Research paper Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing N.B. Marshall a,b , S.K. Oda a , C.A. London a , H.M. Moulton a , P.L. Iversen a , N.I. Kerkvliet c , D.V. Mourich a,b, ⁎ a AVI BioPharma Inc., Corvallis, Oregon 97333, United States b Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, United States c Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331, United States Received 1 May 2007; received in revised form 7 June 2007; accepted 19 June 2007 Abstract Phosphorodiamidate morpholino oligomers (PMO) are synthetic antisense molecules that interfere with translation, pre-mRNA splicing and RNA synthesis. Like other gene-silencing technologies, PMO are poorly taken up by primary leukocytes without the use of physical or chemical delivery techniques. We sought an alternative delivery mechanism of PMO into immune cells that eliminates the need for such manipulations. Here we demonstrate the first use of arginine-rich cell-penetrating peptides (CPPs) to deliver PMO (P-PMO) directly into primary murine leukocytes for inhibition of gene expression and promotion of altered pre- mRNA splicing. We compared the P-PMO delivery efficacy of four arginine-rich CPPs including HIV Tat and penetratin, and one histidine rich CPP, and found that the (RXR) 4 peptide was the most efficacious for PMO delivery and targeted antisense effect. The delivery and antisense effects of P-PMO are time- and dose-dependent and influenced by the activation and maturation states of T cells and dendritic cells, respectively. Targeted expression of several genes using P-PMO is shown including surface signaling proteins (CD45 and OX-40), a cytokine (interleukin-2), and a nuclear transcription factor (Foxp3). Considering the abundance of naturally occurring alternatively spliced gene products involved in immune regulation, P-PMO offer an effective method for modulating gene activity for immunological research and applications beyond traditional antisense approaches. © 2007 Elsevier B.V. All rights reserved. Keywords: Morpholino; Antisense; Splicing; Leukocytes; Cell-penetrating peptides; Delivery; Foxp3; OX-40; CD45; Interleukin-2 1. Introduction Antisense is a method of altering gene expression by introducing RNA, DNA or synthetic oligomers that complement sequences within a targeted mRNA molecule. There are two traditional approaches to dis- rupting mRNA translation using antisense: (i) targeting of the AUG start site and thus sterically blocking ribosomal assembly or scanning and (ii) targeting the mRNA for nuclease degradation using RNase H or Journal of Immunological Methods 325 (2007) 114 – 126 www.elsevier.com/locate/jim Abbreviations: P-PMO; peptide-conjugated phosphorodiamidate morpholino oligomer(s); CPP(s); cell-penetrating peptides; DCs; dendritic cells; MFI; mean fluorescence intensity; Con-A; concanav- alin A; MHC; major histocompatibility complex. ⁎ Corresponding author. AVI BioPharma Inc., 4575 SW Research Way, Corvallis, Oregon 97333, United States. Tel.: +1 541 753 3635; fax: +1 541 754 3545. E-mail address: dmourich@avibio.com (D.V. Mourich). 0022-1759/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2007.06.009