David, A; Srirangalingam, U; Metherell, LA; Khoo, B; Clark, AJL; (2010) Repair of Aberrant Splicing in Growth Hormone Receptor by Antisense Oligonucleotides Targeting the Splice Sites of a Pseudoexon. J CLIN ENDOCR METAB , 95 (7) 3542 - 3546. 10.1210/jc.2009-1968.

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Abstract

Context: The GH receptor (GHR) pseudoexon 6 Psi 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 6 Psi 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 6 Psi 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-GHR6 Psi-L2, respectively) were created by inserting the GHR 6 Psi in a well-characterized splice reporter (Adml-par). For the in vitro splicing assay, the wt- and mtL1-GHR6 Psi-L2 were transcribed into pre-mRNA in the presence of [alpha P-32]GTP and incubated with ASOs in HeLa nuclear extracts. For the cell transfection studies, wt-and mtL1-GHR6 Psi-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-GHR6 Psi-L2 and did not act as an enhancer of 6 Psi inclusion.Conclusions: The exon-skipping ASO approach was effective in correcting aberrant GHR splicing and may be a promising therapeutic tool. (J Clin Endocrinol Metab 95: 3542-3546, 2010)

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