SUN-182 - Minimally Invasive and Sustained Therapy using AAV Vectors Containing Hydroxylase Genes Combined with Coenzymes for a Mouse Model of Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) results from defects in steroidogenic enzymes including both microsomal and mitochondrial cytochrome P450 enzymes, each of which requires specific electron-transfer coenzymes. Microsomal P450 enzymes such as 21-hydroxylase require P450 oxidoreductase (POR) as their electron donor. Mitochondrial P450 enzymes, including 11β-hydroxylase, depend on ferredoxin (FDX) and ferredoxin reductase (FDXR) for electron transfer. 21-hydroxylase deficiency (21-OHD) and 11β-hydroxylase deficiency (11β-OHD) are the two most common forms of CAH and lead to reduced glucocorticoid and mineralocorticoid synthesis with excess androgen production. Although CAH is managed with oral glucocorticoid and mineralocorticoid replacement, achieving optimal hormonal control remains challenging. We previously demonstrated successful gene therapy in CAH mouse models and patient-derived iPS cells (1). Intravenous AAV-mediated delivery of human CYP21A2 in adult 21-OHD patients has been reported (2).
In this study, we evaluated whether AAV-mediated delivery of coenzyme genes could correct steroidogenic abnormalities in CAH mouse models. CRISPR/Cas9 technology was used to generate both 21-OHD and 11β-OHD mouse models. AAV9 vectors were engineered for each CAH subtype. For 21-OHD mice, AAV9 vector expressing both Cyp21a and Por cDNAs under a muscle specific promotor was constructed (AAV9-Cyp21a1-Por). For 11β-OHD mice, a single AAV9 vector expressing a fused Cyp11b1-Fdx-Fdxr cDNA under an adrenal cortex specific promotor was generated. (AAV9-Cyp11b1-Fdxr-Fdx). 1.0X10e12 vector genomes of AAV9-Cyp21a1-Por were injected into the limb muscles of 21-OHD mice. 1.0X10e12 vector genomes of AAV9-Cyp11b1-Fdxr-Fdx injected intravenously into 11β-OHD mice. Serum steroid levels were quantified before and after treatment using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In 21-OHD mice, the P4/DOC ratio decreased approximately by 90% and remained improved for up to 7 months. In 11β-OHD mice, the DOC/B ratio decreased by nearly 99%, comparable to our previous results with direct adrenal injection (1). These findings suggest that the coenzyme-supplemented gene therapy offer a minimally-invasive and sustained treatment strategy for patients with CAH.

Reference: (1) Naiki et al., Hum Gene Ther. 2022 Aug;33(15-16):801-809. (2) Merle et al., J Endo Soc. 2021 Apr-May;5(sup1):A82