Leigh Syndrome Gene-Edited Cell Lines for Drug Discovery

Leigh syndrome (LS) is a rare, progressive neurodegenerative disorder with an estimated incidence of 1 in 40,000 live births (WHO, 2023). It is the most common pediatric mitochondrial disease, typically presenting before age 2. The condition is characterized by bilateral symmetrical necrotic lesions in the basal ganglia, brainstem, and thalamus. Prognosis is poor, with most patients surviving only a few years after onset. No curative therapies exist; current management is supportive. The disease imposes a significant burden on families and healthcare systems due to the need for long-term specialized care.

• • Leigh syndrome is an ideal model for studying mitochondrial dysfunction and oxidative phosphorylation (OXPHOS) defects. Key research advantages include:
• • Defined genetic etiology: mutations in over 75 genes (nuclear and mitochondrial) are known, enabling precise modeling.
• • Strong genotype-phenotype correlations, allowing mechanistic studies of specific biochemical pathways.
• • Publicly available patient-derived cell lines and induced pluripotent stem cell (iPSC) resources (e.g., from the NINDS Human Cell Repository).
• • Open questions remain regarding tissue-specific vulnerability, the role of reactive oxygen species (ROS), and potential therapeutic windows, making it a rich area for functional genomics.

Leigh syndrome arises from defects in mitochondrial energy production, primarily affecting the electron transport chain (ETC). The major pathways involved are:

1. Complex I (NADH:ubiquinone oxidoreductase) deficiency – most common cause (~30% of cases). Mutations in nuclear-encoded subunits (e.g., NDUFS1, NDUFV1) or mitochondrial-encoded MT-ND genes impair NADH oxidation and proton pumping.

2. Complex IV (cytochrome c oxidase, COX) deficiency – caused by mutations in assembly factors like SURF1, SCO2, or COX10. Leads to impaired electron transfer to oxygen.

3. Complex II (succinate dehydrogenase) deficiency – rare, but mutations in SDHA cause isolated complex II deficiency.

4. Pyruvate dehydrogenase complex (PDHC) deficiency – mutations in PDHA1 or PDHX disrupt the link between glycolysis and the TCA cycle, reducing acetyl-CoA production.

These defects result in reduced ATP synthesis, increased ROS production, and eventual neuronal cell death.

The following table summarizes the most frequently mutated genes in Leigh syndrome, based on data from ClinVar and the Mitochondrial Disease Sequence Data Resource (MSeqDR).

Data from ClinVar (2024) and COSMIC (v99).

Mitochondrial dysfunction in Leigh syndrome triggers several downstream signaling cascades:

• • AMPK/mTOR pathway: Low ATP activates AMPK, which inhibits mTORC1, reducing protein synthesis and cell growth. Chronic activation contributes to neurodegeneration.
• • ROS/JNK signaling: Elevated ROS from defective ETC complexes activates JNK and p38 MAPK, promoting apoptosis.
• • HIF-1α stabilization: Impaired OXPHOS can stabilize HIF-1α even under normoxia, leading to metabolic reprogramming toward glycolysis.
• • Mitochondrial unfolded protein response (UPRmt): Accumulation of misfolded proteins in mitochondria activates ATF5 and CHOP, which can be protective or pro-apoptotic depending on severity.
• • Calcium homeostasis: Mitochondrial membrane potential loss disrupts calcium buffering, sensitizing neurons to excitotoxicity.

Commonly used cell models for Leigh syndrome research include:

Organoid models (e.g., midbrain organoids) derived from patient iPSCs recapitulate 3D tissue architecture and allow study of neuronal-glial interactions in a mitochondrial disease context.

Animal models for Leigh syndrome are essential for in vivo validation:

• • Ndufs4 knockout mouse: Global or neuron-specific knockout of Ndufs4 recapitulates Leigh-like encephalopathy, with progressive motor deficits and brain lesions.
• • Surf1 knockout mouse: Shows reduced COX activity and mild neurological phenotype; useful for testing therapies.
• • Mito-mice (e.g., MT-ATP6 mutant): Transmitochondrial mice carrying pathogenic mtDNA mutations; model maternal inheritance.
• • Zebrafish models: Morpholino or CRISPR-mediated knockdown of complex I subunits (e.g., ndufs4) causes developmental defects and reduced locomotion.
• • Drosophila models: Knockdown of complex I genes (e.g., ND-75) leads to shortened lifespan and neurodegeneration.

CRISPR/Cas9-engineered isogenic cell lines provide precise tools to study Leigh syndrome mutations in a controlled genetic background. Examples include:

• • TP53-/- (not LS-specific, but used as a control for genomic stability)
• • SURF1 knockout (e.g., in HEK293T or SH-SY5Y): recapitulates complex IV deficiency.
• • NDUFV1 knockout: models complex I deficiency; used for metabolic flux analysis.
• • MT-ATP6 m.8993T>G knock-in: introduces the common pathogenic mtDNA mutation via mitochondrial-targeted editing.

Commercially available, sequence-verified gene-edited cell models accelerate research by eliminating the need for laborious cloning and validation. These models are available from multiple commercial sources and can be customized for specific mutations.

Knockout and knock-in cell lines enable direct functional validation of Leigh syndrome-associated genes. For example:

• • SURF1 knockout cells show reduced COX activity and increased ROS, confirming the gene's role in complex IV assembly.
• • NDUFS1 knockout cells exhibit decreased oxygen consumption rate (OCR) and ATP levels, linking the mutation to bioenergetic failure.
• • MT-ATP6 knock-in cells allow study of ATP synthase dysfunction in a human neuronal background.

These models are also used in CRISPR activation (CRISPRa) or interference (CRISPRi) screens to identify genetic modifiers of mitochondrial dysfunction.

Isogenic pairs (wild-type vs. mutant) are ideal for high-throughput drug screening:

• • Phenotypic screens: Measure OCR, mitochondrial membrane potential, or cell viability in response to compound libraries.
• • Targeted screens: Test compounds that enhance residual complex activity (e.g., EPI-743, idebenone) in SURF1 or NDUFS1 knockout cells.
• • Resistance modeling: Chronic treatment with mitochondrial toxins (e.g., rotenone) in wild-type cells can select for resistance mutations; isogenic lines help validate these mutations.

CRISPR-engineered cells facilitate identification of biomarkers for Leigh syndrome:

• • Synthetic lethality screens: Identify genes whose knockout is lethal only in a Leigh syndrome genetic background (e.g., NDUFS1 knockout + OXPHOS inhibitor).
• • Secretome analysis: Conditioned media from mutant cells can reveal secreted proteins (e.g., FGF21, GDF15) as potential blood biomarkers.
• • Metabolomics: Isogenic lines enable discovery of metabolic signatures (e.g., lactate/pyruvate ratio, acylcarnitines) that correlate with disease severity.

The following databases provide essential data for Leigh syndrome research: