Weight changes during the experimental period starting from the first day of repeated i

Weight changes during the experimental period starting from the first day of repeated i.v. unmodified AONs exhibit inefficient delivery systemically, leading to dystrophin induction with high variability in skeletal muscles and barely detectable in cardiac muscle. Here, we examined a Morpholino oligomer conjugated with a dendrimeric octaguanidine (Vivo-Morpholino) and exhibited that this delivery moiety significantly improved dystrophin production in both skeletal and cardiac muscles in mice mouse.4,5 More recently, restoration of dystrophin expression by antisense therapy has been demonstrated in DMD patients via intramuscular injection of 2’O methyl phosphorothioate (2OMePS) AONs in the Netherlands.14 Dehydrodiisoeugenol However, exon skipping and dystrophin expression induced by all unmodified antisense chemistries are highly variable, and therapeutic amounts of dystrophin have been achieved only in some skeletal muscles through systemic delivery of Morpholino oligos.15 Furthermore, effective dystrophin expression has not been achieved in the cardiac muscle when unmodified Morpholino oligos were used. This could severely reduce the therapeutic value of antisense therapy for treating DMD as the disease affects all body muscles, and fatality is commonly the direct consequences of the failure of respiratory and cardiac functions.16,17 One approach to improve the efficiency of exon skipping is to enhance the delivery efficiency of oligos by attaching them to cell-penetrating peptides.18,19 Most recently, we and other groups have shown high levels of dystrophin expression in both skeletal and cardiac muscles by Morpholinos tagged with specifically designed arginine-rich peptides.19,20,21 However, long-term repeated applications of peptides required for treating DMD with antisense oligomers risk a potential immune response. Guanidinium head groups of arginine-rich peptides are Dehydrodiisoeugenol principally responsible for effective delivery of Morpholino oligos19 and 8C10 guanidine head groups exhibit the most efficient delivery enhancing effect.22 Here, we report the use of a nonpeptide dendrimeric octaguanidine moietyCtagged morpholino oligomer targeting mouse dystrophin exon 23 (E23) [Vivo-MorpholinoE23 (Vivo-ME23)]. We exhibited that this delivery moiety significantly improved efficiency of ME23-induced skipping of E23 in mice, a model of human DMD. A single intravenous (IV) injection of 6 mg/kg Vivo-ME23 induced functional levels of dystrophin in bodywide skeletal muscles equivalent to that achieved by 300 mg/kg unmodified ME23. Repeated injections of Vivo-ME23 restored homogeneous expression of dystrophin in all skeletal muscles and partial expression in cardiac muscle without eliciting a detectable immune response. This together with the improvement of muscle pathology indicates the feasibility of the use of Vivo-Morpholino as antisense therapy for the treatment of a majority of DMD patients. Effective targeting for dystrophin expression was also exhibited in easy muscles of blood vessels bodywide. Conjugation of morpholinos with the nonpeptide delivery moiety, therefore, also provides promise for universal delivery of oligonucleotides to achieve RNA interference for treating diseases from viral infections to cancers. Results Intramuscular injection of Vivo-ME23-induced dystrophin expression in 90% muscle fibers The mouse exhibits a nonsense-point mutation in E23 of the dystrophin gene and, except for a few revertant fibers or cardiomyocytes, produces no dystrophin in either skeletal or cardiac muscles.5,6,23,24 Our previous studies have shown that a Morpholino oligomer, E23 + 7C18 (referred as ME23), targeting the boundary sequence of E23 (last seven bases) and intron 23 (first 18 bases) in the mouse dystrophin gene was able to induce skipping of E23 and dystrophin expression in muscles of the mice.15 However, restoration of functional amounts of dystrophin in muscles requires repeated injections of large amounts of ME23. Furthermore, dystrophin induction was highly variable within and between the skeletal muscles and was barely detectable in cardiac muscle. We suspect that the low efficiency of antisense effects is primarily due to the poor penetration of Morpholino into cells, severely limiting the amounts of AON gaining access to its intracellular target. To explore whether delivery-enabled Morpholino can improve antisense effects, we FLJ20032 first compared the octa-guanidine-conjugated Dehydrodiisoeugenol ME23 (Vivo-ME23) (Physique 1a) to the unmodified ME23 injected intramuscularly, either alone or with endoporter, an amphiphilic peptide (hereafter referred as ME23 + endoporter). All formulations of.