Exon Skipping in a Dysf-Missense Mutant Mouse Model

Abstract

Limb girdle muscular dystrophy 2B (LGMD2B) is without treatment and caused by mutations in the dysferlin gene (DYSF). One-third is missense mutations leading to dysferlin aggregation and amyloid formation, in addition to defects in sarcolemmal repair and progressive muscle wasting. Dysferlin-null mouse models do not allow study of the consequences of missense mutations. We generated a new mouse model (MMex38) carrying a missense mutation in exon 38 in analogy to a clinically relevant human DYSF variant (DYSF p.Leu1341Pro). The targeted mutation induces all characteristics of missense mutant dysferlinopathy, including a progressive dystrophic pattern, amyloid formation, and defects in membrane repair. We chose U7 small nuclear RNA (snRNA)-based splice switching to demonstrate a possible exon-skipping strategy in this new animal model. We show that Dysf exons 37 and 38 can successfully be skipped in vivo. Overall, the MMex38 mouse model provides an ideal tool for preclinical development of treatment strategies for dysferlinopathy.

Keywords: AAV; MMex38; U7 snRNA; dysferlin; dysferlinopathy; exon skipping; mouse model.

Figure 1

Characteristics of the New Animal Model Dysf-MMex38 (A) The index dysferlinopathy patient harbors the homozygous missense mutation DYSF c.4022T > C in exon 38. MMex38 mice carry the corresponding mutation Dysf c.4079T > C leading to Dysf p.Leu1360Pro (NCBI GenPept: NP_001071162.1, dysferlin isoform 2) ($\stackrel{\wedge}{=}$DYSF p.Leu1341Pro, NM_003494) in the C2E domain (red asterisk) of dysferlin protein. (B) On the protein level, the missense mutated dysferlin detected by Hamlet antibody is markedly reduced (western blot of quadriceps muscle of differently aged mice; α-tubulin used as loading control). Dysf pLeu1360Pro does not localize at the membrane of MMex38 mice. Immunostaining of TA sections of MMex38 and WT mouse with Hoechst and Romeo antibody. Scale bars, 20 μm. (C) Dystrophic changes worsen with age in MMex38 mice (Gomori’s trichrome staining of MMex38 quadriceps muscles). Scale bar, 50 μm. w, weeks of age. (D) Amyloid in MMex38 quadriceps muscle (Congo red staining). Scale bar, 50 μm. (E) Membrane repair after laser wounding is impaired in MMex38 flexor digitorum brevis muscle fibers (number of fibers n = 20 from 3 mice) compared to WT (number of fibers n = 24 from 4 mice) in the presence of Ca²⁺. Fluorescence intensity below wounding site, mean ± SEM; ****p < 0.0001, unpaired t test for the time points above 300 s.

Figure 2

Functional Assessment of Exon 37- and 38-Truncated Dysferlin (A) Schematic mapping of dysferlin domains on exons in pre-mRNA. (B) Dysferlin constructs introduced into dysferlin-null human myoblast via lentiviral transduction. (C) Quantification of the fluorescent signal influx into wounded myotubes over time. For each construct, the following number of myotubes was wounded: hDYSF_Δ37, n = 8; hDYSF_Δ38, n = 15; hDYSF_Δ3738, n = 14; hDYSF_Full, n = 15; non-transduced, n = 14; and EGFP, n = 7. Data points represent means ± SEM; **p < 0.01 and ***p < 0.001, unpaired t test for the time points above 350 s.

Figure 3

Screening of U7 snRNA Antisense Sequences for Exon-Skipping Activity in MDX Mice and C2C12 Cells (A) Antisense sequences of U7 snRNA constructs mapped on the intron 37–38, exon 38, and intron 38–39 of mouse dysferlin pre-mRNA. (B) Primer localization. (C) Detection of exon skipping using RT-PCR and nested PCR in MDX mice. The intermediate-sized bands represent a heteroduplex formed between the full and skipped PCR product. (D) Densitometric quantification of exon skipping in MDX mice. (E) Sequencing of the skipped dysferlin. The presence of the junction between exons 36 and 39 was verified. (F) Detection and densitometric quantification of exon skipping using RT-PCR and nested PCR in C2C12 cells. LD, low dose; HD, high dose.

Figure 4

U7-Based Exon Skipping in MMex38 Mice (A) Detection of exon skipping using RT-PCR and nested PCR for dysferlin mRNA spanning the region between exons 33 and 39. Densitometric quantification of exon skipping in MMex38 mice using ImageJ is shown. (B) Sequencing of the skipped dysferlin. The presence of the junction between the exons 36 and 39 was verified. (C) Detection of dysferlin expression in MMex38 TAs treated with U7 snRNAs using biotin-streptavidin western blot with Hamlet antibody (α-tubulin as loading control). Densitometric quantification of dysferlin as a percentage of WT (C57BL6) using ImageJ is shown. (D) Immunostaining with Hoechst and Romeo antibody of TA sections of MMex38 treated with U7 snRNAs and WT mouse treated with PBS. Scale bars, 20 μm.