Inefficient nuclear delivery of plasmid DNA is thought to activity of apoptotic and lysosomal nucleases; (2) disposal be one of the daunting hurdles to gene transfer, utilizing a of microinjected plasmid DNA was inhibited in cytosolnonviral delivery system such as polycation-DNA complex.depleted cells or following the encapsulation of DNA in Following its internalization by endocytosis, plasmid DNA phospholipid vesicles; (3) generation and subsequent elimhas to be released into the cytosol before its nuclear entry ination of free 3Ј-OH ends could be detected by the tercan occur. However, the stability of plasmid DNA in the minal deoxynucleotidyl transferase-mediated dUTP nick cytoplasm, that may play a determinant role in the transfecend-labeling assay (TUNEL), reflecting the fragmentation tion efficiency, is not known. The turnover of plasmid DNA, of the injected DNA; and finally (4) isolated cytosol, delivered by microinjection into the cytosol, was deterobtained by selective permeabilization of the plasma memmined by fluorescence in situ hybridization (FISH) and brane, exhibits divalent cation-dependent, thermolabile quantitative single-cell fluorescence video-image analysis. nuclease activity, determined by Southern blotting and 32 PBoth single-and double-stranded circular plasmid DNA disrelease from end-labeled DNA. Collectively, these findings appeared with an apparent half-life of 50-90 min from the suggest that the metabolic instability of plasmid DNA, cytoplasm of HeLa and COS cells, while the amount of cocaused by cytosolic nuclease, may constitute a previously injected dextran (MW 70 000) remained unaltered. We prounrecognized impediment for DNA translocation into the pose that cytosolic nuclease(s) are responsible for the nucleus and a possible target to enhance the efficiency of rapid degradation of plasmid DNA, since (1) elimination of gene delivery. plasmid DNA cannot be attributed to cell division or to the Keywords: gene transfer; plasmid DNA; turnover; degradation; DNase; microinjection Introduction Liposome-mediated cellular transfer of plasmid DNA is a promising approach for gene therapy. However, despite the significant amount of lipid/DNA complexes internalized by the target cells, transgene expression remains undesirably low.1 Obstacles to nuclear accumulation of plasmid DNA include: the slow internalization process of the lipid/DNA complex in certain cells; 2 the entrapment of DNA in the endolysosomal compartment; 1,3,4 and the diffusional barrier of the nuclear envelope. 5The underlying mechanism of escape of internalized plasmid DNA from the endo-lysosomes is not fully understood. This process involves the destabilization of the limiting membrane of the endolysosomal compartment, the dissociation of the lipid/DNA complex and the release of plasmid DNA into the cytosol.6-8 Penetration of naked plasmid DNA into the cytosol was verified by using the T7 polymerase transfection system, which Correspondence: GL Lukacs, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canad...

A detergent dialysis procedure is described which allows of up to 70% and permits inclusion of 'fusigenic' lipids such encapsulation of plasmid DNA within a lipid envelope, as dioleoylphosphatidylethanolamine (DOPE). The in vitro where the resulting particle is stabilized in aqueous media transfection capabilities of SPLP are demonstrated to be by the presence of a poly(ethyleneglycol) (PEG) coating. strongly dependent on the length of the acyl chain conThese 'stabilized plasmid-lipid particles' (SPLP) exhibit an tained in the ceramide group used to anchor the PEG polyaverage size of 70 nm in diameter, contain one plasmid mer to the surface of the SPLP. Shorter acyl chain lengths per particle and fully protect the encapsulated plasmid from result in a PEG coating which can dissociate from the digestion by serum nucleases and E. coli DNase I. Encap-SPLP surface, transforming the SPLP from a stable parsulation is a sensitive function of cationic lipid content, with ticle to a transfection-competent entity. It is suggested that maximum entrapment observed at dioleoyldimethylam-SPLP may have utility as systemic gene delivery systems monium chloride (DODAC) contents of 5 to 10 mol%. The for gene therapy protocols. formulation process results in plasmid-trapping efficiencies

Cationic lipid-based delivery systems such as lipoplexes or aphidicolin exhibited 20-fold lower reporter gene activity stabilized plasmid-lipid particles (SPLP) represent a safer than asynchronous control cells upon incubation with lipoalternative to viral systems for gene therapy applications.plexes. When cells arrested in the G1 phase were allowed We studied the impact of cell cycle status on the efficiency to proceed though the cell cycle in the presence of the lipoof transfection of human ovarian carcinoma tumor cells plex or SPLP, transgene expression was found to coincide using two cationic-lipid based delivery systems. Cells with the transition of cells from the G2/M phase into the arrested in the G1 phase of the cell cycle by treatment with G1 phase of the subsequent cell cycle. In addition, higher aphidicolin were compared with an asynchronous dividing levels of reporter gene expression were observed when the population of cells. Treatment of the cells with aphidicolin cells were incubated with lipoplexes or SPLP during, or just had no effect on the rate of internalization of the lipid forbefore, mitosis. These results suggest that it may be possmulated DNA or on the level of gene expression observible to augment cationic lipid-mediated transfection by able in stably transfected cells. However, cells treated with manipulating the cell cycle status of the target cells.

The structure of 'stabilized plasmid-lipid particles' (SPLP) and their properties as systemic gene therapy vectors has been investigated. We show that SPLP can be visualized employing cryo-electron microscopy to be homogeneous particles of diameter 72 ± 5 nm consisting of a lipid bilayer surrounding a core of plasmid DNA. It is also shown that SPLP exhibit long circulation lifetimes (circulation half-life Ͼ6 h) following intravenous (i.v.) injection in a murine tumor model resulting in accumulation of up to 3% of the total injected dose and concomitant reporter gene expression at a distal (hind flank) tumor site. In contrast, i.v. injection of

An ability to generate a well defined lipid-based carrier system for the delivery of plasmid DNA in vivo requires the characterization of factors governing DNA/lipid interactions and carrier formation. We report that a hydrophobic DNA/lipid complex can be formed following addition of cationic lipids to DNA in a Bligh and Dyer monophase consisting of chloroform/methanol/water (1:2.1:1). Subsequent partitioning of the monophase into a two-phase system allows for the extraction of DNA into the organic phase. When using monovalent cationic lipids, such as dimethyldioctadecylammonium bromide, dioleyldimethylammonium chloride, and 1,2-dioleyl-3-N,N,N-trimethylaminopropane chloride, greater than 95% of the DNA present can be recovered in the organic phase when the lipid is added at concentrations sufficient to neutralize DNA phosphate charge. When the polyvalent cationic lipids 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 1- propanaminium trifluoroacetate and diheptadecylamidoglycyl spermidine are used, efficient extraction of the DNA into the organic phase is also achieved when the charge ratio between lipid and DNA is approximately equal. Formation of the hydrophobic DNA complex can only be achieved with cationic lipids. In the absence of added cations or in the presence of excess Ca2+, L-lysine, or poly(L-lysine), 100% of the DNA is recovered in the aqueous fraction. The monovalent cationic lipid/DNA complexes can also be prepared in the presence of detergent; however, low concentrations of NaCl (< 1 mM) lead to dissociation of the complex. Importantly, these results clearly demonstrate that cationic lipid binding does not lead to DNA condensation. The methods described, therefore, enable DNA/lipid complexes to be characterized in the absence of DNA condensation.(ABSTRACT TRUNCATED AT 250 WORDS)