One-year treatment of morpholino antisense oligomer improves skeletal and cardiac muscle functions in dystrophic mdx mice - PubMed (original) (raw)

One-year treatment of morpholino antisense oligomer improves skeletal and cardiac muscle functions in dystrophic mdx mice

Bo Wu et al. Mol Ther. 2011 Mar.

Abstract

Antisense therapy has been successful to skip targeted dystrophin exon with correction of frameshift and nonsense mutations of Duchenne muscular dystrophy (DMD). Systemic production of truncated but functional dystrophin proteins has been achieved in animal models. Furthermore, phase I/II clinical trials in United Kingdom and the Netherlands have demonstrated dystrophin induction by local and systemic administrations of antisense oligomers. However, long-term efficacy and potential toxicity remain to be determined. The present study examined 1-year systemic effect of phosphorodiamidate morpholino oligomers (PMO) treatment targeting mutated dystrophin exon 23 in mdx mice. PMO induced dystrophin expression dose-dependently and significantly improved skeletal muscle pathology and function with reduced creatine kinase (CK) levels by a regimen of 60 mg/kg biweekly administration. This regimen induced <2% dystrophin expression in the heart, but improved cardiac functions demonstrated by hemodynamics analysis. The results suggest that low levels of dystrophin induction may be able to provide detectable benefit to cardiac muscle with limited myopathy. Body weight, serum enzyme tests, and histology analysis showed no sign of toxicity in the mice treated with up to 1.5 g/kg PMO for 6 months. These results indicate that PMO could be used safely as effective drugs for long-term systemic treatment of DMD.

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Figures

Figure 1

Figure 1

Restoration of dystrophin expression after 1-year intravenous (i.v.) treatment of 15 or 60 mg/kg phosphorodiamidate morpholino oligomers (PMO). (a) Detection of dystrophin by immunohistochemistry with rabbit polyclonal antibody P7 against dystrophin. Blue nuclear staining with DAPI. (b) Detection of exon 23 skipping in muscles by reverse transcription-PCR (RT-PCR). Lane 1 size marker. (c) Western blot showed levels of dystrophin expression in muscles [10 µg total protein loaded from tibialis anterior (TA) muscle of C57BL/6 mouse; 50 µg total protein loaded from untreated or PMO-treated mdx mice]. C57-TA, TA muscle from normal C57BL/6 mouse; Cont-TA, TA muscle from untreated mdx mouse. 15 or 60 mg/kg, the muscles from 15 or 60 mg/kg PMO-treated mdx mice. The samples from two different mdx mice treated with 60 mg/kg PMO were shown. (d) α-Actin as loading controls. (e) The percentage of non-centranucleated muscle fibers. Control, muscles from untreated mdx mouse. (n = 10; #P ≤ 0.05 compared with untreated mdx mice. Two-tailed _t_-test).

Figure 2

Figure 2

Examination of pathology, serum, and skeletal muscle function after 1-year treatment of 15 and 60 mg/kg phosphorodiamidate morpholino oligomers (PMO). (a) Histology [hematoxylin and eosin (H&E) staining] of tibialis anterior (TA), diaphragm, liver, and kidney from the normal C57BL/6 mice, untreated mdx mice (control), 15 or 60 mg/kg PMO-treated mdx mice (15 mg/kg, 60 mg/kg). (b) The levels of serum enzymes. Creatine kinase (KU/l), creatinine (mg/l), urea nitrogen (mg/ml), total bilirubin (mg/l), direct bilirubin (mg/dl), alanine transaminase (ALT) (U/dl), alkaline phosphatase (ALP) (U/dl), and γ-glutamyltransferase (GGT) (U/l). A significant reduction in creatine kinase levels was observed in mdx mice treated with 60 mg/kg PMO when compared with untreated mdx mice. (c) Grip strength measurement. Significant improvement was observed in the mice after treatment with 60 mg/kg PMO. KGF, kilogram force. (n = 10; *P ≤ 0.05 compared with C57BL/6 mice; #P ≤ 0.05 compared with untreated mdx mice. Two-tailed _t_-test). Final mean body weight: C57BL/6, 31.3 ± 0.8 g; untreated mdx, 34.1 ± 2.0; 15 mg/kg PMO-treated mdx, 31.8 ± 2.4 g; 60 mg/kg PMO-treated mdx, 33.7 ± 1.6 g. PMO, phosphorodiamidate morpholino oligomers.

Figure 3

Figure 3

Cardiac functions in the mdx mice treated with 15 or 60 mg/kg phosphorodiamidate morpholino oligomers (PMO) (1 year) by hemodynamics at 15 minutes after dobutamine challenges. Significant improvement was observed in (a) stroke volume, (b) cardiac output, (c) end-systolic pressure, (d) ejection fraction, (e) d_p_/d_t_max, (f) d_p_/d_t_min, (g) d_V_/d_t_max, (h) dV/dtmin. (1) Age-matched normal C57BL/6 mice; (2) age-matched untreated mdx mice; (3) 1-year 15 mg/kg PMO-treated mdx mice; (4) 1-year 60 mg/kg PMO-treated mdx mice. (n = 10; *P ≤ 0.05 in comparison with C57BL/6 mice; #P ≤ 0.05 in comparison with untreated mdx mice. Two-tailed _t_-test).

Figure 4

Figure 4

Restoration of dystrophin expression in the mdx mice after 6 months intravenous (i.v.) treatment with phosphorodiamidate morpholino oligomers (PMO) (300 mg/kg or 1.5 g/kg biweekly). (a) Detection of dystrophin by immunohistochemistry with rabbit polyclonal antibody P7. Blue nuclear staining with DAPI. (b) Detection of exon 23 skipping in muscles by reverse transcription-PCR (RT-PCR). Lane 1, size marker. (c) Western blot for dystrophin expression [25 µg total protein loaded from tibialis anterior (TA) muscle of C57BL/6 mouse; 50 µg total protein loaded from untreated or treated mdx mice]. C57-TA, TA muscle from normal C57BL/6 mouse; Cont-TA, TA muscle from untreated mdx mouse. 300 mg/kg, 300 mg/kg PMO-treated mdx mice. 1.5 g/kg PMO, 1.5 g/kg PMO-treated mdx mice. (d) α-Actin as loading controls.

Figure 5

Figure 5

Examination of pathology, serum, and skeletal muscle function after 6 months treatment with 300 mg/kg and 1.5 g/kg phosphorodiamidate morpholino oligomers (PMO). (a) Histology [hematoxylin and eosin (H&E) staining] of tibialis anterior (TA), diaphragm, and kidney from untreated (control), 300 mg/kg and 1.5 g/kg PMO-treated mdx mice (300 mg/kg, 1.5 g/kg). (b) The levels of serum enzymes. Creatine kinase (KU/l), creatinine (mg/l), urea nitrogen (mg/ml), total bilirubin (mg/l), direct bilirubin (mg/dl), alanine transaminase (ALT) (U/dl), alkaline phosphatase (ALP) (U/dl), and γ-glutamyltransferase (GGT) (U/l). (c) Grip strength tests. Significant improvement was observed in the mdx mice treated with 300 mg/kg and 1.5 g/kg PMO. KGF, kilogram force. (n = 10; *P ≤ 0.05 in comparison with C57BL/6 mice; #P ≤ 0.05 in comparison with untreated mdx mice. Two-tailed _t_-test).

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