function of the defective gene (4,5). One example is Duchenne’s muscular dystrophy (DMD), a genetic disease that affects 1 in 3500 young boys worldwide (6). DMD is a neuromuscular disorder caused mainly by nonsense or frame-shift mutations in the dystrophin gene. SCOs are used to induce targeted ‘exon skipping’ and to correct the reading frame of mutated dystrophin mRNA such that shorter, partially-functional dystrophin forms are produced (7). SCOs targeting exon 51 are cur- rently in human clinical trials in various parts of Europe to treat DMD (8,9). However, translating the promising results of SCOs into bed-side drug products requires op- timization of many parameters ranging from enhancement of cellular uptake and biodistribution to pharmaceutical formulation and long-term stability. SCOs are antisense oligonucleotides (ONs) ranging from 15 to 25 bases in length. In contrast to the normal antisense approach, SCOs must not activate RNase H, which would destroy the pre-mRNA target before it could be spliced (4,5). That is why these ONs are of dif- ferent chemical nature than DNA and RNA. 20-O-methyl RNA (20-OMe) and phosphorothioate (PS) RNA are among the chemistries successfully utilized to induce splice-correction (4). Such chemical modiﬁcations impart better annealing with RNA and enhance the serum stabil- ity of the ONs (10). For these reasons PS-20-OMe SCOs are used in this study. From a pharmaceutical perspective, PS-20-OMe SCOs are to be considered class III drugs in the biopharmaceutical classiﬁcation system (11), with high solubility and low permeability owing to their high mo- lecular weight and dense charge distribution (12). That is why several methods have been devised to enhance their uptake into cells. Cationic liposomes have been routinely used as delivery vectors for ONs; however, toxicity remains a signiﬁcant problem, according to several in vivo ﬁndings (13). One class of vectors that appears potent for this purpose without the associated toxicity of cationic liposomes is cell-penetrating peptides (CPPs). CPPs are polybasic and/or amphipathic peptides, usually less than 30 amino acids in length, that possess the ability to translocate across cellular plasma membranes. They have gained much attention in recent years due to their ability to efﬁ- ciently and safely deliver an array of therapeutic cargos, from small molecules to nanopaticles both in vitro and in vivo (14,15). For splice-correction, CPPs covalently conjugated to different SCOs have been reported to suc- cessfully induce splice-correction in different models (7,16). However, despite being less toxic, the transfection efﬁciencies reported have generally been less than those achieved utilizing lipid-based vectors such as (LF2000) (17). Interestingly, some CPPs have been successfully exploited for ON delivery using a non-covalently complex- ation strategy (18). Having net positive charge, such CPPs have been shown to form nano-sized complexes with nega- tively charged ONs, which are efﬁciently internalized by cells presumably via an endocytosis-dependant mechanism (15,18,18,19). The non-covalent complexation strategy has the advantage of avoiding laborious chemical conjugation of the CPP with its cargo. Moreover, it was found that lower concentrations of ONs are generally required to Nucleic Acids Research, 2011, Vol. 39, No. 12 5285 achieve a biological response utilizing the non-covalent complexation (17,19). However, one limitation of this strategy is that much of the delivered cargo becomes entrapped in endosomes following endocytosis. Therefore, several chemical modiﬁcations have been intro- duced into CPPs to enhance endosomal escape (19). N-terminal stearic acid modiﬁcation was shown to enhance both the complexation capacity and endosomal escape properties of certain CPPs (19,20). It has been shown previously by our group that the stearic acid- modiﬁed CPPs, stearyl-TP10 (21) and stearyl-(RxR)4 (17), are able to transfect cells with PS-20-OMe SCOs . However, a drawback with the above-mentioned peptides is that they are generally less efﬁcient than commercial lipid-based vectors such as LF2000, and relatively inefﬁ- cient at transfecting cells in the presence of serum proteins. Although CPPs have been extensively studied as drug delivery vectors, formulation of CPP-based therapeutics into different pharmaceutical forms has never been given the same attention. Pharmaceutical formulation has always a major inﬂuence on the efﬁciency and stability of active pharmaceutical ingredients. In addition, each therapeutic application requires its own pharmaceutical form. Therefore, developing different formulations for CPP-based therapeutics could have great implications on their activity and stability, besides, broadening the range of therapeutic applications that CPPs could be utilized in. Particularly, solid formulation is the most widely used pharmaceutical form. It is used in tablets, capsules, sustained-release formulations, powders for inhalation and powders for injection. In addition, solid formulations are the most stable forms upon storage and transport. Thus, from a translational perspective, it would be of great importance to be able to formulate CPP-based therapeutics as solid form. Here, we describe a new stearylated CPP that we desig- nate PepFect 14 (PF14). It is a modiﬁed version of our previously reported peptide, stearyl-transportan10 (stearyl-TP10) (21), where we utilized ornithines and leucines instead of lysines and isoleucines. PF14 demon- strates remarkable splice-correction activity in two differ- ent cell models including mdx mouse myotubes, a cell culture model of DMD, even in the presence of serum. Furthermore, the feasibility of formulating these nano- complexes into solid formulation was investigated and the efﬁciency and stability of such formulations were assessed. Remarkably, the solid formulations obtained upon drying the nanocomplexes over soluble excipients were as active as the freshly prepared counterpart even when stored at elevated temperatures for several weeks. MATERIALS AND METHODS Oligonucleotide and peptide synthesis PS-20-O-methyl RNA ON (Table 1) was synthesized on an A¨ KTATM oligopilotTM plus 10 synthesizer with OligosyntTM 15 (GE Healthcare, Sweden) pre-packed syn- thesis columns as previously described (22). For 50-labeling, a molar excess of 10 equivalents Cy5-amidite (GE Healthcare, UK) at 0.1 M was used and Cy5-amidite