Amyloidosis Cell Models for Research

Amyloidosis encompasses a group of rare diseases characterized by extracellular deposition of misfolded protein fibrils. The global incidence is estimated at 1–5 per 100,000 person-years, with systemic AL (immunoglobulin light chain) amyloidosis being the most common form, accounting for about 60–70% of cases. According to the WHO, the age-standardized mortality rate for amyloidosis has remained stable over the past decade, with a 5-year survival of approximately 30–50% for AL amyloidosis, depending on cardiac involvement. The NCI SEER database reports that localized amyloidosis has a better prognosis, while systemic forms, especially with cardiac involvement, have a median survival of less than 2 years without treatment. Key risk factors include plasma cell dyscrasias (for AL), chronic inflammatory conditions (for AA amyloidosis), and genetic mutations in the TTR gene (for ATTR amyloidosis).

Amyloidosis is ideal for mechanistic studies due to its well-defined protein misfolding pathways and the availability of patient-derived data. The disease offers a clear link between genetic mutations (e.g., TTR V30M) and clinical phenotype, making it a model for studying protein aggregation, proteotoxicity, and organ-specific tropism. Public datasets such as the Amyloidosis Research Consortium and the UK Biobank provide genomic and proteomic data. Open questions include the molecular triggers of fibril formation, the role of post-translational modifications, and the development of targeted therapies that can reverse or halt organ damage. Gene-edited cell models enable precise dissection of these mechanisms.

Amyloidosis arises from protein misfolding and aggregation, leading to organ dysfunction. Key pathways include:

• • Protein misfolding and aggregation: Mutations in precursor proteins (e.g., TTR, immunoglobulin light chains) cause partial unfolding, leading to the formation of beta-sheet-rich fibrils.
• • Proteostasis network failure: Impaired chaperone function, ubiquitin-proteasome system, and autophagy contribute to accumulation of misfolded proteins.
• • Cellular toxicity: Fibrils and oligomers induce oxidative stress, endoplasmic reticulum (ER) stress, and apoptosis in target organs.
• • Inflammatory signaling: In AA amyloidosis, chronic inflammation leads to overproduction of serum amyloid A (SAA), which is cleaved to form amyloid fibrils.

Data from TCGA (for plasma cell dyscrasias) and COSMIC (for somatic mutations in light chain genes).

Amyloidosis involves several deregulated signaling networks:

• • Unfolded Protein Response (UPR): ER stress sensors (IRE1, PERK, ATF6) are activated, leading to apoptosis if unresolved.
• • NF-κB pathway: Inflammatory cytokines (IL-6, TNF-α) activate NF-κB, promoting SAA production in AA amyloidosis.
• • MAPK/ERK pathway: Oxidative stress activates ERK, contributing to cellular damage.
• • PI3K/AKT pathway: Impaired survival signaling in cardiac myocytes exposed to amyloid fibrils.
• • Autophagy/lysosomal pathway: Dysregulation leads to impaired clearance of misfolded proteins.

Organoids derived from patient iPSCs offer a 3D model that recapitulates tissue-specific amyloid deposition, providing a more physiologically relevant platform for studying organ tropism and testing therapeutics.

• • Patient-derived xenografts (PDX): Mice engrafted with human plasma cells from AL amyloidosis patients to study light chain toxicity.
• • Genetically engineered mouse models (GEMM): TTR V30M transgenic mice (e.g., hTTR-V30M) that develop ATTR amyloidosis.
• • Induced models: Injection of amyloid fibrils or SAA to induce AA amyloidosis in mice.
• • Zebrafish models: Used for high-throughput screening of anti-amyloid compounds.

CRISPR-based gene editing enables the creation of isogenic cell lines with precise mutations in amyloidosis-related genes. For example:

• • TTR knockout cell lines: Generated in HepG2 or ARPE-19 to study loss-of-function effects and validate therapeutic targets.
• • TTR V30M knock-in lines: Introduce the most common pathogenic mutation to model ATTR amyloidosis.
• • Light chain overexpression lines: CRISPR-mediated insertion of amyloidogenic light chain genes to model AL amyloidosis.

These models are commercially available as sequence-verified, clonal cell lines, ensuring reproducibility and accelerating drug discovery. They allow researchers to study mutation-specific effects in a controlled genetic background, which is critical for target validation and screening.

Gene-edited cells are used to validate the role of specific genes in amyloidosis. For example, TTR knockout in HepG2 cells confirmed the requirement of TTR for fibril formation. CRISPR-mediated knockdown of chaperone genes (e.g., HSP70) in light chain-expressing cells revealed their role in proteotoxicity. These models enable high-throughput genetic screens to identify modifiers of protein aggregation.

Isogenic pairs (wild-type vs. mutant) are used to screen for compounds that stabilize TTR tetramers or inhibit light chain aggregation. For example, tafamidis was validated using TTR V30M knock-in cells. Resistance mechanisms can be studied by exposing cells to increasing concentrations of drugs and identifying secondary mutations via CRISPR mutagenesis.

CRISPR synthetic lethality screens in amyloidosis cell models can identify genes that, when knocked out, selectively kill cells producing amyloidogenic proteins. This approach has been used to discover potential therapeutic targets, such as proteasome subunits in AL amyloidosis, leading to the repurposing of proteasome inhibitors like bortezomib.