Investigating antisense oligonucleotides for reducing ACTH production in an in vitro model of Cushing’s disease

Background: Cushing’s disease (CD) is caused by high levels of blood cortisol resulting from excess secretion of adrenocorticotropic hormone (ACTH) from a corticotroph adenoma in the anterior pituitary gland. Clinical features include hypertension, diabetes, osteoporosis, and depression. If untreated CD has an increased mortality of five-fold owing to cardiovascular comorbidities, stroke and raised vulnerability to infection. Transsphenoidal surgery is considered the first-line treatment, but remission is achieved in only 65% of cases and the relapse rate is high. Furthermore, medical treatments are often accompanied by unpleasant side-effects. Antisense therapy is a technique for suppressing gene expression at the level of translation using antisense oligonucleotides (ASOs) against the mRNA of interest. Aims: Using an in vitro AtT-20 cell model, the overall aim of the project was to investigate antisense therapy as a treatment for CD by targeting ASOs against ACTH�encoding Pomc mRNA thereby reducing production of the hormone. Methods: Computer-aided design of POMC ASOs, transfection of ACTH�hypersecreting AtT-20 cells with POMC ASOs, measurement of ACTH by immunoassay and the innate immune response by ELISA, qualitative and quantitative assessment of ASO nuclease degradation, and statistical analysis using ANOVA and Student’s t tests. Results: Following ASO design guidelines and using ASO design programs, four POMC ASOs targeted at different exon sites were selected for experimentation. The ASOs were used unmodified, with a phosphorothioate-modified backbone, or with 2’- O-methyl- or locked nucleic acid (LNA)-modified end-nucleotides. All POMC ASOs significantly reduced ACTH secretion from AtT-20 cells when compared with untreated cells or control ASOs (14-59% of normal ACTH levels; ANOVA, P > 0.05). LNA�modified POMC ASOs were the most effective when used at lower concentrations (1 nM) and over time (five days). Modified ASOs were more stable to nuclease degradation. None of the ASOs appeared to stimulate the expression of cytokines associated with the innate immune response.