SAT264 Effects Of Coenzymes In Gene Therapy For Congenital Adrenal Hyperplasia With AAV Vectors Into Model Mice

Disclosure: Y. Naiki: None. M. Miyado: None. R. Horikawa: None. N. Katsumata: None. S. Takada: None. M. Fukami: None. Congenital adrenal hyperplasia (CAH) is due to defects of steroid synthetic enzymes, which includes microsomal and mitochondrial P450s and they need coenzymes respectively. Microsomal P450s include 21-hydroxylase need P450 oxidoreductase which coded by POR and ferredoxin and ferredoxin reductase, which coded by FDX and FDXR, respectively, work for mitochondrial P450s include 11β-hydroxylase. 21-hydroxylase deficiency (21-OHD), in which CYP21A2 is mutated or deleted, and 11β-hydroxylase deficiency (11β-OHD), which caused by mutated CYP11B1 are the top two cause of CAH. CAH are treated with oral steroid supplementation, but optimal control remains difficult. Thus, new therapeutic approaches are still needed. We succeeded gene therapy for model mice of CAH and iPS cell from CAH patients (1). In this study, we examined the effects of induction of coenzyme genes with an AAV vector into a model mouse. A 21OHD mouse were made by breeding with H-2aw18 haplotype mice and treated with subcutaneous corticosteroid injection until 3 weeks of age. A 11β-OHD mouse were made by using the CRISPR/Cas9 method. A type 9 AAV (AAV9) containing Cyp21a cDNA (AAV9-Cyp21a1) was constructed with pAAV-CMV-shuttle. AAV9 containing Por cDNA (AAV9-Por), Cyp11b1 cDNA (AAV9-Cyp11b1), Fdx (AAV9-Fdx), Fdxr (AAV9-Fdxr) were also made. An AAV9-Cyp21a1 and AAV9-Por were mixed and injected into limbs muscles of a 21-OHD mouse and AAV9-Cyp11b1 mixed with AAV9-Fdx and AAV9-Fdxr injected into limbs muscles of 11β-OHD mouse. Serum progesterone (P4) and deoxycorticosterone (DOC) concentrations for 21-OHD mouse and DOC and corticosterone (B) concentrations for 11β-OHD mouse were measured before and after injection. P4 and DOC concentrations were measured by LC-MS/MS. An AAV9-Cyp21a1 and AAV9-Por injected mouse showed improved steroid synthesis up to 7 months after AAV9 induction. Comparing with a mouse injected only AAV9-Cyp21a1, significant reduction of P4/DOC was observed after 4 weeks of injection but there was no difference in the effectiveness of Cyp21a1 and Por combined treatment at the end of observation. Instead of a 21-OHD mouse, a 11β-OHD mouse did not show significant improvement by AAV mixture injected. These results indicate that extra-adrenal induction of Cyp21a1 and Por may ameliorate steroid metabolism in 21-OHD model mice. Although this coenzyme combined therapy showed some effects, but no long-term improvement of the efficacy exhibited. Instead of microsomal P450s, extra-adrenal induction with coenzyme genes did not work enough for mitochondrial P450s defects. Coenzymes of P450s are necessary for their activity, but our results suggest that the coenzyme combined gene therapeutic strategies are needed to be improved. Reference: (1) Naiki et al., Hum Gene Ther. 2022 Aug;33(15-16):801-809. Presentation: Saturday, June 17, 2023