Plasmid-Mediated Gene Therapy in Mouse Models of Limb Girdle Muscular Dystrophy

Figures

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  Figure 1. DNA Delivery of GFP, Luciferase, and CAPN3 Genes to Mouse Muscle (A) Electroporation device. Photograph of syringe attached to Ichor TriGrid for DNA delivery and electroporation. The syringe needle, which delivers the DNA, is surrounded by 4 small electrodes. (B) GFP delivery: Whole mount of GFP fluorescence seen in quadriceps muscle from C57BL/6 mouse injected with GFP plasmid, followed by electroporation. (C) Cross-section of quadriceps muscle seen in (B), showing GFP fluorescence in transfected muscle fibers. Scale bar, 100 um. (D) Comparison of delivery with and without electroporation: luciferase live imaging of Capn3 null mice that received CAPN3 plasmid DNA injected in the gastrocnemius muscle of Capn3 null mice. Left panel, left legs received electroporation, while right legs did not. Right panel, luciferase radiance values revealed that electroporation was associated with an increase of approximately 2 orders of magnitude compared to non-electroporated muscles. Each dot represented one mouse, n = 4 per group. (E) Western blot analysis of protein extracted from treated gastrocnemius muscles from the mice in (D). Upper row, levels of 100 kDa CAPN3 protein made. Lower row,…

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  Figure 2. Gene Therapy in Dysferlin-Deficient BL/AJ Mice (A) Western blot from quadriceps muscles of BL/AJ mice injected with plasmids DYSF-CPL and DYSF-CFL, encoding dysferlin with and without follistatin. After one month, muscles were harvested from representative quadriceps receiving DYSF-CPL or DYSF-CFL, as well as samples from untreated BL/AJ (HBSS) and wild-type C57BL/6 controls. (B) Densitometry on the dysferlin bands from western blots of the experiment described in (A). The value for the wild-type C57BL/6 was set at 1.0. Untreated BL/AJ animals (HBSS) had significantly less dysferlin than wild-type, while animals injected with DYSF-CFL had DYSF levels that were similar to those observed in C57BL/6 wild-type mice. Mice injected with DYSF-CPL showed a significantly increased amount of DYSF compared to wild-type. Data are mean ± SEM with n = 3 and *p < 0.05. (C) Immunohistochemistry on cross-sections of muscles treated with DYSF-CPL or DYSF-CFL. We stained for dysferlin (green) to identify protein delivered by gene therapy and laminin (red) to identify all muscle fibers. Representative sections from untreated BL/AJ and wild-type C57BL/6 are shown for comparison. In wild-type muscle,…

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  Figure 3. Gene Therapy in Sgca null Mice (A) Time course of development of centronucleation in gastrocnemius muscles of untreated Sgca null mice. CNF, centronucleation frequency. Each dot represents one mouse, and n = 4–6 per group. (B) Time course of development of Evans blue dye permeability in gastrocnemius muscles of untreated Sgca null mice. Each dot represents one mouse, and n = 6 per group. (C) Luciferase live imaging of representative animals in Sgca null mice. Image was taken 1 month after injection in gastrocnemius muscles of HBSS alone or SGCA + FST plasmids. (D) Western blot of same experiment described in (C). An antibody against the FLAG tag fused to the SGCA gene on the plasmid was used to detect expression of human SGCA. Protein extracted from C57BL/6 mice was used as the negative control for FLAG tag (lane 1). GAPDH was used as a loading control. (E) Western blot of follistatin delivery, from same experiment described in (C). Representative animals received the SGCA and FST plasmids, FST alone positive control (lane 2), or SGCA (negative control, lane 3). (F) ELISA of follistatin delivery, from same experiment described in (C). C57BL/6 and HBSS represent negative controls,…