HUMAN GENE THERAPY 16:000–000 (November 2005)© Mary Ann Liebert, Inc.Poly(ethylene imine)–Polyethylene Glycol CopolymersFacilitate Efficient Delivery of Antisense Oligonucleotides toNuclei of Mature Muscle Cells of mdx MiceSHASHANK R. SIRSI, JASON WILLIAMS, and GORDON J. LUTZABSTRACTAntisense oligonucleotides (AO) can facilitate dystrophin expression via targeted exon skipping in culturedcells of Duchenne muscular dystrophy (DMD) patients and in the mouse model of DMD (mdx mice). How-ever, the lack of effective means to deliver AO to myonuclei remains the foremost limitation to their useful-ness in DMD gene therapy. In this study we show that copolymers of cationic poly(ethylene imine) (PEI) andpolyethylene glycol (PEG) facilitated efficient cellular uptake and nuclear delivery of AO in mature skeletalmuscle fibers isolated from mdx mice. Confocal analysis of dual fluorescently tagged PEG–PEI–AO polyplexes,24 hr after transfection, showed that the copolymer and AO were colocalized within punctate membrane-as-sociated structures. Importantly, AO was efficiently translocated into myonuclei, whereas the copolymer wasmostly excluded. The morphology of all transfected myofibers was perfectly maintained with no indication ofdamage or cytotoxicity. Quantitative fluorescence analysis showed that transfection with PEG–PEI–AO re-sulted in a 6-fold higher uptake of AO into myonuclei compared with transfections of AO alone. Interestingly,transfections with rhodamine-labeled PEG–PEI copolymers yielded an approximately 2-fold higher uptake ofAO into myonuclei compared with transfections of unlabeled copolymers. Attempts to further increase AOdelivery by addition of insulin–transferrin–selenium (ITS) to the medium showed no further improvement inAO delivery. Dose–response analysis indicated saturation of endocytotic uptake of the polyplex. Overall, weconclude that PEG–PEI copolymers represent high-capacity, nontoxic carriers for efficient delivery of AO tonuclei of mature myofibers.1OVERVIEW SUMMARYAntisense oligonucleotides (AO) containing 2-O-methylmodifications can facilitate dystrophin expression via mod-ulation of pre-mRNA splicing and may represent agents forpotential treatment of Duchenne muscular dystrophy(DMD). However, better methods of comprehensively de-livering AO to myonuclei must first be developed. In thispaper we show that PEG–PEI copolymers function as high-capacity carriers that facilitate efficient delivery of AO tonuclei of mature myofibers isolated from mdx mice. Confo-cal microscopy of muscle cells 24 hr after transfection withdual fluorescently labeled PEG–PEI–AO polyplexes re-vealed that AO was efficiently translocated into myonuclei,whereas the copolymer was almost entirely excluded fromentering the nuclei. An important aspect of this study wasthe finding that the polymer-mediated delivery of AO to my-onuclei was accomplished without any indication of cyto-toxicity. We conclude that PEG–PEI–AO polyplexes mayprove to be effective and safe agents for induction of dys-trophin expression and treatment of DMD.INTRODUCTIONAVARIETY OF CHEMICAL MODIFICATIONSto the basic structureof small antisense oligonucleotides (AO) has greatly ex-panded their target specificity, functionality, and resistance todegradation, and thus improved their potential usefulness (Mar-cusson et al., 1999; Dias and Stein, 2002; Opalinska andDepartment of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA 19102.AU1Gewirtz, 2002; Kurreck, 2003; Goodchild, 2004; Ravichandranet al., 2004; Shi and Hoekstra, 2004). Chemically modified AOare becoming increasingly valuable agents for gaining knowl-edge of protein function from genomic and proteomic studies(Goodchild, 2004; Ravichandran et al., 2004) as well as fortreating diseases through modulation of pre-mRNA splicing, al-ternative splicing, and exon skipping (Sazani and Kole, 2003;Bremmer-Bout et al., 2004; Goodchild, 2004; Ravichandran etal., 2004; van Deutekom, 2005).Studies have shown that AO containing 2-O-methyl modifi-cations can facilitate exon skipping of dystrophin pre-mRNAand produce dystrophin expression in mdx mice (Mann et al.,2001; Aartsma-Rus et al., 2002; Gebski et al., 2003; Wells etal., 2003) and in cultured cells of Duchenne muscular dystro-phy (DMD) patients (van Deutekom et al., 2001; Aartsma-Ruset al., 2003). This AO-based approach represents an attractivealternative to virus-based gene replacement strategies, as viralvectors continue to be plagued by complications arising fromhost immunogenic response, uncontrolled insertion into the hostgenome, and spontaneous mutagenesis (Marshall, 1999;Gilchrist et al., 2002; Cerletti et al., 2003; Jiang et al., 2004;Zaiss and Muruve, 2005). However, the lack of effective meansto deliver AO to the nuclei of mature myofibers remains theforemost limitation to their usefulness as a nonviral alternativefor DMD gene therapy.Several excellent reviews have defined the barriers that mustbe overcome for successful delivery of oligonucleotides to tar-get cell nuclei, and have outlined progress in carrier-mediatednonviral delivery to overcome those barriers (Hughes et al.,2001; Dass, 2002; Dias and Stein, 2002; Merdan et al., 2002;Wiethoff and Middaugh, 2003; Roth and Sundaram, 2004; Shiand Hoekstra, 2004). The rate-limiting factors in biopolymer-mediated delivery are further complicated in mature skeletalmuscle cells, relative to most other cell types, because of theirhighly ordered membrane structure and reduced rate of endo-cytosis. Alone, small oligonucleotides have low transfection ef-ficiency and are rapidly degraded by nucleases, necessitatingthe use of carrier molecules.For carrier-mediated transfection of small oligonucleotides, themajority of studies have relied on cationic lipoplexes (Brazas andHagstrom, 2005), some of which are commercially available. Al-though significant progress has been made in improvinglipoplexes for oligonucleotide delivery (Hughes et al., 2001; Ul-rich, 2002), cytotoxicity and serum reactivity continue to hindertheir usefulness especially for use in vivo (Semple et al., 2005).The synthetic polymer poly(ethylene imine) (PEI) is wellknown as an efficient nucleotide carrier because of its cationicnature, enabling it to bind both to the negatively