Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias

Abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3) are rare recessive disorders of lipoprotein metabolism due to mutations in MTTP and SAR1B genes, respectively, which lead to defective chylomicron formation and secretion. This results in lipid and fat-soluble vitamin malabsorption, which induces severe neuro-ophthalmic complications. Currently, treatment combines a low-fat diet with high-dose vitamin A and E supplementation but still fails in normalizing serum vitamin E levels and providing complete ophthalmic protection. To explore these persistent complications, we developed two knock-out cell models of FHBL-SD1 and FHBL-SD3 using the CRISPR/Cas9 technique in Caco-2/TC7 cells. DNA sequencing, RNA quantification and Western blotting confirmed the introduction of mutations with protein knock-out in four clones associated with i) impaired lipid droplet formation and ii) defective triglyceride (-57.0 +/- 2.6% to -83.9 +/- 1.6%) and cholesterol (-35.3 +/- 4.4% to -60.6 +/- 3.5%) secretion. A significant decrease in alpha-tocopherol secretion was also observed in these clones (-41.5 +/- 3.7% to -97.2 +/- 2.8%), even with the pharmaceutical forms of vitamin E: tocopherol-acetate and tocofersolan (alpha-tocopheryl polyethylene glycol succinate 1000). MTTP silencing led to a more severe phenotype than SAR1B silencing, which is consistent with clinical observations. Our cellular models thus provide an efficient tool to experiment with therapeutic strategies and will allow progress in understanding the mechanisms involved in lipid metabolism.

Automated summary

Abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3) are rare recessive disorders caused by mutations in the MTTP and SAR1B genes, respectively. These mutations lead to defective chylomicron formation and secretion, resulting in lipid and fat-soluble vitamin malabsorption and severe neuro-ophthalmic complications. Current treatment options are not fully effective in normalizing serum vitamin E levels and providing complete ophthalmic protection.