5284–5298 Nucleic Acids Research, 2011, Vol. 39, No. 12 doi:10.1093/nar/gkr072 Published online 23 February 2011 PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation Kariem Ezzat1,*, Samir EL Andaloussi2, Eman M. Zaghloul2, Taavi Lehto3, Staffan Lindberg1, Pedro M. D. Moreno 2 , Joana R. Viola2, Tarek Magdy1, Rania Abdo 1 , Peter Guterstam1, Rannar Sillard1, Suzan M. Hammond 4 , Matthew J. A. Wood 4 , Andrey A. Arzumanov5, Michael J. Gait5, C. I. Edvard Smith2,*, Mattias Ha¨ llbrink1,6 and U¨lo Langel1,3,* 1Department of Neurochemistry, Stockholm University, SE-106 91 Stockholm, 2Department of Laboratory Medicine, Karolinska Institutet, SE-141 86 Huddinge, Sweden, 3Tartu University, Institute of Technology, 504 11 Tartu, Estonia, 4Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, 5Medical Research Council, Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK and 6Cepep 2, SE-104 30, Stockholm, Sweden Received August 28, 2010; Revised January 24, 2011; Accepted January 27, 2011 ABSTRACT Numerous human genetic diseases are caused by mutations that give rise to aberrant alterna- tive splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the pheno- type in experimental models. However, transla- tional approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Non-covalent PF14- SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector LipofectamineTM 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of in- corporating this delivery system into solid formula- tions that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms. INTRODUCTION Despite the tremendous success of basic biomedical research in the last decades, new-drug output from pharmaceutical companies has been constant over the last 60 years and mostly based on small molecules (1). This gap between discoveries and therapeutics has recently led to intense interest in translational research; to transform biomedical discoveries into commercializable drug products (2). One group of gene modulating agents showing great potential as therapeutic drug products is splice-correcting oligonucleotides (SCOs). Recent studies using high-throughput sequencing indicate that 95–100% of human pre-mRNAs have alternative splice forms (3). Mutations that affect alternative pre-mRNA splicing have been linked to a variety of cancers and genetic diseases, and SCOs can be used to silence mutations that cause aberrant splicing, thus restoring correct splicing and *To whom correspondence should be addressed. Tel: +468164264; Fax: +468161371; Email: kariem@neurochem.su.se Correspondence may also be addressed to C. I. Edvard Smith. Tel: +46858583651; Fax: +46858583650; Email: edvard.smith@ki.se Correspondence may also be addressed to U¨ lo Langel. Tel: +468161793; Fax: +46816137; Email: ulo@neurochem.su.se Present address: Peter Guterstam, GE Healthcare Bio-Sciences, SE-751 84 Uppsala, Sweden. The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.