5288 Nucleic Acids Research, 2011, Vol. 39, No. 12 according to the protocols for transfection and diluted in Opti-MEM with 10% serum into a ﬁnal volume of 500 ml. Samples were assessed in disposable low-volume cuvettes. All data was converted to ‘relative intensity’ plots from where the mean hydrodynamic diameter was derived. Zeta potential was measured in OptiMEM supplemented with 10% FCS or in 0.01 mM KCl. Measurements were performed utilizing the abovementioned instrument, set to automode and a number of ﬁve runs. Stability study Lactose solid dispersion formulations at a concentration of 3.33% were prepared according to the protocol for transfection and stored either at room temperature or in ovens of adjusted temperatures (40 and 60 C). Samples were monitored for 8 weeks and tested at 0, 2, 4 and 8 weeks time points. Statistics RESULTS Values in all experiments are represented as mean ± SEM of at least three independent experiments done in dupli- cate. Differences were considered signiﬁcant at *P < 0.05 using two-tailed Student’s t-test. PF14 efﬁciently induces SCO-mediated splice-correction in serum-free and serum-containing media in HeLa pLuc705 cell model To screen for the most optimal peptide-SCO ratio for splice-correction activity, PF14-SCO complexes were formed at different MRs. MR 3, 5, 7 and 10 were tested in serum-free medium and MR 3, 5, 10 and 20 were tested in serum-containing medium with dose titrations using 200, 100 or 50 nM SCO. LF2000 was used as positive control in all experiments according to manufacturer’s protocol utilizing the highest dose (200 nM SCO). The assay we used is based on the HeLa pLuc705 cell-line stably transfected with a luciferase-encoding gene inter- rupted by a mutated b-globin intron 2. This mutation causes aberrant splicing of luciferase pre-mRNA resulting in the synthesis of non-functional luciferase (26). Masking the aberrant splice site with PS-20-OMe SCO redirects splicing towards the correct mRNA and consequently restores luciferase activity. We found that splice- correction was induced in a dose-dependent manner at all the tested MRs and using MR5 resulted in the highest splice-correction activity in both types of media. Remarkably, in serum-free medium, PF14 at MR5 exceeded LF2000 by several times (Figure 1A). Interestingly, in serum-containing medium, PF14 at MR5 was capable of retaining >75% of its activity observed in serum-free conditions, and also signiﬁcantly surpassing LF2000 (Figure 1B). EC50 and kinetics of PF14-SCO mediated splice-correction Next, RT–PCR was conducted to assess the EC50 of PF14 nanocomplexes. EC50 is the concentration of SCO at which 50% splice-correction occurs. It is a very important parameter in the characterization of a delivery vector, because it allows direct comparison of the efﬁciencies of different vectors tested using the same model. RT–PCR was utilized to detect the quantity of corrected full-length luciferase mRNA after dose titration with different SCO concentrations complexed with PF14 at MR5. We found that PF14-SCO nanocomplexes have very low EC50 values ( 100 nM) in both serum-free and serum-containing medium (Figure 2A). In order to assess the kinetics of splice-correction, cells were transfected with PF14 at MR5 using 200 nM SCO and then the percent of splice-correction was monitored over a time interval ranging from 2 h to 48 h. More than 60% splice-correction was observed already after 8 h (Figure 2B). This shows that, although PF14 forms stable and highly active nanocomplexes with SCOs, the SCO cargo is released quickly inside the cells resulting in an early onset of the biological activity. The splice- correction activity reached its peak after 24 h before it started to decline within 48 h (Figure 2B). PF14 efﬁciently induces SCO-mediated splice-correction in serum-free and serum-containing media in differentiated mdx mouse myotubes The leading cell-model system for development of drugs and drug delivery systems for treatment of DMD is the H2K mdx mouse myotubes, which carries a point mutation in exon 23 of the dystrophin gene (25). PF14- SCO nanocomplexes at MR5 (Figure 3A and B) or MR7 (Figure 3C) were incubated with mouse mdx muscle cells at various concentrations in the presence or absence of 10% FBS. Signiﬁcant amounts of exon skipping were observed, which increased in a concentration-dependent manner. For MR5, the levels were as good as treatment with the same concentration of SCO using LF2000, both in OptiMEM (serum-free conditions), and at MR7, PF14 was higher than LF2000 (Figure 3B and C). The effect of added 10% FBS was small on exon skipping at both MRs and especially at MR7, showing that PF14 activity is not substantially affected by serum. PF14-SCO nanocomplexes are internalized via endocytosis Subsequently, Cy5-labeled SCOs were used to determine the relation between the quantity of SCOs taken up by the cells and the biological activity. PF14 mediated uptake of Cy5-labeled SCO in a dose-dependent manner, which was congruent to the splice-correction data obtained simultan- eously (Figure 4A). However, in the presence of chloro- quine, the splice-correction activity increased, while the uptake remained nearly unchanged. This discrepancy between uptake and biological activity in the presence of chloroquine, a well-known endosomolytic agent, suggests the involvement of endocytic vesicles in the uptake of the nanocomplexes. Endocytic uptake was further supported by the signiﬁcant decrease in activity after incubation at 4 C which is known to inhibit energy-dependant uptake mechanisms like endocytosis (Figure 4B). To further corroborate these results, live cell confocal imaging