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

CONTEXT The GH receptor (GHR) pseudoexon 6Psi 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 6Psi 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 6Psi 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-GHR6Psi-L2, respectively) were created by inserting the GHR 6Psi in a well-characterized splice reporter (Adml-par). For the in vitro splicing assay, the wt- and mtL1-GHR6Psi-L2 were transcribed into pre-mRNA in the presence of [alpha(32)P]GTP and incubated with ASOs in HeLa nuclear extracts. For the cell transfection studies, wt- and mtL1-GHR6Psi-L2 cloned into pcDNA 3.1 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-GHR6Psi-L2 and did not act as an enhancer of 6Psi inclusion. CONCLUSIONS The exon-skipping ASO approach was effective in correcting aberrant GHR splicing and may be a promising therapeutic tool.