Inhibition of the IKK/NF-B pathway by AAV gene transfer improves muscle regeneration in older mdx mice

The IB kinase (IKKα, Β and the regulatory subunit IKKγ) complex regulates nuclear factor of B (NF-B) transcriptional activity, which is upregulated in many chronic inflammatory diseases. NF-B signaling promotes inflammation and limits muscle regeneration in Duchenne muscular dystrophy (DMD), resulting in fibrotic and fatty tissue replacement of muscle that exacerbates the wasting process in dystrophic muscles. Here, we examined whether dominant-negative forms of IKKα (IKKα-dn) and IKKΒ (IKKΒ-dn) delivered by adeno-associated viral (AAV) vectors to the gastrocnemius (GAS) and tibialis anterior (TA) muscles of 1, 2 and 11-month-old mdx mice, a murine DMD model, block NF-B activation and increase muscle regeneration. At 1 month post-treatment, the levels of nuclear NF-B in locally treated muscle were decreased by gene transfer with either AAV-CMV-IKKα-dn or AAV-CMV-IKKΒ-dn, but not by IKK wild-type controls (IKKα and Β) or phosphate-buffered saline (PBS). Although treatment with AAV-IKKα-dn or AAV-IKKΒ-dn vectors had no significant effect on muscle regeneration in young mdx mice treated at 1 and 2 months of age and collected 1 month later, treatment of old (11 months) mdx with AAV-CMV-IKKα-dn or AAV-CMV-IKKΒ-dn significantly increased levels of muscle regeneration. In addition, there was a significant decrease in myofiber necrosis in the AAV-IKKα-dn- and AAV-IKKΒ-dn-treated mdx muscle in both young and old mice. These results demonstrate that inhibition of IKKα or IKKΒ in dystrophic muscle reduces the adverse effects of NF-B signaling, resulting in a therapeutic effect. Moreover, these results clearly demonstrate the therapeutic benefits of inhibiting NF-B activation by AAV gene transfer in dystrophic muscle to promote regeneration, particularly in older mdx mice, and block necrosis. © 2010 Macmillan Publishers Limited All rights reserved

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-Dmdmdx/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

Postopek izdaje gradbenega dovoljenja

This is the author accepted manuscript. The final version is available from Palgrave Macmillan via the DOI in this record

A specific nanobody prevents amyloidogenesis of D76N β2-microglobulin in vitro and modifies its tissue distribution in vivo

Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of β2-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type β2-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The β2-microglobulin -binding nanobody, Nb24, more potently inhibits D76N β2-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In β2-microglobulin knock out mice, the D76N β2-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type β2-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis