Manipulating splicing by inducing terminal intron retention

Regulation of gene expression can occur at the pre-mRNA level by alternative splicing. As a type of alternative splicing, intron retention received attention only in recent years. Specifically, there are few reports on endogenous terminal intron retention event in different gene transcripts and only one report of antisense oligonucleotide induced retention of the terminal intron in SMN transcripts. So far, there is no report of induced terminal intron retention other gene transcripts. Terminal intron retention is proposed to be inducible in human gene transcripts using antisense oligonucleotides, with an effect on expression at the mRNA and/or protein levels. Five gene transcripts, LMNA, LMNC, ITGA4, SOD1, and DMD, were selected and for each transcript, the terminal intron/exon splice site and exon-internal sequences of the last exon were targeted for antisense oligonucleotide annealing. It was found that terminal intron retention is inducible in LMNC and ITGA4 transcripts, and possibly LMNA transcripts. Inclusion of the terminal intron decreases full-length LMNC and ITGA4 mRNA expression whereas its effect on protein expression is undetermined. Using the PMO chemistry resulted in a reduced effect on terminal intron retention compared to when 2’-OMeAOs were used. In addition, in both LMNC and ITGA4 transcripts, terminal intron retention led to approximately 3% to 13% decrease in full-length mRNA expression. Changing transfection parameters such as antisense oligonucleotide concentrations, transfection reagent, and transfection duration can also influence transfection outcome. Possible features that may predict terminal intron retention, including but not limited to, splicing motifs, intron length and splice site strengths, were compared between transcripts with and without retained terminal intron. However, no specific features for retention of the terminal intron were observed. Overall, this project shows that terminal intron retention is inducible in human gene transcripts other than SMN transcripts, but not in all gene transcripts examined