SAT-425 Gene Knockout Cells For Personalized Models Of P450 Oxidoreductase Deficiency

Abstract Disclosure: A. Matveeva: None. T. Du Toit: None. A.V. Pandey: None. Introduction: P450 oxidoreductase (POR) is a flavoprotein that facilitates electron transfer from NADPH to microsomal cytochrome P450 proteins (CYPs) for metabolism of steroids, including sex hormones biosynthesis in adrenal glands and gonads. POR deficiency can lead to metabolic disorders in humans including disrupted sexual development, skeletal abnormalities, and congenital adrenal hyperplasia. To date, over 200 mutations in POR that cause loss of function have been discovered, with a few of them being characterized using purified protein systems. To gain a deeper understanding of POR activity within the living cell context, further studies using human cell lines are essential. Methods: The genomes of the NCI - H295R (adrenal carcinoma cells) and HEK 293T (human embryonic kidney cells) were edited using CRISPR/Cas9 system with sgRNAs targeting 4th exon of the POR gene. Resulting cells were subcloned to obtain monogenic cultures representing unified genotype. To evaluate the status of POR, we employed Sanger sequencing of POR gene, Western blot analysis, and the assays on POR function. For HEK 293T cells, the CYP17A1 and CYP21A2 activity were measured using thin layer chromatography. For NCI - H295R cells, the full steroid profile was obtained using LC-MS/MS. Results: POR gene knockout cell models were developed using human adrenal carcinoma and embryonic kidney cell lines. Adrenal - derived POR knockout cells were characterized by the significant inhibition of aldosterone, cortisol, and testosterone pathways indicating reduced activities of CYP21A2 and CYP17A1 - two key metabolic partners of POR in steroid biosynthesis. The Progesterone levels were significantly higher in POR knockout adrenal cells and 17OH-Progesterone levels were comparable to controls. In HEK293T cells, loss of POR showed 3.6 % of wild-type activity towards CYP21A2 and 0% activity towards CYP17A1. Conclusion: The generated cell lines resemble the steroid abnormalities found in patients with POR deficiency and have the advantage of lacking endogenous POR activity, which removes background interference. It allows us to study the patient-specific POR mutations, supporting the creation of personalized disease models. By reflecting individual genetic profiles, these cell models offer valuable insights into the complex metabolic processes in the adrenal glands while also serving as a screening platform to characterize the impact of the POR mutations on steroidogenesis and drug response. These models can greatly improve both the diagnosis and treatment of POR deficiency. This study was funded by the Swiss National Science Foundation grant number 310030M_204518 to Amit V Pandey (www.pandeylab.org) Presentation: Saturday, July 12, 2025