Restoration of Adrenal Steroidogenesis by Adenovirus-Mediated Transfer of Human Cytochrome P450 21-Hydroxylase Into the Adrenal Gland of 21-Hydroxylase-Deﬁcient Mice

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Gene Therapy (1999) 6, 1898–1903  1999 Stockton Press All rights reserved 0969-7128/99 $15.00 http://www.stockton-press.co.uk/gt Restoration of adrenal steroidogenesis by adenovirus-mediated transfer of human cytochrome P450 21-hydroxylase into the adrenal gland of 21-hydroxylase-deﬁcient mice

T Tajima1, T Okada2, X-M Ma1, WJ Ramsey2, SR Bornstein1 and G Aguilera1 1Developmental Endocrinology Branch, National Institute of Child Health and Human Development and 2Clinical Gene Therapy Branch, National Human Genome Research Center, NIH, Bethesda, MD, USA

21-Hydroxylase deficiency, a potentially fatal disease due injection. Adrenal morphology of 21OH− mice showed lack to deletions or mutations of the cytochrome P450 21- of zonation, and hypertrophy and hyperplasia of adreno- hydroxylase gene (CYP21), causes congenital adrenal cortical mitochondria with few tubulovesicular christae. hyperplasia (CAH) with low or absent glucocorticoid and These morphological abnormalities were markedly mineralocorticoid production. The feasibility of gene ther- improved 7 days after hAdCYP21 gene therapy. Plasma apy for CAH was studied using 21OH-deficient mice corticosterone increased from undetectable levels to (21OH−) and a replication-deficient adenovirus containing values similar in wild-type mice by 7 and 14 days, declining the genomic sequence of human CYP21 (hAdCYP21). over the next 40 days. This is the first demonstration that Intra-adrenal injection of hAdCYP21 in 21OH− mice a single intra-adrenal injection of an adenoviral vector induced hCYP21 mRNA with the highest expression from encoding CYP21 can compensate for the biochemical, 2 to 7 days before a gradual decline. 21OH activity meas- endocrine and histological alterations in 21OH-deficient ured in adrenal tissue increased from undetectable to lev- mice, and shows that gene therapy could be a feasible els found in wild-type mice 2 to 7 days after AdhCYP21 option for treatment of CAH.

Keywords: gene therapy; adenovirus; 21-hydroxylase; mice; adrenal; CAH

Introduction human disease, the accumulated precursor is progesterone and no androgenization occurs in this model. Also, Congenital adrenal hyperplasia (CAH) due to 21- the deletion of the gene for complement component C4 hydroxylase (21-OH) deficiency is a relatively common makes the 21-hydroxylase-deficient mouse different from autosomal recessive disease in humans, occurring in one the human disease. However, the presence of severe gluc- 1 per 14 000 live births. In this genetic disease, mutations ocorticoid and mineralocorticoid deficiency makes this or deletion of the cytochrome P450 21 hydroxylase gene mouse an ideal animal model to explore the possibility (CYP21) causes glucocorticoid and mineralocorticoid of replacing the defective enzyme in the adrenal by gene deficiency, leading to an excess of ACTH and androgen therapy. Adenoviral vectors have been used to transfer production and adrenal hyperplasia. The classical form genes to cells in order to correct for genetic deficiencies in of the disease which is characterized by severe salt wasting animal models and humans.8–11 While these vectors have 1–3 can be fatal if not promptly diagnosed and treated. several advantages, such as their capacity to infect non- Current steroid replacement regimens have serious prob- dividing cells, and the possibility of obtaining high virals lems with dose adjustment and patient compliance, and titers, their use in gene therapy is limited because of sev- development of new therapeutic strategies for treatment ere immune reactions following administration in of this disease is needed for improving the clinical vivo.12,13 Since steroids are anti-inflammatory,14 we 4,5 management of these patients. sought the possibility that the high content of cortico- A naturally occurring strain of mouse with deletion of steroids in the adrenal gland may prevent the immune the CYP21 and complement 4 component genes has reaction to adenovirus injection and make it possible to impaired 21-OH activity and glucocorticoid production use these vectors for gene therapy in the adrenal. leading to hyperproduction of ACTH with adrenocortical hyperplasia and accumulation of precursor steroids.6,7 Since the mouse adrenal lacks 17-hydroxylase, unlike the Results To determine the ability of adenoviral vectors to trans- duce genes in the adrenal we performed preliminary Correspondence: G Aguilera, Section on Endocrine Physiology, Develop- mental Endocrinology Branch, NICHD, NIH, Bldg 10 Rm 10n262, 10 experiments using an adenoviral construct encoding E. Center Drive MSC 1862, Bethesda, MD 20892-1862, USA coli β-galactosidase under control of the Rous sarcoma Received 5 March 1999; accepted 23 June 1999 virus promoter (AV1.LacZ4). Intra-adrenal injection of AV-mediated induction of CYP21 in 21OH-deficient mice T Tajima et al 1899

Figure 1 Dark ﬁeld photographs of adrenal sections from 21-OH-deﬁcient mice, untreated (a), or 7 days after intra-adrenal injection of adCYP21 (b) hybridized with 35S-labeled oligonucleotides probes complementary to human CYP21 (×50). Light microscopy photograph showing silver grains overlying adrenocortical cells is depicted in (c) (×400).

AV1.LacZ4 induced β-galactosidase staining in the adrenal cortex and medulla up to 40 days after injection, with- out any detectable inflammatory reaction. Therefore, we engineered an adenoviral vector encoding human CYP21, and used the 21-OH-deficient mouse to test the feasibility of adenovirus gene therapy for treatment of enzymatic deficiency in the adrenal. The effectiveness of adCYP21 to induce 21-OH activity was first evaluated by analysis of the conversion of 3H-progesterone to 3H-deoxycorticosterone, by primary pituitary cell cultures, which are devoid of endogenous activity. Cells were exposed to 50 plaque-forming units (p.f.u.) per cell adCYP21 or AV1.LacZ4 for 4 h, and maintained from 4 to 30 days before measuring 21-OH activity. Deoxycorticosterone formation was negligible in nontransfected pituitary cells, or in cells transduced with AV1.LacZ4 at any time- point. In contrast, 4, 7 and 14 days after transduction with adCYP21, cell cultures showed marked 21-OH activity with 74.5 ± 4.6, 47.0 ± 5.1 and 26.6 ± 4.1 percent of progesterone being converted to deoxycorticosterone. Activity continued declining with time but it was still present 30 days after transduction. Based on these in vitro results, we performed intra- adrenal injection of adCYP21 in 21-OH-deficient mice and evaluated 21-OH expression and the biological effects in the adrenal. In situ hybridization showed human CYP21 mRNA in the adrenal cortex and medulla of adCYP21 injected mice but not in AV1.LacZ4 injected control adrenals. Human CYP21 mRNA was expressed in cell clusters comprising about 30% of the adrenal cortex and medulla, with the highest expression occurring 2 to 7 days after injection, and declining but being clearly detectable after 14 days (Figure 1). In vitro analysis of 21-OH activity in adrenal quarters of 21-OH-deficient mice showed that the conversion of 3 3 3 H-progesterone to H-deoxycorticosterone or H-cortico- Figure 2 (a) Serum corticosterone levels before and after intra-adrenal sterone was absent in untreated mice or after intra-adre- injection of adCYP21 in 21-hydroxylase-deficient mice. The mean and s.e. nal injection of AV1.LacZ4. In contrast, adrenals from of values in wild-type mice are shown by the shaded area. (b and c) Time- course of 21-OH activity after intra-adrenal injection of adCYP21 in 21- mice injected with adCYP21 converted progesterone to 3 3 deoxycorticosterone and to corticosterone by 2 to 7 days OH-deficient mice. The formation of H-deoxycorticosterone (b) and H- corticosterone (c) was measured after 2 h incubation of adrenal quarters after injection (Figure 2b and c) to levels similar to those with 1 µm 3H-progesterone, extraction and TLC separation of the steroids observed in adrenals of wild-type mice. Forty days after in the supernatant. The shaded area represents the mean and s.e. of values injection, deoxycorticosterone production had decreased obtained in wild-type mice. AV-mediated induction of CYP21 in 21OH-deficient mice T Tajima et al 1900 but conversion to corticosterone was still at the levels inflammatory responses. This makes the adrenal an seen in adrenals from wild-type mice, indicating a immune privileged organ and an ideal candidate for decrease in 21-OH activity and that all deoxycortico- gene therapy. sterone formed was immediately used as substrate for Although technically difficult (especially in mice), it is 11-hydroxylase. necessary to replace the CYP21 gene or other defective The expression and activity of 21-OH in treated mice steroidogenic enzymes directly in the adrenal. This will was sufficient for production and release of cortico- place the transferred gene in close proximity to other sterone to the circulation in vivo. HPLC analysis showed enzymes responsible for substrate production and for low plasma corticosterone levels (4.7± 0.9 ng/ml) in converting the product (deoxycorticosterone in case of homozygous 21-OH-deficient mice as compared with CYP21) to continue the synthesis of the end-point ster- 44.4 ± 5.6 ng/ml in age-matched controls. After intra- oids, glucocorticoids and mineralocorticoids. Since the adrenal injection of adCYP21, plasma corticosterone lev- rate-limiting steps of the steroidogenic pathway are the els did not change significantly after 4 days, but increased intramitochondrial transport of cholesterol and side chain to levels similar to those in wild-type mice by 7 and 14 cleavage enzyme, which are under the control of ACTH, and 30 days (78 ± 30, 86.0 ± 28 and 36.5 ± 1.0, it is unlikely that overexpression of CYP21 would have respectively) (Figure 2a). any adverse effects on plasma glucocorticoid levels. Thus, We have recently shown marked morphological alter- the increase in circulating corticosterone following resto- ations in adrenal of newborn 21-OH-deficient mice, ration of the adrenal capacity to convert progesterone to including lack of normal zonation and ultrastructural glucocorticoids by gene therapy, would re-establish the abnormalities of the mitochondria.15 Adrenals from negative feedback of ACTH secretion and the capacity to untreated adult mice in these studies showed similar respond to stress. Because of limited availability of 21- alterations. Under light microscopy, the adrenal cortex OH-deficient mice, in these experiments it was not pos- was enlarged, with no clear zonation, a thin capsule, and sible to measure the responsiveness of the hypothalamic fasciculata type cells extending from the capsule to the pituitary adrenal axis to stress or circadian variations of medulla. Numerous chromaffin cells were observed plasma glucocorticoids. However, the fact that 21-OH traversing the cortex as demonstrated by tyrosine activity in adrenal quarters of treated mice reached the hydroxylase staining (Figure 3a). Seven days after gene levels of wild-type mice, suggests that the adrenals of therapy by intra-adrenal injection of adCYP21, the adre- these mice have the capacity to respond to stimulation. nal showed a thick capsule, differentiated zona glomeru- It should be noted that blood samples for plasma cortico- losa, and tyrosine hydroxylase staining corresponding to sterone measurements were obtained under stress con- adrenal medullary cells confined to the center of the ditions (tail clipping under CO2 sedation), and that the organ (Figure 3b). At light microscopy, no evidence of levels were similar in wild-type and treated 21-OH- inflammatory reaction was observed at any time-point deficient mice. Although, it is clear that circulating gluco- after intra-adrenal injection of AV1.LacZ4 or adCYP21. corticoids in 21-OH-deficient mice can reach stress levels At the ultrastructural level, adrenocortical cells in after gene therapy, further studies are needed to deter- untreated mice had increased smooth endoplasmic retic- mine the effects of treatment on the hypothalamic pitu- ulum, hypertrophied and hyperplastic mitochondria with itary adrenal axis responsiveness to stress, and sensitivity protrusion of finger-like projections from one mitochon- of the ACTH responses to glucocorticoid feedback. The drion into an invagination in a neighboring mitochon- volume of the plasma samples did not allow measure- drion. Cells located in the subcapsular region displaying ment of plasma renin activity or aldosterone to evaluate fasciculata-type round hyperplastic mitochondria and the effects of intra-adrenal injection of adCYP21 on salt few tubulovesicular-christae (Figure 3c). Gene therapy and water metabolism. However, the presence of CYP21 induced dramatic improvement in adrenal ultrastructure mRNA in the subcapsular area as well as the improve- in these mice; 7 days after ad CYP injection, mitochon- ment of the adrenocortical structure with differentiation drial size became normal, and subcapsular cells showed of zona glomerulosa type cells observed after gene ther- elongated tubulolamellar christae, typical of the zona glo- apy suggest that the adrenal has also recovered its merulosa. Fasciculata and reticularis cells showed normal capacity to synthesize mineralocorticoids. tubulovesicular mitochondria and SER (Figure 3d). In normal conditions, the high intra-adrenal levels of glucocorticoids rather than circulating steroid are Discussion involved in adrenal differentiation and regulation of cat- echolamine biosynthetic enzymes in chromaffin cells.17 This study shows that recombinant adenoviral vectors Therefore, systemic administration of the gene resulting can be used to correct deficiencies of steroidogenic in little, if any, expression of the enzyme in the adrenal enzymes by administration directly into the adrenal would be unlikely to provide adequate levels of circulat- gland. Adenoviral vectors have the advantage that they ing glucocorticoids in case of increased demand, and can be produced at high titers and can be transduced into would be insufficient for local adrenal regulation. The nondividing cells.8 However, since these vectors also mitochondrial abnormalities in 21-OH-deficient mice deliver large amounts of viral sequences they often cause resemble those described in humans with CAH,18 and host immune reactions and tissue damage, an affect differ from the increase in mitochondrial vesicular mem- which can be prevented by immunosuppressants includ- branes that occurs following ACTH or CRH adminis- ing glucocorticoids.12,13,16 The lack of any detectable tration.19,20 The remarkable improvement of adrenal inflammation in the adrenal following intra-adrenal injec- structure and function after gene therapy contrasts with tion of the adenoviral constructs suggests that in contrast the lack of change observed by suppressing excessive to other tissues, the high levels of endogenous steroids ACTH secretion by corticosterone replacement for 7 days in this tissue may have a protective effect preventing (not shown). This suggests that the alterations in adrenal AV-mediated induction of CYP21 in 21OH-deficient mice T Tajima et al 1901

Figure 3 (a and b) Paraffin sections of mouse adrenal from 6-week-old 21-OH-deficient mice immunostained for tyrosine hydroxylase (×50). In untreated mice (a), adrenals have a thin capsule and no clear zonation. Fasciculata cells reach up to the capsule (arrows) and a normal zona glomerulosa is absent. Chromaffin tissue traverses the cortex and formation of the medulla is incomplete. Fourteen days after transduction of AD-hCYP21 (b), the adrenals have formed a thick capsule and the cortex can be clearly differentiated into three zones including the zona glomerulosa. The medulla is in the center of the gland. CAP, capsule; ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis; ZM, zona medullaris. (c and d) Electron micrographs (×15 000) of adrenocortical cell of 21-OH-deficient mice, untreated and 14 days after transduction of ad-hCYP21. In untreated mice (c) adrenal cells demonstrate an increase in smooth endoplasmic reticulum (SER) and hypertrophy of mitochondria (MIT). Alterations of internal membranes, lipidic inclusions and intermitochondrial herniations (arrow) can be demonstrated. After treatment in (d), the cytoplasm contains ample and large SER and the cells form filopodia (arrow). Mitochondria are round with normal tubulo-vesicular inner membranes. AV-mediated induction of CYP21 in 21OH-deficient mice T Tajima et al 1902 morphology are not solely due to excess ACTH pro- been described previously25 and was kindly provided by duction and supports the view that high intra-adrenal Dr Bruce Trapnell (Genetic Therapy, Gaithersburg, MD, glucocorticoids acting in an autocrine or paracrine man- USA). ner are necessary for a normal adrenocortical structure and migration of chromaffin cells. It is also possible that CYP21 transduction in cultured cells accumulation of abnormal steroids contributes to adrenal Rat pituitary cells were isolated by trypsin digestion and abnormalities in 21-OH-deficient mice. cultured in 24-well plates as previously described.26 Cells It is noteworthy that the striking effects of gene therapy were exposed for 4 h to adCYP21, 50 p.f.u., washed and on adrenal structure and function occur with only about cultured for 2 to 30 days before measurement of 21-OH 30% of adrenocortical cells expressing CYP21. This sug- activity by their ability to convert 3H-progesterone to gests that functional recovery of only a proportion of the 3-deoxycorticosterone. Parallel wells were transduced adrenal cells can lead to production of sufficient gluco- with 50 p.f.u. of adLac-Z in the same experimental con- corticoids to improve adrenal zonation and differen- ditions to evaluate gene transfer efficiency by measure- tiation, and that it would not be necessary to express the ment of the number of cells showing β-galactosidase enzyme in the whole adrenal cortex to obtain thera- staining. peutic benefits. Although the adenoviral vector effectively transduced In vivo procedures the missing CYP21 gene into the adrenal and restored Female 21-OH-deficient mice kindly provided by Dr T normal steroidogenic capacity and anatomic structure of Shiroishi, Institute of Genetics, Mishima, Japan) were the adrenal, expression of the transferred gene started to mated with male C57BL10J mice (Jackson Labs, Bar decline by 14 days. Typically, the duration of expression Harbor, ME, USA). All dams received injections of 5 µg with these vectors is limited because of lack of integration dexamethasone from gestational day 20 until delivery to of the transferred gene to the cell genome.8 Studies using prevent death at birth. Pups were treated with cortico- different viral vectors and stronger promoters to transfer sterone (5 µg per day) fludrocortisone (0.025 µg per day) the CYP21 gene into the adrenal of CYP21[−] mice are until day 14, followed by corticosterone in the drinking currently being performed to increase the magnitude and water until day 21. After this age mice were maintained duration of expression. in standard conditions with regular rodent diet pellets In summary, we have shown that a single intra-adrenal and water ad libitum. Homozygous 21-OH-deficient mice injection of an adenoviral vector encoding CYP21 induces were identified by Southern blot analysis of genomic compensation of biochemical, endocrine and histological DNA and the presence of high plasma progesterone lev- alterations in 21-OH-deficient mice. While development els. Bilateral intra-adrenal microinjections of adCYP21 or of alternative viral vectors can improve the efficiency and ad-LacZ were performed in 2- to 3-month old mice, after duration of gene transfer in the adrenal, the present data surgical exposure of the adrenals via a dorsal approach show that gene therapy could be a feasible option for under ketamine/xylazine anesthesia. Two µl containing treatment of CAH. 108 p.f.u. of adCYP21were injected into each adrenal at the rate of 0.4 µl/min using a Hamilton syringe and a PE-10 polyethylene tubing attached to a 32-gauge stain- Materials and methods less steel needle. Plasma corticosterone levels were measured by high-performance liquid chromatography Adenovirus construction (HPLC) at days 4, 7, 14 and 30 after injection. Mice were The construction of AVC2.null has been described else- killed by decapitation from 2 to 40 days after injection where.21 Briefly, the virus consists of a ‘left viral arm’ and the adrenals removed for determination of human containing 400 bp of the left terminus of Ad5, the CYP21-OH expression, 21-OH activity or histological immmediate–early promoter of cytomegalovirus, and a analysis. All animal protocols were approved by the multiple cloning site. The ‘right viral arm’ contains the Animal Care Users Committee, NICHD. SV40 early intron and polyadenylation site connecting to viral sequences derived from pJM17, nt 3328 to the right Plasma steroid levels viral terminus.22,23 A 3.1 kb DNA fragment containing the Blood was collected into non-heparinized capillary tubes

full genomic sequence of the human CYP21-OH was after tail clipping under CO2 sedation. Serum cortico- obtained by PCR and cloned into pBluescript SK+ sterone levels were measured quantitatively by HPLC as (Stratagene, La Jolla, CA, USA). The DNA fragment previously described.27 Serum progesterone concen- extends from 45 bases upstream of the genomic cyp21 trations were determined using commercial kit reagents transciptional start site and extends 2700 bases down- from Diagnostic Systems Laboratories (Webster, TX, stream, including all nine exons and introns with the USA). exclusion of the last 250 bases of 39 untranslated region (including the Cyp 21 polyadenylation site). The CYP21 21-OH expression and activity gene was excised from pBluescript by digestion with XbaI The presence of human CYP21-OH was determined by and ClaI and ligated directly into AVC2.null using the in situ hybridization using 12 µm cryostat sections and a DNA–protein complex directional ligation technique of specific 35S-labeled oligonucleotide complementary to Okada et al.21 The DNA–protein complex was introduced base pairs 350 to 386 of exon 2 of the human 21-OH gene, into 293 cells by calcium phosphate cotransfection, and according to previously described procedures.28 21-OH viral isolates screened by PCR and selected clones ampli- activity was determined by the ability of cell cultures or fied by standard techniques.24 AV1.lacZ4, a first-gener- adrenal quarters to convert 3H-progesterone to 3H-deoxy- ation adenoviral vector expressing E. coli β-galactosidase corticosterone and corticosterone. 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