Mechanisms for the Differential Antisense Effect by Systemic Treatment of Unmodified AONs

Antisense therapy for treating DMD relies on the rescue of dystrophin expression throughout the body musculature. It was expected that systemic delivered AON could restore dystrophin expression evenly in all muscles and muscle fibers. When 2OMePS AON targeting mouse exon 23 was delivered i.v., systemic effect was evident (Lu et al. 2005). However it was a surprise to see that dystrophin expression induced by systemically delivered 2OMePS AON was highly variable with very limited number of fibers expressing near normal levels of dystrophin by immunostaining. Variation was observed between individual muscles and within muscles as groups of dystrophin positive fibers can be seen right adjacent to completely dystrophin negative fibers. The mechanism(s) responsible for such disparity is not understood, but is of great importance if we are to achieve therapeutic effect in the body-wide affected muscles in DMD patients. The focal distribution of positive fibers in the same muscle suggests that the differential induction is unlikely to be related to the fiber types and this is supported by immunohistochemistry with antibodies specific to fiber types and dystrophin (Lu et al. 2005). The efficiency of dystrophin induction is not simply related to the levels of fiber maturation as fibers of all calibers were found to express similar levels of dystrophin. This is also supported by the fact that both neonatal myosin positive and negative fibers can be either dystrophin positive or negative. Since the characteristic pathological feature of the DMD muscles and muscles in the mdx mice is the cycles of muscle degeneration and regeneration, the focal variations in dystrophin induction is therefore proposed to be related to the stages of the pathological cycles of dystrophic muscles. Studies of dystrophin induction by AON and muscle damage indicated by Evans blue staining suggested that mature fibers expressing high levels of dystrophin are most frequently seen neighboring to or in the areas with clear leakage of Evans blue dye (Fig. 6.2). Fibers of small caliber are also more frequently observed with high levels of dystrophin induction. These results suggest that higher efficiency in dystrophin induction is related to a more permeabilized vasculatures system and fiber membrane. This leads to the hypothesis that delivery efficiency of the negatively charged 2OMePS AON in vivo is closely related to the process of muscle degeneration and the delivery mechanism of 2OMePS AON is likely to be a process of passive diffusion. The repulsion between the like-charges of 2OMePS AONs and the molecules at the surface of cell membrane would be expected to hinder efficient delivery of the AON into target cells with intact membrane, and thus the lower efficiency of antisense effect in those fibers with minimum membrane permeabilization.

This together with other factors prompted the examination of the charge-neutral PMO for systemic effect of exon skipping and dystrophin induction. The lack of charge is expected to have much less impediment to cell surface contact and may thus allow PMO enter muscle fibers more efficiently, particularly those with sA A >i



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Fig. 6.2 Correlation between antisense oligomer-induced dystrophin expression (green fluorescence membrane labeling) and muscle damage indicated by Evans Blue staining (red labeling). Focal clusters of fibers expressing dystrophin localize in the areas with trace but clear observable staining with the Evans Blue. The dye is given 24 h before the experiment is terminated and 7 days after the treatment with an antisense oligomer for dystrophin exon 23 skipping in the mdx mouse. (a) TA muscles; (b) diaphragm leaky membrane as seen in the dystrophic muscles. This hypothesis however would predict a similar pattern of dystrophin induction by PMO in dystrophic muscles. Indeed, single i.v injection of low doses of PMO produced highly variable dystrophin expression in all muscles (Alter et al. 2006). PMO is highly stable in biological system, for example, without any clear degradation in serum for at least 24 h (Arora et al. 2004). This together with the fact that most if not all fibers in DMD muscles will undergo cycles of degeneration provides the possibility that regular injections could effectively deliver PMO into majority of muscle fibers with the production of therapeutic levels of dystrophin. This was demonstrated by regular i.v. injections in the mdx mice as we discussed in the previous section. The higher efficiency in dystrophin induction by PMO compared to 2OMePS AON is most likely a combinatorial effect of the stable oligomers, the high binding affinity to target sequence and the higher mobility of the charge-neutral molecules within the extracellular matrix and through the cell membrane.

The dependence on muscle damage for effective delivery of AONs including charge-neutral PMO has an advantage of limiting the amount of AON entering untargeted and undamaged nonmuscle cells, thus diminishing possible side effects. At the same time however, this limitation presents a potential barrier for effective treatment of DMD (Alter et al. 2006). This model would predict that muscle fibers rescued earlier by PMO-induced exon skipping, might have to reenter a myopathic state before they could again be protected by further entry of antisense oligomers. Such a requirement for recurring cycles of rescue and degeneration in treated muscles would severely limit the value of antisense therapy for DMD patients.

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