Preclinical Assessment of Mitochondrial-Targeted ND4 Gene Therapy for Leber Hereditary Optic Neuropathy

The purpose of this study was to establish preclinical safety data to advance mitochondrial targeted AAV-delivered human ND4 (MTSAAV/hND4) for treatment of Leber Hereditary Optic Neuropathy (LHON).

MTSAAV/hND4 was bilaterally injected into mouse eyes to evaluate potential adverse effects on visual function using serial pattern electroretinograms (PERGs) and spectral-domain optical coherence tomography (SD-OCT). In rats, MTSAAV/hND4 was administered unilaterally to assess toxicity, biodistribution, and neutralizing antibody levels. Control groups include PBS-injected and untreated naïve mice and rats.

No significant adverse effects on visual function were observed in mice treated with MTSAAV/hND4. Retinal thickness remained largely stable, with only a transient, benign increase in RNFL-IPL thickness observed in the low-dose group at 1 month. Postmortem analysis showed no significant differences in RGC density among MTSAAV/hND4-injected mice, PBS-injected controls, and naïve controls (P = 0.214), and axons morphology and distribution were preserved. In rats, viral DNA was primarily detected in the injected eyes with copy number of 4.63 × 10⁴ at day 7 and 4.94 × 103 at day 90 per 100 ng of genomic DNA. Viral DNA levels in non-ocular tissues were low (<500 copies). Histopathological analysis showed no vector-related toxicity. At 6 months post-injection, overall hematological parameters remained within normal limits. All MTSAAV/hND4-injected rats seroconverted by day 90, with neutralizing antibody titers reaching 1:1280.

Intravitreal delivery of MTSAAV/hND4 in rodents resulted in favorable biodistribution, with vector DNA largely confined to the injected eye and minimal systemic exposure. No vector-related adverse effects were observed based on functional, anatomic, and histopathological assessments, supporting the ocular safety of this gene therapy strategy. These findings provide preclinical evidence supporting the advancement of MTSAAV-mediated ND4 gene therapy toward clinical trials for LHON.