Endocrine Abstracts

Introduction: There are two isoforms of the insulin receptor (IR), generated by the alternative splicing of exon 11 to give IR-A (exon 11-) and IR-B (exon 11+) isoforms. The differing distribution and function of the isoforms has been postulated to play a role in the pathogenesis of diabetes mellitus, myotonic dystrophy and thyroid carcinoma. Previous work investigating IR alternative splicing has identified hormonal factors, splicing elements and factors which modulate the relative ratios of the isoforms.

Objectives: 1. Construct 2′O-methyl antisense oligonucleotides (ASOs) to influence the alternative splicing of IR.

2. Identify intronic and exonic sequences involved in alternative splicing of IR exon 11.

Methods: ASOs were constructed to bind to the 3′ splice-site (3), 5′ splice-site (5), branch point sequence (B), combinations of 2 splice-sites (designated 3B, 5B and 53), and sequences within exon 11 predicted to enhance its inclusion. These were transfected into HepG2 cells for 48 hours, total RNA harvested, and RT-PCR was performed to amplify the IR isoform cDNAs, which were separated by PAGE and quantified.

Results: The IR-B isoform constitutes approximately 28% of the total IR mRNA in mock-transfected cells. ASOs targeted to single splice-sites did not exert any significant effect on IR alternative splicing. However, combination ASOs targeted to two splice-sites significantly reduced the proportion of the IR-B isoform in the order: 53 (16%) >5B (18%) > 3B (21%). ASOs targeted to sequences in the 3′ half of exon 11 resulted in a significant knockdown of IR-B (11%), this effect being enhanced by an ASO spanning the entire exon (4%).

Conclusions: a. IR alternative splicing can be influenced by synthetic ASOs.

b. Exon 11 contains sequences that favour its inclusion. Interference with the recognition of these elements causes exon 11 skipping.

c. Targeting combinations of 2 splice-sites causes exon 11 skipping.