Effect of nuclear factor κB inhibition on serotype 9 adeno-associated viral (AAV9) minidystrophin gene transfer to the mdx mouse.

Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery.

Systemic delivery of NEMO binding domain/IKKγ inhibitory peptide to young mdx mice improves dystrophic skeletal muscle histopathology.

The activation of nuclear factor κB (NF-κB) contributes to muscle degeneration that results from dystrophin deficiency in human Duchenne muscular dystrophy (DMD) and in the mdx mouse. In dystrophic muscle, NF-κB participates in inflammation and failure of muscle regeneration. Peptides containing the NF-κB Essential Modulator (NEMO) binding domain (NBD) disrupt the IκB kinase complex, thus blocking NF-κB activation. The NBD peptide, which is linked to a protein transduction domain to achieve in vivo peptide delivery to muscle tissue, was systemically delivered to mdx mice for 4 or 7 weeks to study NF-κB activation, histological changes in hind limb and diaphragm muscle and ex vivo function of diaphragm muscle. Decreased NF-κB activation, decreased necrosis and increased regeneration were observed in hind limb and diaphragm muscle in mdx mice treated systemically with NBD peptide, as compared to control mdx mice. NBD peptide treatment resulted in improved generation of specific force and greater resistance to lengthening activations in diaphragm muscle ex vivo. Together these data support the potential of NBD peptides for the treatment of DMD by modulating dystrophic pathways in muscle that are downstream of dystrophin deficiency.

Skeletal muscle glucose transporter protein responses to antenatal glucocorticoids in the ovine fetus.

We investigated the effects of maternal antenatal dexamethasone (Dex) treatment given as a single course (4 doses) or multiple courses (20 doses) on fetal skeletal muscle glucose transporter (GLUT) protein concentrations at 70% of gestation (106 to 107 days with term being 145 to 150 days) in the ovine fetus. Antenatal corticosteroid administration was associated with a decrease in endogenous fetal plasma cortisol concentrations (P < 0.05), fetal hyperglycemia (P < 0.02) and hyperinsulinemia (P < 0.05). These metabolic/hormonal changes were associated with a decrease in fetal body weight (P < 0.05) in the multiple course Dex group compared with the multiple course placebo group. These perturbations were associated with an increase in fetal skeletal muscle GLUT 1 concentrations that mediate basal glucose transport in the extensor digitorum lateralis and extensor digitorum longus muscles (P < 0.05) 18 h after the last dose of Dex was given in the single course group. However, in the multiple course Dex group, a small increase in GLUT 1 was observed only in the biceps femoris. In contrast, both single and multiple courses of antenatal Dex were associated with an increase in the extensor digitorum lateralis and biceps femoris muscle GLUT 4 (insulin-responsive) concentrations (P < 0.05). We conclude that antenatal corticosteroids perturb fetal glucose/insulin homeostasis, which is associated with increases in fetal skeletal muscle glucose transporters to compensate for and attenuate the associated catabolic fetal state. These changes consist of an increase in proteins that mediate basal glucose transport (GLUT 1) to meet immediate energy requirements of the fetal skeletal muscle with an increase in basal insulin sensitivity (GLUT 4) to compensate for the Dex-induced catabolic state after exposure to multiple courses of Dex.