Oral Presentation ESA-SRB-APEG-NZSE 2022

Recombinant AAV-mediated liver-targeted dual adrenal enzyme gene delivery in a model for congenital adrenal hyperplasia (#250)

Lara Graves 1 2 3 , Sundar Koyyalamudi 1 , Tiffany Wotton 4 , Shubha Srinivasan 1 3 , Samantha Ginn 2 5 , Ian E Alexander 2 3 6
  1. Institute of Endocrinology and Diabetes, The Children's Hospital at Westmead, Westmead, NEW SOUTH WALES, Australia
  2. Gene Therapy Research Unit, Children's Medical Research Institute and Sydney Children's Hospitals Network, Westmead, NSW, Australia
  3. Children’s Hospital Westmead Clinical School, University of Sydney, Westmead, New South Wales, Australia
  4. Newborn Screening, The Children's Hospital at Westmead, Westmead, New South Wales, Australia
  5. School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia
  6. Discipline of Genetic Medicine, Children’s Hospital Westmead Clinical School, University of Sydney, Westmead, NSW, Australia

Despite the development of steroid replacement therapy in the 1950s(1-3), excess morbidity and mortality remains for people with congenital adrenal hyperplasia (CAH)(4), and alternative treatment options are sought. A recombinant adeno-associated viral (rAAV) vector could deliver wild-type CYP21A2, however one caveat is that rAAV vector genomes are predominantly episomal, therefore not replicated during cell division(5). The adrenal cortex continuously renews itself from progenitor cells(6). As a result, episomal gene therapy used in the adrenal cortex will only have a transient effect(7). A potential alternative is to induce ectopic expression of 21-hydroxylase in a stable organ extra-adrenally. We have shown that ectopic expression of 21-hydroxylase in the murine liver can restore aldosterone production and improve corticosterone (major glucocorticoid in the mouse(8))(9). This study sought to further restore corticosterone through the delivery of both CYP21A2 and CYP11B1 to the murine liver in 21-hydroxylase deficient mice.

Two vectors were produced: rAAV8-CYP21A2 and rAAV8-CYP11B1, both with a liver-specific promoter. Mice were injected intravenously with either rAAV8-CYP21A2 alone or with a stoichiometric mix of both vectors and harvested four weeks later. Using liquid chromatography tandem mass spectrometry, steroid profiles were compared between wild-type, and untreated, single enzyme (CYP21A2) treated and dual enzyme (CYP21A2 and CYP11B1) treated Cyp21a1-/- mice.

The median serum corticosterone was higher in dual enzyme vector treated than single vector treated Cyp21a1-/- mice, with dual vector treated corticosterone levels for both sexes restored to that of wild-type males (Figure 1).

Ectopic expression of enzymes CYP21A2 and CYP11B1 in hepatocytes of 21-hydroxylase deficient mice increased production corticosterone further than ectopic CYP21A2 expression alone. While further studies are necessary, ectopic 21-hydroxylase expression could be an alternative genetic therapeutic option that overcomes the limitations of episomal gene therapy in a rapidly turning over organ such as the adrenal cortex.

 

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  1. Bartter FC, Albright F, Forbes AP, Leaf A, Dempsey E, Carroll E. The effects of adrenocorticotropic hormone and cortisone in the adrenogenital syndrome associated with congenital adrenal hyperplasia: an attempt to explain and correct its disordered hormonal pattern. J Clin Invest. 1951;30(3):237-51
  2. Wilkins L, Lewis RA, Klein R, Gardner LI, Crigler JF, Jr., Rosemberg E, et al. Treatment of congenital adrenal hyperplasia with cortisone. J Clin Endocrinol Metab. 1951;11(1):1-25.
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  5. Alexander IE, Cunningham SC, Logan GJ, Christodoulou J. Potential of AAV vectors in the treatment of metabolic disease. Gene Ther. 2008;15(11):831-9.
  6. Chang SP, Morrison HD, Nilsson F, Kenyon CJ, West JD, Morley SD. Cell proliferation, movement and differentiation during maintenance of the adult mouse adrenal cortex. PLoS One. 2013;8(12):e81865.
  7. Markmann S, De BP, Reid J, Jose CL, Rosenberg JB, Leopold PL, et al. Biology of the Adrenal Gland Cortex Obviates Effective Use of Adeno-Associated Virus Vectors to Treat Hereditary Adrenal Disorders. Hum Gene Ther. 2018;29(4):403-12.
  8. Nandi J, Bern HA, Biglieri EG, Pieprzyk JK. In vitro steroidogenesis by the adrenal glands of mice. Endocrinology. 1967;80(4):576-82.
  9. Graves, LE, Koyyalamudi, S, Wotton, T, Srinivasan, S, Ginn, S and Alexander, IE. Inducing ectopic 21-hydroxylase expression in a mouse model for congenital adrenal hyperplasia through hepatocyte-targeting gene addition, Emerging Investigators Session presented at: Australasian Paediatric Endocrine Group Annual Scientific Meeting; 2021 Nov 22-23; Virtual Event