6589 Antisense Oligonucleotides for Reducing ACTH Secretion in a Model of Cushing's Disease

Abstract Disclosure: M.S. Varughese: None. H. Eltumi: None. E.H. Kemp: None. J.D. Newell-Price: None. Antisense Oligonucleotides for Reducing ACTH Secretion in a Model of Cushing’s Disease Background: Cushing’s disease (CD) is a multi-system disorder with high morbidity and mortality characterised by pathologically raised serum cortisol due excess expression of proopiomelanocortin (Pomc) and overproduction of ACTH by a pituitary corticotroph adenoma. Trans-sphenoidal adenomectomy is the treatment of choice, but there is a 30% recurrence rate over time. Advances in medical therapy are needed. Antisense oligonucleotides (ASOs) can silence mRNA translation by specific base-pairing. Locked nucleic acid (LNA) and 2’-O-methyl (OMe) ribose terminals make ASOs more effective by improving affinity and resistance to nuclease degradation. Aims: To investigate the effectiveness of LNA- and OMe-modified anti-Pomc ASOs at reducing ACTH secretion from murine adrenotropic tumour (AtT20) cells. To analyse the nuclease resistance of Pomc ASOs and their ability to drive an immune response. Methods: Two ASOs (ASO2 and ASO3) were designed against Pomc with phosphorothioate backbones modified with either LNA or OMe ribose terminals. These were used in lipofectamine-mediated cell transfections of AtT20 cells and ACTH in the culture medium measured by immunoassay. ASO stability was assessed by nuclease degradation assays and immunogenicity by cytokine ELISAs. Results: When compared with untreated AtT20 cells, LNA- and OMe-modified Pomc ASOs at 1 nM significantly suppressed ACTH secretion for up to 120 h and 96 h, respectively (n = 4; Unpaired t tests, all P values < 0.05). In contrast, control treatments, including scrambled and mismatched ASOs, did not cause a significant reduction. For comparison, 1 nM of ASO2-OMe, ASO2-LNA, ASO3-OMe and ASO3-LNA lowered ACTH at 24 h by 62%, 71%, 63% and 79%, respectively. Modified Pomc ASOs showed 14-31% degradation over a 120-min incubation period with 3’- and 5’-exonucleases, whilst equivalent unmodified Pomc ASOs were completely degraded. No secretion of IFN-α, IFN-β, TNF-α, IL-6 or IL-1β was detected following transfection of AtT20 cells with Pomc ASOs. Conclusions:Pomc ASOs caused significant reduction of ACTH secretion from AtT-20 cells. LNA-modified Pomc ASOs were more effective than those with OMe modifications, with neither stimulating a detectable immune response. These ASOs hold promise as a systemic therapy for Cushing’s disease. Presentation: 6/3/2024