and expression of mutant ND4. Plasmid maps of (a) the mutant mitochondrial ND4+mCherry and (b) test article, P1ND4v2, are shown. Four months after dual viral injections, confocal laser scanning ophthalmoscopy shows mCherry fluorescence (arrowheads) in ScAAV2-GFP- and ScAAV2-HSP-ND4(G11778A)+mCherry-injected eyes (c, d) and in test article, ScAAV2-P1ND4v2-, along with ScAAV2-HSP-ND4(G11778A)+mCherry-injected eyes (e, f). mCherry fluorescence was absent in uninjected control (g). Confocal microscopy of retinal flat mounts performed 4 months after injection of ScAAV2-HSP-ND4(G11778A)+mCherry shows immunofluorescence of FLAG (h), Thy1.2 (i), porin (j), and merged image of FLAG, porin, and DAPI (k). A 3D rendered view of retinal longitudinal sections shows perinuclear mCherry expression (l) in RGCs (m), mitochondrial porin (n), and a merged image of mCherry, Thy1.2, porin, and DAPI (o). iTR, inverted terminal repeat; ATP1pre, ATP 1 leader sequence; HSP, mitochondrial heavy strand promoter; ONL, outer nuclear layer; INL, inner nuclear layer; RGC, retinal ganglion cell layer. Scale bars: 25 μm.

Figure 2

Rescue of visual function. PERG analysis of uninjected age-matched control mice and mice injected with ScAAV2-HSP-ND4(G11778A)+mCherry and mock treated with ScAAV2-GFP or test article, ScAAV2-P1ND4v2, performed at 2 months (2m) 6 months (6m), or 12 months (12m) post injection. Bar plots of PERG amplitudes (a) and latencies (b) are shown (mean ± SE). Representative PERG waveforms are shown at (c) 2 months, (d) 6 months, and (e) 12 months post injection, n = 24, *P = 0.01 to 0.05, **P = 0.001 to 0.01, ***P < 0.001. m PI, months post injection.

Figure 3

In vivo optical coherence tomography (OCT) imaging. Serial OCT imaging of a mock-treated mouse eye shows the nerve fiber layer to the inner boundary of the inner nuclear layer demarcated by yellow lines (a) and a corresponding 3D thickness map (b) at 4 months PI, a two-dimensional image (2D) (c) and 3D map (d) at 8 months PI, and a 2D (e) and 3D map (f) at 12 months PI. Also shown are 2D (g) and 3D maps (h) of a rescued mouse eye injected with the test article at 4 months PI, 2D (i) and 3D maps (j) at 8 months PI, and a 2D image (k) and 3D map (l) at 12 months PI (m). The bar plot shows average thickness measurements from the nerve fiber layer to the inner boundary of the inner nuclear layer (as marked in the 2D images) at 8 and 12 months PI (mean ± SE); n = 26, *P = 0.01 to 0.05, ***P < 0.001. m PI, months post injection.

Figure 4

Histological and ultrastructural studies of retina. One year after intravitreal injection, light microscopic analysis of toluidine blue–stained longitudinal retinal sections showed (a) loss of RGCs with mock treatment (ScAAV2-GFP). (b) With ScAAV-P1ND4v2 rescue from the disease-inducing ScAAV2-HSP-ND4(G11778A)+mCherry, there were more RGCs. (c) Transmission electron microscopy identified apoptotic RGCs in mock-treated mice. (d) Transmission electron micrographs of mice treated with P1ND4v2 and also injected with ScAAV2-mtND4FLAG+mCherry had RGCs with a characteristic elliptical and lighter nucleus typical of RGCs. (e) Bar plot of RGC numbers counted on toluidine blue–stained light microscopic images is shown (mean ± SE). Scale bars: 20 μm (a, b); 2 μm (c, d). ONL, outer nuclear layer; INL, inner nuclear layer; RGC, retinal ganglion cell layer. n = 10, ***P = 3.65 × 10−5.

Figure 5

Ultrastructural studies of rescue. Transmission electron micrographs of the (a) optic nerves of mock-treated mice with reduced axon density, (b) swollen axons with thin myelin lamellae, (c) prominent astroglial processes where axons were lost, and (d) degenerating axons exhibiting Wallerian degeneration (arrows). (e) Axons were more numerous with P1ND4v2 treatment. (f) Enlarged image showing an inflammatory cell exhibiting phagocytosis of myelin (arrow). (g, h) Astroglial processes and axonal degeneration were less with P1ND4v2 treatment. (i) Bar graph of axon numbers is shown (mean ± SE). Scale bar: 25 μm (a, e); 2 μm (b–d, f–h). g, glia; ax, axons; IC, inflammatory cell. n = 10, **P = 0.00758.