Figure 2

Minigene therapeutic approaches with U1 adaptations to correct the pathogenic effect of different donor ss mutations on the HGSNAT gene. (A), (D) and (G) Schematic illustrations of base pairing between the wild type U1 (U1-WT) and the 5’ ss of wild type and mutant exons 2, 6 and 15 of the HGSNAT gene. The position of each mutation in the 5’ ss is marked in grey and it is in italics. The different U1 snRNAs used for each mutated 5’ ss of HGSNAT (designated as U1-sup, for suppressor) are also shown. The U1 sequence changes are illustrated in bold. (B), (C), (E), (F), (H) and (I) RT-PCR analysis of COS-7 cells in which splicing reporter minigenes for each mutation were co-transfected with the different modified U1 constructs as appropriate. Neither the U1-WT nor the various adapted U1s showed an effect on the splicing of exon 2, 6 or 15 wild type minigenes (B, E and H). In the case of the mutant minigenes, some of the U1 co-transfections [U1-sup2, 3 and 4 (C); U1-sup6 (F); U1-sup7, 8 and 9 (I)] changed the splicing pattern, not to the correct one, but mainly towards an alternative pattern using a “gt” 5’ ss immediately downstream of the constitutive one. Importantly, the wild type minigene for exon 2 (Mini WT ex2) shows an extra lower band due to the amount of empty vector added to adjust the total quantity of DNA. Sequencing results for all RT-PCR products are illustrated by schematic drawings. M: molecular weight marker; NT: non-treated cells; C-: negative control.