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.
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.
Authors: Pavla Hujová; Přemysl Souček; Lucie Grodecká; Hana Grombiříková; Barbora Ravčuková; Pavel Kuklínek; Roman Hakl; Jiří Litzman; Tomáš Freiberger Journal: J Clin Immunol Date: 2020-01-25 Impact factor: 8.317
Authors: Tatiana V Novoselova; Shoshana R Rath; Karen Carpenter; Nicholas Pachter; Jan E Dickinson; Glynis Price; Li F Chan; Catherine S Choong; Louise A Metherell Journal: J Clin Endocrinol Metab Date: 2014-12-02 Impact factor: 5.958