Endocrine Abstracts

Background: The growth hormone receptor (GHR) 6Ψ pseudoexon mutation (A to G at ds-1) is one of the most frequent mutations causing GH insensitivity. It causes aberrant mRNA splicing, leading to activation of a pseudoexon and insertion of 36 additional amino acids, resulting in a functionless receptor. Although IGF-I remains the mainstay of treatment for these patients we investigated the ability of RNA antisense oligonucleotides (ASOs) to correct aberrant GHR splicing using an in vitro splicing assay and a cell transfection system.

Methods: Three ASOs targeting the donor (ASO5′), acceptor (ASO3′) and branch site (ASObr) of the GHR pseudoexon were designed. The wild type and mutant DNA minigenes (wt and mt L1-GHR6Ψ-L2) were created by inserting the pseudoexon between exons L1 and L2 of a well-characterised splice reporter (Adml-par). For the in vitro splicing assay the wt and mt L1-GHR6Ψ-L2 were transcribed into mRNA in the presence ³²P-GTP. Radiolabeled minigenes were incubated in a splice reaction with HeLa nuclear extracts and ASOs (0–250 nM) at 30 °C for 1 h. Reactions were separated by PAGE before autoradiography. For transfection studies wt and mt L1-GHR6Ψ-L2 cloned into pcDNA 3.1 were transfected with ASOs (0 to 250 nM) into HEK293 cells. After 48 h, RNA was extracted and radiolabeled RT-PCR products separated by PAGE and quantified.

Results: ASO3′ induced an almost complete pseudoexon skipping in vitro and in HEK293 cells. This effect was dose-dependent and maximal at 125–250 nM. ASO5′ produced modest pseudoexon skipping, whereas ASObr had no effect. Targeting of two splice elements was less effective than one. ASObr was tested on the wt L1-GHR6Ψ-L2 and did not act as an enhancer of pseudoexon inclusion.

Conclusion: The ASO approach was effective in correcting aberrant GHR splicing and may be a promising therapeutic tool.