Lysosomal Storage Disease Cell Models for Research

Lysosomal storage diseases (LSDs) are a group of over 50 rare inherited metabolic disorders caused by defects in lysosomal function, leading to accumulation of undegraded substrates. The combined incidence is estimated at 1 in 5,000 live births, but individual disorders are rare. For example, Gaucher disease has a prevalence of 1 in 40,000 to 60,000 in the general population, but is more common in Ashkenazi Jews (1 in 800). Fabry disease affects approximately 1 in 40,000 males. Pompe disease occurs in about 1 in 40,000 births. These disorders are progressive and often fatal, with symptoms ranging from hepatosplenomegaly, skeletal abnormalities, and neurological degeneration to cardiac and renal failure. The clinical burden is significant, with many patients experiencing reduced quality of life and premature death. According to the World Health Organization (WHO), rare diseases collectively affect millions worldwide, and LSDs represent a substantial proportion. Early diagnosis and treatment are critical, but many disorders lack effective therapies, highlighting the need for research.

LSDs are ideal for mechanistic studies because they are monogenic disorders with well-defined enzyme deficiencies. This allows for precise genetic modeling using CRISPR. The availability of patient-derived cell lines and public datasets, such as those from the National Center for Biotechnology Information (NCBI) and the Human Gene Mutation Database, facilitates research. Open questions include the pathophysiology of neurodegeneration, genotype-phenotype correlations, and the development of therapies beyond enzyme replacement. Gene-edited cell models provide a powerful tool to dissect disease mechanisms and test novel therapeutic strategies.

LSDs arise from defects in lysosomal enzymes, transporters, or accessory proteins, leading to substrate accumulation. Key pathways include:

1. Enzyme deficiency: Mutations in genes encoding lysosomal hydrolases (e.g., GBA in Gaucher, GLA in Fabry) result in reduced or absent enzyme activity.

2. Substrate accumulation: Undegraded substrates (e.g., glucocerebroside, globotriaosylceramide) accumulate in lysosomes, causing cellular dysfunction.

3. Secondary pathways: Accumulation triggers inflammation, autophagy impairment, and endoplasmic reticulum stress, contributing to cell death.

4. Organ-specific effects: Different substrates affect specific tissues, leading to varied clinical presentations.

Data from ClinVar and COSMIC.

LSDs affect multiple signaling pathways:

• • Autophagy: Impaired autophagic flux due to lysosomal dysfunction leads to accumulation of damaged organelles.
• • Inflammatory signaling: Substrate accumulation activates Toll-like receptors and inflammasomes, promoting chronic inflammation.
• • Calcium signaling: Lysosomal calcium release is disrupted, affecting cellular signaling.
• • mTOR pathway: Nutrient sensing is altered, impacting cell growth and metabolism.
• • Apoptosis: Accumulation triggers cell death pathways, particularly in neurons.

Organoids derived from patient iPSCs offer a more physiologically relevant model, recapitulating tissue-specific pathology.

• • Genetically engineered mouse models (GEMMs) with targeted mutations in Gba, Gla, Gaa, or Npc1 recapitulate disease phenotypes.
• • Patient-derived xenograft (PDX) models are less common for LSDs but can be used for certain hematological manifestations.
• • Induced models using chemical inhibitors (e.g., conduritol B epoxide for Gaucher) can mimic enzyme deficiency.

CRISPR-Cas9 technology enables the creation of isogenic cell lines with precise gene knockouts or knock-ins. For example:

• • GBA knockout cell lines: Used to study glucocerebrosidase deficiency and test chaperone therapies.
• • GLA knock-in models with specific missense mutations (e.g., p.N215S) to mimic Fabry disease.
• • GAA knockout lines for Pompe disease to evaluate gene therapy approaches.
• • NPC1 mutant lines to investigate cholesterol trafficking.

These models are commercially available and sequence-verified, providing a reliable and reproducible platform for drug discovery and functional genomics.

Knockout and knock-in lines are used to validate the function of genes implicated in LSDs. For example, GBA knockout lines have been used to confirm the role of glucocerebrosidase in Parkinson's disease risk. Similarly, NPC1 knockout lines have helped elucidate the role of NPC1 in viral entry and cholesterol transport.

Isogenic pairs (wild-type vs. knockout) are ideal for high-throughput screening of compounds that rescue the phenotype. For instance, screening for small molecules that increase enzyme activity in GBA knockout cells can identify potential therapeutics. Resistance to enzyme replacement therapy can be modeled by exposing cells to increasing concentrations of the enzyme and selecting for resistant clones.

CRISPR-based synthetic lethality screens can identify genes that, when silenced, are lethal in the disease context but not in normal cells. This approach has been used to find novel targets for LSDs, such as genes involved in substrate clearance. Additionally, secretome analysis of gene-edited cells can identify potential biomarkers for early diagnosis.