Endocrine Abstracts

JOINT2046

Despite life-saving corticosteroid treatment, individuals with congenital adrenal hyperplasia (CAH) have increased morbidity and mortality. Gene therapy has the potential to restore physiological corticosteroid production, however, optimisation of the technology is required. Recombinant adeno-associated virus (rAAV) is the most common vector used in in vivo gene delivery. rAAV-based gene addition strategies have been explored in CAH, however due to adrenocortical cellular renewal, resultant phenotypic changes were temporary. Here, we introduced targeted editing events in the 21-hydroxylase locus (Cyp21a1) in the murine adrenal gland in vivo through rAAV-delivered genomic editing, with phenotypic effect to 15 weeks. Homology-independent targeted integration was utilised. A single-guide RNA (sgRNA) was designed targeting the first intron and a matching donor cassette was designed capturing the endogenous Cyp21a1 promoter. Both vectors were pseudo-serotyped with AAV-Rh10. Adult 21-hydroxylase deficient mice (C57BL/10SnSlc-H-2^aw18) were treated intravenously with the Staphylococcus aureus Cas9 (SaCas9)/sgRNA (2.5×10e12vg/mouse) and/or donor (1×10e12vg/mouse) vectors and harvested 4 (short-term) or 15 (long-term, dual vector only) weeks later. Sanger sequencing demonstrated editing events at the desired locus within the Cyp21a1 gene in the dual vector-treated mice. Correctly oriented donor inserts were detected in 7% of total Cyp21a1 alleles and 39% of total Cyp21a1 transcripts in the short-term mice. Treated mice had improved corticosterone production, with 6.7-fold (males) and 9-fold (females) increases. There was no phenotypic benefit in the control groups that received a single vector. Four female mice were harvested at 15 weeks after treatment to determine durability of effect, as the adrenocortical turnover period has been estimated at 3 months. Correctly oriented donor inserts accounted for 5% of the total alleles and 25% of the total expressed transcripts in the adrenal. These mice had persistence of improvement in corticosterone, with no statistical difference between the serum corticosterone level in the group harvested at 4 weeks compared with the group harvested at 15 weeks. Renin expression was lower in the long-term group compared with the short-term group. There was no statistically significant difference in serum aldosterone, serum progesterone, aldosterone synthase expression or adrenal size between these treated groups. We demonstrated that rAAV delivery of genomic editing reagents to the adrenal gland resulted in targeted editing events that conferred durable phenotypic benefit. Targeted editing events in the Cyp21a1 gene is possible despite challenges faced with the homologous pseudogene. This strategy could be adapted for use in the human locus once rAAV tropism for the human adrenocortical progenitor cells is determined.