title: Repair of aberrant splicing in growth hormone receptor by antisense oligonucleotides targeting the splice sites of a pseudoexon.
abstract: BACKGROUND
  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).
  
  
  METHODS
  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.affiliation: Centre for Endocrinology, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, EC1M 6BQ London, UK. a.david@qmul.ac.uk
authors.forename: Alessia
authors.initials: A
authors.lastname: David
authors.forename: Umasuthan
authors.initials: U
authors.lastname: Srirangalingam
authors.forename: Louise A
authors.initials: LA
authors.lastname: Metherell
authors.forename: Bernard
authors.initials: B
authors.lastname: Khoo
authors.forename: Adrian J L
authors.initials: AJ
authors.lastname: Clark
mesh_terms: D017398:Alternative Splicing; D002460:Cell Line; D005091:Exons / Q000235:genetics*; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D011986:Receptors, Somatotropin / Q000235:genetics*; D014162:Transfection
chemical_list: D016376:Oligonucleotides, Antisense; D011986:Receptors, Somatotropin