Endocrine Abstracts (2008) 15 P236

Locked nucleic acid modified siRNAs maintain efficacy of gene silencing, but have reduced serum stability in a model of Cushing's disease

Alia Munir, Adim Eltobgi, Lee Abbott & John Newll-Price

University of Sheffield, Sheffield, UK.

Introduction: POMC is activated in ACTH-dependent Cushing’s Syndrome. We have previously shown that RNA interference (RNAi) targeting POMC coding and promoter sequences induces silencing of POMC and a reduction in ACTH. We have modified our most potent exonic sequence with locked nucleic acid (LNA) chemistry. This is purported to increase serum stability. Here we test whether the LNA confers stability without compromising siRNA silencing potency.

Method: The LNA-modified short interfering RNA (siLNA) targeted to POMC was transfected in to cultured murine pituitary cells (AtT20 D16:16). Transfection efficiency was established with scrambled fluorescent siRNAs using fluorescent activated cell sorting (FACS) and confocal microscopy.

Cells were incubated with the LNA-modified siRNA complexes and cultured for 24 h and 5 days. RT-PCR and RT-qPCR were used to measure relative POMC mRNA suppression. Media collected was analysed using an ACTH IRMA assay.

In addition serum stability of the siRNA and siLNA, was assessed by incubation at 37  °C in 10% fetal calf serum (FCS) over 0–48 h.

Results: Transfection efficiency was measured at >80% on both FACS analysis and microscopy.

We demonstrate an efficient rate of suppression of POMC with the siLNA. At 24 h POMC mRNA was knocked down by 80% and at 5 days by 66%. ACTH levels were reduced by 30% and 59% respectively.

Interestingly the siLNA conferred poorer stability, degrading at 6 h, compared with the unmodified siRNA, which surprisingly remained stable at 48 h in the same conditions.

Conclusions: We have demonstrated that chemically modified siRNAs produce potent and sustained knockdown of murine POMC mRNA. However the LNA modification did not confer increased serum stability in this case. This is of clinical relevance when translating to an in vivo model.

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