Arterial Calcification, Generalized, of Infancy, 2 (GACI2) Cell Models for Research

Arterial Calcification, Generalized, of Infancy, 2 (GACI2) is an ultra-rare autosomal recessive disorder characterized by extensive calcification of the internal elastic lamina of muscular arteries and stenosis due to myointimal proliferation. The exact incidence is unknown, but it is estimated to affect fewer than 1 in 1,000,000 live births. Most cases present in infancy with heart failure, hypertension, and ischemic complications. Prognosis is poor, with many patients dying within the first year of life. There is no approved therapy; management is supportive, focusing on controlling calcification and blood pressure. The disease is caused by mutations in the ENPP1 gene (GACI1) or ABCC6 gene (GACI2). This review focuses on GACI2 (ABCC6-related).

GACI2 is an ideal model for studying ectopic calcification, phosphate metabolism, and vascular biology. The disease is monogenic, making it amenable to gene editing. Public datasets from ClinVar and the 1000 Genomes Project provide mutation frequencies. Open questions include the exact mechanism by which ABCC6 deficiency leads to calcification, the role of circulating factors, and potential therapeutic targets. Gene-edited cell models are essential for functional validation.

The primary pathway involves ABCC6, an ATP-binding cassette transporter primarily expressed in the liver. ABCC6 exports ATP, which is metabolized to pyrophosphate (PPi), a potent inhibitor of hydroxyapatite crystal formation. Loss of ABCC6 function leads to reduced plasma PPi levels, promoting vascular calcification. Additionally, ABCC6 deficiency may affect the expression of genes involved in calcification, such as osteopontin and matrix Gla protein. The pathway can be summarized as:

1. ABCC6 transports ATP out of cells.

2. Extracellular ATP is converted to AMP and PPi by ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1).

3. PPi inhibits calcium-phosphate precipitation.

4. ABCC6 deficiency reduces PPi, leading to uncontrolled calcification.

Data from ClinVar and literature. Most mutations are private, with no common founder mutation.

The primary deregulated network is the PPi/ATP metabolism axis. Key nodes include:

• • ABCC6: transporter, loss of function.
• • ENPP1: enzyme that generates PPi, often mutated in GACI1.
• • ANKH: transporter of PPi, may modulate severity.
• • Tissue-nonspecific alkaline phosphatase (TNAP): degrades PPi, increased activity may exacerbate calcification.

Secondary pathways include inflammation and oxidative stress, which may be upregulated in response to calcification.

Organoids: Liver organoids derived from patient iPSCs can model ABCC6 deficiency and are useful for studying hepatic secretion of factors.

• • Abcc6 knockout mouse: recapitulates ectopic calcification, especially in arteries and eyes.
• • Abcc6 mutant rat: similar phenotype.
• • Induced models: using CRISPR to introduce patient-specific mutations in mice.
• • PDX models are not applicable for GACI2 as it is not a cancer.

CRISPR-Cas9 gene editing enables the creation of isogenic cell lines with ABCC6 knockouts or patient-specific point mutations. For example, a HepG2 ABCC6 knockout line can be generated to study the effect on PPi secretion. Similarly, a HEK293 line with a common mutation (e.g., R1141X) can be created. These models are commercially available from various sources, sequence-verified, and can be used for drug screening and mechanistic studies.

Knockout and knock-in lines are used to validate the function of ABCC6 and identify modifier genes. For example, a genome-wide CRISPR screen in ABCC6 knockout cells can identify genes that, when knocked out, rescue or exacerbate the calcification phenotype.

Isogenic pairs (wild-type vs. knockout) are used to screen for compounds that increase PPi levels or inhibit calcification. Resistance models can be developed by exposing cells to increasing concentrations of a drug to identify mechanisms of resistance.

CRISPR synthetic lethality screens can identify genes that are essential only in ABCC6-deficient cells, providing potential therapeutic targets. Secreted proteins from knockout cells can be analyzed to discover biomarkers for disease monitoring.