Development of Two In Vitro ND1-LHON Models for Evaluating Gene Therapy Efficacy

The purpose of this study was to address the lack of effective treatments for NADH-ubiquinone oxidoreductase chain 1 (ND1)-related Leber hereditary optic neuropathy (LHON), this study aimed to (1) establish in vitro models mimicking mitochondrial dysfunction in LHON and (2) evaluate the therapeutic potential of recombinant adeno-associated virus (AAV)-mediated ND1 gene therapy (rAAV-ND1).

Two in vitro models were developed: (1) transmitochondrial cybrid cells carrying the m.3460G>A mutation in the ND1 gene; and (2) patient-derived induced pluripotent stem cells (iPSC)-differentiated retinal ganglion cells (RGCs). Mitochondrial function was assessed via measurements of oxygen consumption and adenosine triphosphate (ATP) production. The efficacy of rAAV-ND1 was tested by infecting both models to rescue mitochondrial deficiency.

Our two LHON models - ND1-mutant cybrid cells and patient-derived iPSC-RGCs - successfully recapitulated characteristic mitochondrial dysfunction, demonstrating impaired oxidative phosphorylation and reduced ATP production. Through qPCR and subcellular fractionation analyses, we confirmed dose-dependent ND1 transgene expression and proper mitochondrial localization. Notably, rAAV2-ND1 treatment effectively restored mitochondrial function in both models: in ND1-cybrids, it recovered spare respiratory capacity to 85% of the control levels, enhanced complex I activity from 65.5% to 90.5%, and increased ATP production from 47.6% to 69.5%; whereas in ND1-RGCs, it also ameliorated bioenergetic deficits, partially reversing SRC reduction, and improving ATP-linked respiration.

The study demonstrates the utility of transmitochondrial cybrids and iPSC-derived RGCs as reliable in vitro models for studying ND1-related LHON. The rAAV-ND1 gene therapy effectively restored mitochondrial function, highlighting its potential as a treatment for LHON caused by ND1 mutations. These findings underscore the value of in vitro systems for evaluating therapies when robust animal models are unavailable.