Targeted Skipping of Human Dystrophin Exons in Transgenic Mouse Model Systemically for Antisense Drug Development

Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs). However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD/mdx mouse, a transgenic model carrying the full-length human dystrophin gene with mdx background, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD/mdx mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD/mdx mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement

Mutations alter secretion of fukutin-related protein

Mutations in the fukutin-related protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) as well as other severe muscle disorders, including Walker-Warburg syndrome, muscle-eye-brain disease, and congenital muscular dystrophy type 1C. The FKRP gene encodes a putative glycosyltransferase, but its precise localization and functions have yet to be determined. In the present study, we demonstrated that normal FKRP is secreted into culture medium and mutations alter the pattern of secretion in CHO cells. L276I mutation associated with mild disease phenotype was shown to reduce the level of secretion whereas P448L and C318Y mutations associated with severe disease phenotype almost abolished the secretion. However, a truncated FKRP mutant protein lacking the entire C-terminal 185 amino acids due to the E310X nonsense mutation was able to secrete as efficiently as the normal FKRP. The N-terminal signal peptide sequence is apparently cleaved from the secreted FKRP proteins. Alteration of the secretion pathway by different mutations and spontaneous read-through of nonsense mutation may contribute to wide variations in phenotypes associated with FKRP-related diseases

Effect of Vivo-PMO for human dystrophin exon 50 skipping systemically in the hDMD/<i>mdx</i> mice.

<p>TA, tibialis anterior; Quad, quadriceps; Gastro, gastronemium; Abdom, abdominal; Diaph, diaphragm. The signal intensity of the bands representing human dystrophin exon 50 skipping was 70±13% and 5±1%, 20±5% and 0% in body-wide skeletal muscles and cardiac muscles treated with Vivo-hE50AO23PMO and Vivo-hE50AO5PMO respectively (measured with NIH ImagJ) (A). Results are presented as mean values ± SE with statistical analysis (t-test). *<i>P</i><0.01 was obtained by comparison of the Vivo-hE50AO23PMO group with the hE50AO5PMO group. (n = 5) (B). Immunohistochemical staining shows similar intensity for dystrophin after the Vivo-PMO treatments in all muscles tested. (C). Serum enzyme tests show a slight increase in the levels of creatine kinase in the mice treated with Vivo-PMOs.</p

Effect of Vivo-PMO for dystrophin exon skipping in tibialis anterior (TA) muscles of normal C57 mice and in the hDMD/<i>mdx</i> mice.

<p>(A). The signal intensity of the bands representing mouse dystrophin exon 23 skipping with Vivo-PMOE23 from normal C57 mice was 70±7%. (B). Signal intensity of the bands representing human dystrophin exon 50 skipping after Vivo-hE50AO23PMO treatment from the hDMD/<i>mdx</i> mice was 65±7%, significantly higher when compared with 25±6%, 34±4% detected after Vivo-hE50AO12PMO and Vivo-hE50AO5PMO treatment. Results from 2 TA muscles for each Vivo-PMO are presented. Signal intensity was measured with NIH Image J. Results are presented as mean values ± SE with statistical analysis (t-test). *<i>P</i><0.01 was obtained by comparison of the group(s) with PMOE23 and hE50AO12PMO group in (A) and (B) respectively (n = 5). (C). H&E staining of the TA muscles treated with Vivo-PMOs. No change in muscle histology was detected. Control, saline injected TA muscle.</p

AOs target human dystrophin exon 50 in C2C12hE50 GFP reporter myoblasts 48 hours after treatment.

<p>(A). RT-PCR for the detection of GFP/hE50 mRNA in the C2C12hE50 cells. Left lane is the 100bp size marker. Con, Control sample without PMO treatment; 12, 5, 23 are the hE50AOPMOs. The bands marked with +E50 representing normal dystrophin mRNA containing E50; The bands marked with −E50, representing dystrophin mRNA with hE50 skipped. The band representing hE50 skipping was most strongly detected in the cells treated with hE50AO23PMO. (B) and (C) cells treated with the hE50AOPS and hE50AOPMO respectively. (D). FACS analysis for the GFP positive cells treated with the 3 hE50AOPMOs and the control (without AO treatment).</p

RT-PCR and Nested-PCR for the detection of hE50 skipping in the normal human myoblasts and the DMD-derived skin fibroblasts.

<p>(A). Samples from the normal human myoblasts cells treated with 2OMePS AOs; (B). Samples from the normal human myoblasts cells treated with PMOs. +hE50 representing normal dystrophin mRNA containing hE50; −hE50, representing dystrophin mRNA with hE50 skipped. Left lane is the 100 bp size marker; (C). A sequence showing skipping of the hE50 from the PCR product detected as –hE50 in the hE50AO24PMO treated normal human myoblasts cells in (B). (D). The bands marked with +hE50+hE51 representing normal human dystrophin mRNA (NhM) from normal human myoblasts; the bands marked with +hE50−hE51 representing mRNA from the skin fibroblasts of a DMD patient with exon 51 deletion; the bands marked with −hE50 and –hE51 representing mRNA from the skin fibroblasts with exon 50 skipped (both exons are absent). Control, without AO treatment.</p

List of AO compounds, the sequence and size of AOs and the summary of their exon skipping effect.

<p>PS, 2OMePS AO. − and + within the Target column indicate the intron and exon sequences respectively; the numbers represent the first and last nucleotides of the oligonucleotide sequences. Percentage of GFP positive cells with 2OMePS AOs is the average from the flow cytometry analysis. Results from PMO treatment are presented as mean values ± SE with statistical analysis (ANOVA) since a completed set of exon skipping efficiency analysis including the in vivo test was conducted with those PMOs.</p>*<p><i>P</i><0.01 was obtained by comparison of the groups with the hE50AO28PMO groups (n = 3). GFP, GFP reporter C2C12hE50 cells; hM, normal human myoblasts. DMD, DMD patient-derived skin fibroblasts with exon 51 deletion. Scores: -, no positive signal above background is observed for either GFP expression or signals representing mRNA with exon 50 skipping when compared to the samples of negative controls. Cells were treated with 1 µM AOs. N/D. Muscles of hDMD/<i>mdx</i> mice were treated with 10 µg Vivi-PMO.</p

Dose dependent human dystrophin exon 50 skipping with the hE50AO23PMO in normal human myoblast and skin fibroblasts of a DMD patient with exon 51 deletion.

<p>The signal intensity of the strongest bands representing exon 50 skipping was 71% and 65% in the normal human myoblas (A) and skin fibroblasts (B) respectively (measured with NIH ImagJ). (C). C2C12hE50 reporter myoblasts. (D). FACS analysis of the C2C12hE50 reporter cells treated with the hE50AO23PMO with the concentrations specified.</p

Fukutin-related protein localizes to the Golgi apparatus and mutations lead to mislocalization in muscle in vivo

Mutations in the fukutin-related protein gene (FKRP) are associated with a spectrum of diseases from mild limb-girdle muscular dystrophy type 2I to severe congenital muscular dystrophy type 1C, muscle–eye–brain disease (MEB), and Walker–Warburg syndrome (WWS). The effect of mutations on the transportation of the mutant proteins may constitute the underlying mechanisms for the pathogenesis of these diseases. Here we examined the subcellular localization of mouse and human normal and mutant FKRP proteins in cells and in muscle in vivo. Both normal human and mouse FKRPs localize in part of the Golgi apparatus in muscle fibers. Mutations in the FKRP gene invariably altered the localization of the protein, leading to endoplasmic reticulum retention within cells and diminished Golgi localization in muscle fibers. Our results therefore suggest that an individual missense point mutation can confer at least two independent effects on the protein, causing (1) reduction or loss of the presumed glycosyltransferase activity directly and (2) mislocalization that could further alter the function of the protein. The complexity of the effect of individual missense point mutations may partly explain the wide variation of the FKRP-related myopathies