AAV-delivered hepato-adrenal cooperativity in steroidogenesis: Implications for gene therapy for congenital adrenal hyperplasia

Abstract

Despite the availability of life-saving corticosteroids for 70 years, treatment for adrenal insufficiency is not able to recapitulate physiological diurnal cortisol secretion and results in numerous complications. Gene therapy is an attractive possibility for monogenic adrenocortical disorders such as congenital adrenal hyperplasia; however, requires further development of gene transfer/editing technologies and knowledge of the target progenitor cell populations. Vectors based on adeno-associated virus are the leading system for direct in vivo gene delivery but have limitations in targeting replicating cell populations such as in the adrenal cortex. One strategy to overcome this technological limitation is to deliver the relevant adrenocortical gene to a currently targetable organ outside of the adrenal cortex. To explore this possibility, we developed a vector encoding human 21-hydroxylase and directed expression to the liver in a mouse model of congenital adrenal hyperplasia. This extra-adrenal expression resulted in reconstitution of the steroidogenic pathway. Aldosterone and renin levels normalized, and corticosterone levels improved sufficiently to reduce adrenal hyperplasia. This strategy could provide an alternative treatment option for monogenic adrenal disorders, particularly for mineralocorticoid defects. These findings also demonstrate, when targeting the adrenal gland, that inadvertent liver transduction should be precluded as it may confound data interpretation.

Keywords: adeno-associated virus; congenital adrenal hyperplasia; gene addition; gene therapy; steroidogenesis.

Graphical abstract

Figure 1

Study set up to assess hepato-adrenal cooperativity in steroidogenesis (A) Hypothesis: in 21-hydroxylase deficiency, the precursor steroid (progesterone in the mouse, 17-hydroxyprogesterone in the human) accumulates and enters the systemic circulation where it will circulate to the liver. Recombinant AAV-derived human 21-hydroxylase expressed in the liver will convert progesterone to deoxycorticosterone, which will then enter the systemic circulation, reaching the adrenal gland. Enzymes downstream of 21-hydroxylase will be unaffected and will be able to complete steroidogenesis. (B) Recombinant AAV vector genome; liver-specific enhancer-promoter (ApoE-hAAT), human 21-hydroxylase cDNA (hCYP21A2), and bovine growth hormone poly-adenylated tail (polyA). The scale bar represents 500 base pairs. (C) Prior to treatment, dried whole blood was collected on to filter paper. Mice (n = 5 male, n = 5 female) were administered the purified vector intravenously via the tail vein and harvested 4 weeks later. AAV, adeno-associated virus.

Figure 2

Robust delivery to and expression of human CYP21A2 in the murine liver (A) Vector DNA detected in the livers of treated mice. (B) The ratio of vector-derived mRNA transcripts to murine albumin transcripts. (C) Vector copy number vs. vector expression in males (red) and females (black). (D) IHC staining demonstrated CYP21A2 protein (red) in the liver from a representative treated male −/− mouse with 20.5 vcn/diploid nucleus and transcript mRNA ratio 0.88. Scale bar represents 100 µm. DAPI nuclear staining in blue. vcn, vector copy number; IHC, immunohistochemistry; −/−, homozygous 21-hydroxylase deficiency; ♂, male; ♀, female; Rx, homozygous 21-hydroxylase deficient mice that were treated with vector; CYP21A2, human 21-hydroxylase; mAlb, murine albumin; ns, not significant. Individual data points are shown, and error bars represent median and interquartile range. ∗p < 0.05 on Mann-Whitney U test.

Figure 3

Improvement in steroidogenesis following hepatic CYP21A2 expression (A) Serum aldosterone. (B) Serum corticosterone. In the call-out box, +/+ controls were removed to demonstrate more clearly the change in serum corticosterone levels in −/− treated vs. −/− untreated. (C) Dried whole blood corticosterone before treatment and at harvest in males (red) and females (black). (D) Serum progesterone. (E) Renin expression demonstrated as the ratio of renal renin (Ren1) mRNA transcripts to Tbp transcripts. +/+, wild-type 21-hydroxylase sufficiency; −/−, homozygous 21-hydroxylase deficiency; ♂, male; ♀, female; Rx, homozygous 21-hydroxylase deficient mice that were treated with vector; Ren1, renin; Tbp, TATA-box binding protein; ns, not significant. Individual data points are shown, and error bars represent median and interquartile range. ∗p < 0.05; ∗∗p < 0.01 on Mann-Whitney U test. ∗∗∗p < 0.001 on two-way ANOVA.

Figure 4

Reduction in adrenal hyperplasia after hepatic CYP21A2 expression (A) Absolute bilateral adrenal mass. (B) Macroscopic photographs of representative adrenal glands after fat dissection. +/+, wild-type 2-hydroxylase sufficiency; −/−, homozygous 21-hydroxylase deficiency; ♂, male; ♀, female; Rx, homozygous 21-hydroxylase-deficient mice that were treated with vector. Individual data points are shown, and error bars represent as median and interquartile range. ∗∗p < 0.01 on Mann-Whitney U test.