Mitochondrially Targeted Gene Therapy Rescues Visual Loss in a Mouse Model of Leber's Hereditary Optic Neuropathy

Abstract

Leber's hereditary optic neuropathy (LHON) is a common mitochondrial genetic disease, causing irreversible blindness in young individuals. Current treatments are inadequate, and there is no definitive cure. This study evaluates the effectiveness of delivering wildtype human NADH ubiquinone oxidoreductase subunit 4 (hND4) gene using mito-targeted AAV(MTSAAV) to rescue LHOH mice. We observed a declining pattern in electroretinograms amplitudes as mice aged across all groups (p < 0.001), with significant differences among groups (p = 0.023; Control vs. LHON, p = 0.008; Control vs. Rescue, p = 0.228). Inner retinal thickness and intraocular pressure did not change significantly with age or groups. Compared to LHON mice, those rescued with wildtype hND4 exhibited improved retinal visual acuity (0.29 ± 0.1 cy/deg vs. 0.15 ± 0.1 cy/deg) and increased functional hyperemia response (effect of flicker, p < 0.001, effect of Group, p = 0.004; Interaction Flicker × Group, p < 0.001). Postmortem analysis shows a marked reduction in retinal ganglion cell density in the LHON group compared to the other groups (Effect of Group, p < 0.001, Control vs. LHON, p < 0.001, Control vs. Rescue, p = 0.106). These results suggest that MTSAAV-delivered wildtype hND4 gene rescues, at least in part, visual impairment in an LHON mouse model and has the therapeutic potential to treat this disease.

Keywords: LHON; MTSAAV; gene therapy; human ND4.

Figure 1

Mito-targeted wtND4 rescue mutND4-induced RGC dysfunction. (A) Study design and timeline for the retinal structure and function follow-up. (B) Pre-injection and post-injection IOPs were similar in the study groups. (C) FERG amplitudes temporarily increased in all study groups after intravitreal injections and then returned to physiological level. (D) Inner retina thickness and total retinal thickness were temporarily increased in all study groups after intravitreal injections and then returned to baseline levels. (E) PERG amplitudes temporarily decreased in all study groups and progressively recovered the physiological level in Control and Rescue study groups, but not in the LHON group. (F) PERG latencies physiologically increased with age in all study groups.

Figure 2

Wildtype ND4 improves the visual acuity. (A) The PERG signal decreases with increasing spatial frequency. The linear extrapolation of PERG amplitude to the noise level represents the retinal acuity, which is higher in the Control group (0.242 cy/deg) and in the Rescue group (0.293 cy/deg), compared to the LHON group (0.153 cy/deg). (B) Flickering light superimposed on the PERG stimulus causes reduction of PERG amplitude (autoregulatory adaptation), which is present in both Naïve and Control group (34% amplitude change). Flicker adaptation is lost in the LHON group, but it is restored (30%) in the Rescue group.

Figure 3

Wildtype ND4 prevents RGC loss in LHON mice. (A–D) Representative immunostaining of retinal whole-mount using RBPMS (a pan-RGC marker) showed RGCs distribution in Naïve (A), Control (B), LHOH (C), and Rescue (D) mice. (E) The mean RGC density at the endpoint is lower in the LHON group compared to Naïve, Control, and Rescue groups. (F) The probability distribution of RGC density in the LHON group is shifted to the left compared to overlapping distributions of Control and Naive groups. The Rescue group appears to have a bimodal distribution, with one peak in the LHON range and another peak in the range of Controls. Scale bar = 10 μm.

Figure 4

Wildtype ND4 protects optic nerve degeneration. (A–D) Representative transmission electron micrographs of the retrobulbar optic nerve showed the axon distribution in Naïve (A), Control (B), LHON (C), and Rescue (D) mice. (E) The distribution of axon density shows higher density in the Naïve group compared with the other three groups. (F) The main distribution of axon diameter is similar in all groups, but the Naïve group has a tail of axons smaller than 0.3 µm. Scale bar = 1 μm.

Figure 5

Wildtype ND4 preserves mitochondrial and RGC axonal integrity. Integrity scoring for the axon (A) and mitochondria in the optic nerve (B). LHON rescue group has similar healthy axons and mitochondria as the age Control and negative Control groups. (N = 5 per group, 15-month-old).

Figure 6

Block diagram for binocular PERG recording. Pattern stimuli with two slightly different reversal rates are generated on two identical LED displays and presented separately to each eye. PERG signals are recorded continuously by means of subcutaneous needle electrodes (active, snout; reference, back of the head; ground, tail) and fed to one-channel acquisition system. The PERG signals for each eye are desynchronized by phase locking averaging method and PERG waveforms are displayed on the screen of a laptop that controls the stimulation/acquisition box.