ANTISENSE & NUCLEIC ACID DRUG DEVELOPMENT 6:169-175 (1996) Mary Ann Liebert, Inc. A Simple Method for Delivering Morpholino Antisense Oligos into the Cytoplasm of Cells MICHAEL PARTRIDGE, ALEXANDRA VINCENT, PAULA MATTHEWS, JOHN PUMA, DAVID STEIN, and JAMES SUMMERTON ABSTRACT We report a simple and effective means for delivering Morpholino antisense oligos into the cytosol of cultured anchorage-dependent animal cells. This method, referred to as scrape-loading, is carried out in a matter of seconds, uses a common inexpensive laboratory implement, and has minimal detrimental impact on the cel[L Using this delivery method, a Morpholino oligo present at 0.1 μM and 1 μM in the extracellular medium inhibited its targeted genetic sequence within cultured Hela cells at levels of 56% and 85%, respectively. Lack of inhibition by two control Morpholino oligos at concentrations up to 3 μM indicates good sequence specificity by this structural type. Also described is a test system for simple, rapid, and sensitive quantitation of antisense activity in cultured cells. INTRODUCTION DELIVERY OF ANTISENSE OLIGOS into the cytosolic/nuclear compartment of mammalian cells has proven to be one of the greatest challenges in the antisense field. Typically, in cultured cells, antisense oligos are endocytosed, but then they appear to remain largely in the endosornal/lysosomal compartment until degraded or exocytosed (Shoji et al., 1991; Oberhauser and Wagner, 1992; Wagner et al., 1993; Bennett, 1993; Stein and Cheng, 1993; Tonkinson and Stein, 1994; Bonham et al., 1995). In either case, most or all of the endocy- tosed oligos fail to gain access to their targeted genetic sequences within the cell. Various strategies have been devised to achieve cytosolic delivery of antisense oligos. Some of the more effective include direct microinjection (Wagner et al., 1993) and complexing with cationic liposomes (Colige et al., 1993; Bennett et al., 1994). Although these methods appear to work, microinjection is tedious and cationic liposomes are somewhat toxic to cultured animal cells. In 1984, McNeil et al. reported a method for delivering macromolecules into adherent cells, which they refer to as scrape-loading. This method entails gently scraping adherent cells from the surface of a culture dish with a rubber policeman in the presence of the macromolecules to be loaded into the cells. These workers suggest that scraping cells from the surface to which they are adhered generates transient holes in the plasma membrane at those sites that were tightly adhered to the surface. They postulate that macromolecules in the medium enter the cytosol by passage through such holes (McNeil et al., 1984). Scrape-loading has also been used for delivering plasmid DNA into cultured cells (Fechheimer et al., 1987). Although this scrape-loading procedure appears primarily suited for research applications with anchorage-dependent cultured cells, a recent report from research workers at Amgen (Farrell et al., 1995) suggests that an analogous scrape-loading of antisense oligos may occur in smoothmuscle cells permeabilized in the course of balloon angioplasty, a procedure comnionly used to open partially occluded arteries. Herein we describe the use of scrape-loading for delivering into cultured animal cells a novel antisense structural type, which we refer to as Morpholino (Summerton, 1992; ANTIVIRALS Inc. Technical Reports 2 and 3, 1993; Surnmerton and Weller, 1993). As illustrated in Figure 1, Morpholino oligos have a backbone consisting of 6-membered morpholine rings joined by nonionic phosphorodiamidate'intersubunit linkages. Entry of these oligos was assessed using cells stably transfected with plasmids designed for simple, rapid, and sensitive quantitation of antisense activity within cells. MATERIALS AND METHODS Test system The two plasmids used in these studies contain the mouse mammary tumor virus (MMTV) promoter controlling an ANTIVIRALS Inc., Corvallis, OR 97333. 169