Progressive muscle degeneration and wasting leads to pre-mature death in DMD patients.1,2
The accumulation of fibrotic and necrotic tissue in dystrophic muscle exacerbates the wasting process. Fibrosis is the final result of chronic inflammation mediated, in part, by the pathological activation of NF-κB, in both muscle-infiltrating immune cells and damaged skeletal muscle fibers.19
In our previous studies, AAV-mediated delivery of a mini-dystrophin gene resulted in efficient and functional recovery of dystrophic skeletal muscle in mdx
mice; however, this approach was not sufficient to reverse the muscle wasting process or restore a normal life-span in a severe murine model, the dKO mouse.9–11
Despite noted successes in pre-clinical mouse studies, AAV mini-dystrophin gene transfer has limitations of partial transduction of muscle fibers and incomplete reversal of the dystrophic phenotype in muscle tissue. Thus, the development of methods to improve regeneration and block chronic inflammation in dystrophic muscle would not only offer direct therapeutic potential for DMD, but could also enhance the efficacy of gene therapy approaches.
In prior studies, blockade of the NF-κB signaling pathway with pharmacological agents has demonstrated success in delaying progression of the pathological features of dystrophy in mdx
Small molecules, including the use of an IKK inhibitory peptide, used to block pathological activation of NF-κB have improved muscle regeneration. However, the use of small molecules requires chronic, systemic treatment and raises concerns for potential systemic side effects. In contrast, a local gene transfer approach with gene transduction and transgene expression limited to muscle could impart a greater therapeutic effect with minimal adverse side effects. Therefore, inhibition of IKK/NF-κB signaling by a gene transfer-mediated approach could result in reducing progressive muscle loss and weakness. Tas, S et al. used AAV to successfully deliver IKKβ-dn (K > M) to the synovium, resulting in reduced severity of inflammation in adjuvant-induced arthritis (AA) in vivo
and proinflammatory cytokine production in human rheumatoid arthritis (RA) synovial tissues ex vivo
Thus, in this study, we tested AAV-mediated gene transfer of IKKβ-dn on muscle regeneration and necrosis in both young and old mdx
mice. Moreover, since the role of IKKα in regulating muscle degeneration and regeneration as well as inflammation has not been characterized, we also examined the effect of IKKα-dn on dystrophic pathology.
mouse has a nonsense mutation in exon 23 of the dystrophin gene, resulting in premature chain termination of the dystrophin protein.30
Progressive pathological muscle necrosis and regeneration,31,32
are most marked between 1 and 2 months of age. 33
The satellite cell pool is diminished with advancing age of mdx
mice due to the ongoing cycles of muscle degeneration and regeneration. 26
Hence, the AAV mediated IKKα-dn and IKKβ-dn treatment was first performed in young mdx mice. As summarized in , we demonstrated that the level of myofiber necrosis (mice treated beginning at 1 month and 2 months of age) was decreased in the AAV-IKKα-dn (19.7% and 15.0%) and AAV-IKKβ-dn (17.8% and 13.1%) treated muscle of young mdx mice as compared to age-matched control groups treated with PBS (26.9%and 24.5%), AAV-IKKα-wt (25.3% and 26.3%) and AAV-IKKβ-wt (23.9% and 23.3%). This result suggests that the local treatment of the AAV-IKKα-dn and AAV-IKKβ-dn can reduce the course of muscle necrosis.
In contrast to the effect on muscle fiber necrosis, there was no significant increase in muscle regeneration in mdx
mice (treated at 1 and 2 months of age) with AAV-IKKα-dn (29.8% and 20.4%) or AAV-IKKβ-dn (29.5% and 20.2%) when compared to controls treated with PBS (30.8% and 19.5%), AAV-IKKα-wt (29.0% and 18.7%) and AAV-IKKβ-wt (27.3% and 22.5%). The absence of an effect on regeneration in mdx
mice treated at a young age was surprising because of prior studies showing that 3 times weekly treatment with a peptide inhibitor of IKK, resulted in a significant enhancement of muscle regeneration. 22
One possible explanation for the differing results may lie in the difference between a peptide-based therapeutic that would be expected to act immediately and an AAV vector-based therapeutic that requires time, up to two weeks, for transgene expression.
In contrast to the results in young mdx mice, we observed improved skeletal muscle regeneration in mdx mice treated at 11 months of age. As described in , there were significant increases in muscle regeneration in mdx mice (treated at 11 months of age) with AAV-IKKα-dn (4.3%) or AAV-IKKβ-dn (9.9%) when compared to controls treated with PBS (1.4%), AAV-IKKα-wt (0.9%), and AAV-IKKβ-wt (0.8%). This result suggests that inhibition of pathological activation of NF-κB is effective at a time when the regenerative capacity of dystrophic muscle is significantly reduced. This result is of potential significant importance for its ultimate utility in the clinical treatment of dystrophic muscle since the dystrophic process in human DMD muscle is already well advanced even early in life.
The ability of dominant negative IKKα and IKK β to reduce necrosis in old mdx muscle was also examined. Reduced levels of necrosis were observed in older mdx mice treated with AAV-IKKα-dn (15.4%) and AAV-IKKβ-dn (15.1%) compared to age-matched control groups treated with PBS (34.0%), AAV-IKKα-wt (35.6%) and AAV-IKKβ-wt (34.9%).
By using the ubiquitous CMV promoter for expression of the therapeutic constructs, the potential for transduction and expression in inflammatory cells, which originate from hematopoietic stem cells, recruited to dystrophic muscle tissue was preserved. Because the inhibition of NF-κB activity could benefit dystrophic muscle through effects on inflammatory cells as well as muscle fibers, the observed therapeutic effects may reflect a combination of both mechanisms.
Although IKKβ participates the “classical” activation of NF-κB, IKKα is a component of “alternative” activation of NF-κB.34
Interestingly, our results suggest that targeting IKKα also inhibits the level of nuclear NF-κB in dystrophic muscle and enhances muscle regeneration. This result is in contrast to a previous study 35
showing that transfection of the IKKα-dn plasmid into the C2C12 cell line had no significant effect on pro-inflammatory gene expression following LPS-stimulation of myotubes. However, our results demonstrate that following AAV-mediated gene transfer to muscle in vivo
, IKKα-dn is as effective in improving pathology as IKKβ-dn.
In summary, AAV-mediated gene transfer of dominant negative IKK, either IKKβ or IKKα, to block pathological NF-κB signaling is a promising strategy for the treatment of DMD. The current studies suggest effectiveness as an independent therapeutic, especially when the dystrophic process is more advanced. Furthermore, combination therapy for DMD with mini-dystrophin gene transferor stem cell transplantation and inhibition of NF -κB activation may be synergistic and will be explored in future studies.