Investigation of siRNA as a Treatment for Cushing's Disease

Abstract

Cushing's disease results from a pituitary corticotroph adenoma that secretes adrenocorticotrophic hormone (ACTH). High levels of ACTH stimulate the synthesis of excess cortisol from the adrenals, the effects of which include hypertension, diabetes, depression, and weight gain. If left untreated, Cushing's disease carries a 50% five-year mortality rate. At present, there is no suitable medical therapeutic and transsphenoidal surgery, which is the first line treatment, has a high rate of recurrence. RNA-interference is a cellular mechanism that enables gene silencing at the level of mRNA. It can also be used to reduce gene expression by employing small interfering (si)RNAs that target the specific gene of interest. The proopiomelanocortin (POMC) peptide is encoded by the POMC gene and is processed to release ACTH as well as other hormones.

Aims: The first aim of the current project was to test the hypothesis that POMC-targeting siRNAs could be an effective medical therapy for Cushing's disease; post-transcriptional knock-down of POMC expression could reduce ACTH secretion and thereby cortisol production. Current challenges to using siRNAs as a medical therapy include the lack of targeted delivery to the required cell type. The second aim of this project was investigate if the corticotroph-specific corticotrophin-releasing hormone receptor 1 (CRHR1) could be used as a means of specific delivery of POMC siRNAs if they were conjugated that to an anti-CRHR1 antibody.

Methods: A murine AtT-20 cell line, an ACTH hypersecreting in vitro model of Cushing's disease, was used to assess how effective POMC-targeted siRNAs could be at decreasing ACTH secretion. Following transfection of AtT20 cells with POMC siRNAs, the ACTH concentration in the culture medium was measured by immunoassay. Activation of the innate immune system by POMC siRNAs was analysed by measuring for interferon (IFN)-α, IFN-β, interleukin (IL)-1β, IL-6, and tumour-necrosis factor (TNF)-α by ELISA. Analysis of CRHR1 expression in AtT20 cells and in a CHO FlpIN-CRHR1 cell line was undertaken using RTPCR, cAMP-stimulation in response to CRH, and indirect fluorescence-activated cell sorting (FACS).

Results: Three POMC-specific siRNAs affected a reduction in the secretion of ACTH following transfection of AtT20 cells; the concentration of ACTH in the culture medium was 15.6% (8,868 pg/ml), 18.1% (10,717 pg/ml), and 10.6% (6,037 pg/ml), respectively, of that secreted from untreated AtT20 cells (56,978 pg/ml). ACTH levels were reduced statistically significantly when compared to untreated cells (One-way ANOVA, P <0.0001). None of the POMC siRNAs appeared to trigger the innate immune response in AtT20 cells; the secretion of IFN-α, IFN-β, IL-1β, IL-6, and TNF-α was not detected. The cell surface expression of CRHR1 on AtT20 cells was detected, but was at too low a level for effective detection by the available antiCRHR1 antibodies. A CHO-FlpIN-CRHR1 cell line was isolated and shown to express the receptor by indirect FACS using a receptor-specific mouse antibody.

Conclusions: siRNAs targeting POMC reduced ACTH secretion from AtT20 cells. The POMC siRNAs did not appear to be immunostimulatory. The cell surface expression of CRHR1 AtT20 cells was confirmed. This receptor might be useful as a target for the specific delivery of POMC siRNAs to corticotrophs if they are conjugated to a specific anti-CRHR1 antibody or to CRH peptide.