LBODP008 Gene Therapy For Congenital Adrenal Hyperplasia With AAV Vectors Into Fibroblasts, IPS Cells And Model Mice

BACKGROUND: Congenital adrenal hyperplasia (CAH) is due to defects of steroid synthetic enzymes, which includes microsomal and mitochondrial P450s. Microsomal P450s include 21-hydroxylase and 17α-hydroxylase/17-lyase. 21-hydroxylase deficiency (21-OHD), in which CYP21A2 is mutated or deleted, is the most common cause of CAH and results in underproduction of glucocorticoids and mineralocorticoids, and overproduction of androgens. 11β-hydroxylase deficiency (11β-OHD), which is a defect of a mitochondrial P450, is the second common cause of CAH. Patients with CAH are treated with oral steroid supplementation, but optimal control of blood steroid levels remains difficult. Thus, new therapeutic approaches are still needed. Intravenous adeno-associated virus (AAV)-mediated administration of human CYP21A2 into adult 21-OHD patients has been reported at the ENDO 2021 meeting. In this study, we examined the effects of induction of causative genes with an AAV vector into fibroblasts from patients and iPS cells and adrenal glands of model mice. METHODS: Fibroblasts from CAH patients were subjected to primary culture. The cells were infected with a serotype-2 AAV vector (AAV2) containing defected genes and cultured in steroid substrate-containing medium for 24 hours. We measured steroid metabolites in the medium by liquid chromatography tandem mass spectrometry and evaluated gene expressions by RT-PCR. In addition, iPS cells were established from fibroblasts of 11β-OHD and differentiated into adrenocortical cells with retinoic acid and cAMP. They were infected with a serotype-9 AAV vector (AAV9) containing CYP11B1 cDNA. Steroid metabolites in the culture medium were measured and gene expressions were evaluated by RT-PCR. 11β-OHD model mice were made by a gene-editing method. We injected AAV9 containing Cyp11b1 cDNA into the adrenal gland and measured serum DOC and corticosterone levels before and every 4 weeks after injection. RESULTS: AAV2 infected fibroblasts from 21-OHD and 17α-hydroxylase/17-lyase deficiency can metabolite steroid precursors but failed to convert DOC to corticosterone in 11β-OHD. Differentiation into adrenocortical cells from iPS cells of 11β-OHD was confirmed by expression of CYP21A2. AAV9 gene induction of adrenal cortical cells successfully induced 11β-hydroxylase activity. 11β-OHD mice also showed improved steroid synthesis after AAV9 adrenal induction. CONCLUSION: These results indicate that extra-adrenal induction of CYP21A2 may ameliorate steroid metabolism in 21-OHD patients. In contrast, 11β-OHD needs AAV9 adrenal induction to treat the steroid abnormality. Our results suggest that specific gene therapeutic strategies are needed to be adapted for each type of CAH. Presentation: No date and time listed