Endocrine Abstracts

RNA Interference as therapy for Cushing's disease

A Munir

University of Sheffield, Sheffield, UK.

---

Hypothesis: That siRNAs could be translated into systemic therapy for Cushing’s disease in man.

Background and importance: Cushing’s disease is an uncommon but devastating condition, with a mean age of onset of 36 years, and a fivefold excess mortality when inadequately treated. Major complications include diabetes, hypertension, osteoporosis, life-threatening infection, depression and psychosis. No established ACTH-lowering medical therapy exists.

Ninety-five percentage of cases are caused by a tiny benign corticotrope adenomas of the pituitary gland, often only millimetres in diameter, and in 40% of cases are invisible on MRI. These adenomas express the pro-hormone ‘pro-opiomelanocortin’ (POMC), which undergoes post-translational processing to ACTH, before secretion into the systemic circulation. ACTH drives the adrenal glands to synthesise and secrete excessive amounts of the hormone cortisol, which causes the clinical condition of Cushing’s disease.

Invasive neurosurgery aimed at removal of the pituitary tumour, causes long-term remission in only 50% of patients with Cushing’s disease and results in hypopituitarism in approximately half. Hypopituitarism is associated with a twofold excess mortality. Bilateral adrenalectomy is effective treatment but is complicated by life-long hypoadrenalism (twofold excess mortality) and the development of an expanding, and pituitary mass (Nelson’s syndrome) in up to 50% of cases. Use of adrenal steroidogenesis inhibitors is not effective in long-term management.

Unmet clinical need: Patients with Cushing’s disease have inadequate therapeutic options. A reliable ACTH-lowering therapy would represent a significant advance.

Work leading up to project: POMC is expressed from a tightly tissue-restricted promoter active in the anterior pituitary – a highly vascular organ that lies outside the blood brain barrier. Using a careful bio-informatic approach I have designed in house three unique short interfering RNAs (siRNAs), with homology to mouse and human genomes with no predicted off-target effects, that cause remarkably long-lived silencing of POMC in vitro: in AtT20 (mouse corticotrope) cells a single treatment reduced POMC mRNA (assessed by qPCR) by 95% at 24 h, and 80% at 10 days (P<0.001 and P<0.005), whilst secreted ACTH levels in the culture media were reduced by 56 and 80%, respectively (P<0.05). Specificity of effect has been established according to published guidelines, including siRNAs with 1–3 mismatched nucleotides having no effect, and constant expression of β-actin as a housekeeping gene. Delivery was achieved by lipid-based and novel nanopolymer technologies. Furthermore six siRNAs targeting the POMC promoter region reduced mRNA levels by 90 and 56% at 96 and 120 h respectively (P<0.0001 and P<0.001) without de novo methylation, as assessed by bisulphite sequencing. Following ethical approval I have then established a pituitary culture methodology and then used these siRNAs in human pituitary tumour cells from patients undergoing pituitary surgery. Cultured ex vivo, corticotrope cells demonstrated 40% knockdown of ACTH secretion after a single SiRNA treatment, but there was no effects on other pituitary hormones in prolactin or growth hormone-secreting tumours cultured in the same fashion. I have then tested these siRNAs in vivo, using a validated mouse model of Cushing’s syndrome with AtT20 cell tumours grown as subcutaneous xenografts in nude mice, and shown a 40% fall in high levels of circulating ACTH up to six days after a single systemic administration of the siRNAs by tail vein injection.

Conclusions: I have shown, for the first time, significant reduction of secreted ACTH levels caused by siRNAs administered to human corticotrophe cells in vitro, and when administered systemically in an in vivo model of Cushing’s disease. These data support the hypothesis that these siRNAs could be translated into a novel, effective and long-lasting therapy for Cushing’s disease. Further in vivo experiments are in progress.

Note: The above abstract does not appear in the print version of the abstract book.

---

Endocrine Abstracts (2010) 21 YEP2