Repair of aberrant splicing in growth hormone receptor by antisense oligonucleotides targeting the splice sites of a pseudoexon

Abstract

Context: The GH receptor (GHR) pseudoexon 6Ψ defect is a frequent cause of GH insensitivity (GHI) resulting from a non-functioning GH receptor (GHR). It results in a broad range of phenotypes and may also be present in patients diagnosed as idiopathic short stature. Objective: Our objective was to correct aberrant GHR splicing and inclusion of 6Ψ using exon-skipping antisense oligonucleotides (ASOs). Design and Setting: Three ASOs binding the 5′ (ASO-5), 3′ (ASO-3), and branch site (ASO-Br) of 6Ψ were tested in an in vitro splicing assay and a cell transfection system. The wild-type (wt) and mutant (mt) DNA minigenes (wt- and mtL1-GHR6Ψ-L2, respectively) were created by inserting the GHR 6Ψ in a well-characterized splice reporter (Adml-par). For the in vitro splicing assay, the wt-and mtL1-GHR6Ψ-L2 were transcribed into pre-mRNA in the presence of [α32P]GTP and incubated with ASOs in HeLa nuclear extracts. For the cell transfection studies, wt- and mtL1-GHR6Ψ-L2 cloned into pcDNA3.1 were transfected with ASOs into HEK293 cells. After 48 h, RNA was extracted and radiolabeled RT-PCR products quantified. Results: ASO-3 induced an almost complete pseudoexon skipping in vitro and in HEK293 cells. This effect was dose dependent and maximal at 125-250 nM. ASO-5 produced modest pseudoexon skipping, whereas ASO-Br had no effect. Targeting of two splice elements simultaneously was less effective than targeting one. ASO-Br was tested on the wtL1-GHR6Ψ-L2 and did not act as an enhancer of 6Ψ inclusion. Conclusions: The exon-skipping ASO approach was effective in correcting aberrant GHR splicing and may be a promising therapeutic tool. Copyright © 2010 by The Endocrine Society.