AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1

Figure 1.. Experimental strategies for AON-mediated DMPK splice-switching. (A, B) Schematic outline of human DMPK pre-mRNA (not to scale) and two distinct AON-based strategies for testing therapeutic potential of DMPK splice-switching. (C, D) Representative RT-PCR analyses of AON-induced e5 (C) and e15 (D) skipping in control fibroblasts (non-DM1) transfected with indicated amounts of 2′-Ome-PS AONs targeting respective exons. Two lanes (C) or three lanes (D) represent distinct samples, while %Δe5 and %Δe15 indicate percentages of e5 or e15 spliced out, respectively. Schematics of distinct PCR amplicons and primer locations are indicated. Gel images shown in C and D were cropped to show only selected samples. In D, note the appearance of several e16-containing (Δe15/ΔCUG) splice isoforms differing in the alternative inclusion of e13 and e14. Chromatograms presented in panel D show sequencing results across indicated splice-junctions (vertical red dashed line) of three distinct e16-containing DMPK splice products.

Figure 2.. AON-mediated skipping of constitutive exons fails to reduce DMPK RNA. (A) Quantitation of total DMPK mRNA by qPCR (real-time PCR) expression analyses of the 3′UTR fragment demonstrates lack of significant DMPK transcript reduction upon e5 skipping in control and DM1-affected fibroblasts. DMPK expression is shown relative to GAPDH, increasing doses of AON targeting e5 (AON-4), control scrambled sequence AON (scr-1) are indicated. (B) Representative results of western blot analyses showing DMPK protein level in control fibroblasts dosed with 75 nM of indicated AONs. Three lanes indicate individual samples. Quantitation is shown to the right. Whole-membrane scan of DMPK western blot is shown in Supplementary Figure S4C.

Figure 3.. AON-mediated skipping of e15 triggers accumulation of spliced out pre-mRNA fragment containing CUG-expansions and mitigates nuclear retention of DMPK. (A) RT-PCR quantitation of total DMPK mRNA in DM1-1000CUG fibroblasts 72 h post transfection with indicated AONs (PCR amplicon within 3′UTR). Mock, scrambled sequence AON (scr-1) and gapmer AONs were used as controls and representative RT-PCR image is shown below bar graph. (B) Representative immunoblots showing no changes in DMPK protein expression in DM1 fibroblasts 48 h (upper gel) and 84 h (bottom gel) post AON dosing. (C) qPCR quantitation of i14/e15/CUG spliced-out pre-mRNA fragment vs total DMPK pre-mRNA in DM1-1000CUG fibroblasts 72 h after dosing the cells with e15 splice-switching AON-8 either alone or in a combination with i14-1 and i14-2 gapmer AONs targeting distinct regions of i14. Scrambled-sequence AON scr-1 and gapmer AONs were used as controls. i14/e15/CUG pre-mRNA was amplified with primers located within i14 and e15 (upstream of CUG-repeat region), primers for DMPK pre-mRNA amplification were located within i9 and e10. (D) Outline and results of the firefly luciferase reporter assay modeling the DM1…

Figure 4.. DM1 spliceopathy can be reversed by gapmer-mediated degradation of DMPK pre-mRNA, but not by AON-induced skipping of e15/CUG. (A) Representative RT-PCR products showing splicing changes in indicated alternative exons of MBNL1, MBNL2 and NCOR2 in DM1-1000CUG fibroblasts, 72 h post transfection with indicated reagents. Each lane represents individual biological sample and vertical dashed line marks boundaries of two electrophoretic separations. (B) Quantitation of splicing changes based on three distinct RT-PCR analyses of indicated alternative exons in DM1-1000CUG fibroblasts transfected with indicated amounts of e15 splice-switching AON-8 (green), scrambled control AON scr-1 (gray), gapmers Gap i14-1 and Gap i14-2 targeting distinct sequences within DMPK i14 (violet) or combination of gapmers and AON-8 (checked). Mock samples from unaffected (white) and DM1-affected (red) fibroblasts are shown for reference. Black horizontal dashed line marks the percentage of alternative exon inclusion in non-DM1 mock controls, while red dashed line – in untreated DM1-1000CUG mock controls. Statistical significance relative to DM1 mock samples as well as between indicated experimental samples is…

Figure 5.. 2′-Ome-PS-induced splicing abnormalities correlate with aberrant expression and splicing of MBNL1 and CELF1. (A, B) Representative immunoblot analyses of MBNL1 and CELF1 protein level relative to GAPDH in DM1-unaffected (A) or DM1-1000CUG (B) fibroblasts transfected with indicated amounts of distinct 2′-Ome-PS AONs targeting DMPK e5. Mock (lipofectamine) and scrambled AON (scr-1) were used as controls. Each lane represents an individual sample. Quantitation is shown to the right. Horizontal dashed line marks the protein level in untreated controls. (C) RT-PCR quantitation of CELF1 total mRNA and the alternative splicing of MBNL1 e1 in non-DM1 fibroblasts transfected with indicated amounts of 2′-Ome-PS AONs. AON scr-1 was used as a control. Horizontal dashed lines mark the percentage of CELF1 level or MBNL1 e1 inclusion in mock samples, respectively. Statistical significance relative to mock is indicated.

Figure 6.. Targeting DMPK with intronic gapmers, but not splice-switching AONs, diminishes CUGexp foci numbers in DM1 fibroblasts. Representative FISH analyses of CUGexp foci in DM1-1000CUG fibroblasts upon AON-induced skipping of DMPK e5 or e9 (A), and upon skipping of e15/CUG either alone or in a combination with Gap i14-1 treatment (B). Scrambled sequence AONs (scr-1 and scr-2) were used as controls. Statistical significance is indicated in the legend.