15 targeted gene transfer : a practical guide based on experience with lipid-based plasmid delivery systems.

The overall goal of gene therapy is to cure or stabilize a disease process that results from the production of a mutant protein (for example, the chloride channel protein important in cystic fibrosis) or overproduction of a normal protein (such as the products of certain oncogenes). We can achieve this goal by replacing the defective gene or by reducing the overexpression of the target gene using an antisense strategy, thus reducing the production of the diseasepromoting protein (1,2). For either method, it is critical to transfer DNA into target cells in a concentration high enough to be effective in modifying the disease. DNA must be delivered to the desired cell population in an intact state, whereby it can be efficiently transcribed and ultimately translated. The method of gene transfer must be highly efficient and nontoxic, and the delivery system must be relatively easy to prepare and administer (3). There is a great deal of optimism surrounding the development of gene therapy as an effective strategy for management of many different human diseases. The active agent used to procure gene therapy is likely to consist of oligonucleotides, ribozymes, or a DNA sequence that can be transcribed into a message capable of eliciting a therapeutic response. Unlike conventional small-molecule therapeutics however, gene therapy requires the use of a carrier system to deliver the active agent directly into the target cell population.

A multi-step lipid mixing assay to model structural changes in cationic lipoplexes used for in vitro transfection.

Formation of liposome/polynucleotide complexes (lipoplexes) involves electrostatic interactions, which induce changes in liposome structure. The ability of these complexes to transfer DNA into cells is dependent on the physicochemical attributes of the complexes, therefore characterization of binding-induced changes in liposomes is critical for the development of lipid-based DNA delivery systems. To clarify the apparent lack of correlation between membrane fusion and in vitro transfection previously observed, we performed a multi-step lipid mixing assay to model the sequential steps involved in transfection. The roles of anion charge density, charge ratio and presence of salt on lipid mixing and liposome aggregation were investigated. The resonance-energy transfer method was used to monitor lipid mixing as cationic liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na(2)HPO(4). Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentration increased, lipid mixing and aggregation increased, but with Na(2)HPO(4) only aggregation occurred. NaCl (150 mM) reduced the extent of lipid mixing. Transfection studies suggest that the presence of salt during complexation had minimal effects on in vitro transfection. These data give new information about the effects of polynucleotide binding to cationic liposomes, illustrating the complicated nature of anion induced changes in liposome morphology and membrane behavior.

Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates.

The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.

Cationic liposome--plasmid DNA complexes used for gene transfer retain a significant trapped volume.

The goal of this study is to determine whether cationic liposomes retain any trapped volume after their complexation to plasmid DNA. This serves two purposes: to further the understanding of the physical nature of liposome/plasmid DNA complexes used in gene therapy and to investigate the potential for codelivery of other encapsulated molecules with the liposome-DNA complexes. Cationic liposomes composed of N,N-dioleoyl-N,N-dimethylammonium chloride and dioleoylphosphatidylethanolamine (DODAC/DOPE, 50/50 mol %) encapsulating an aqueous trap marker were used to prepare liposome-DNA complexes at various charge ratios. The trapped volume before and after DNA binding was measured by two methods: dialysis and filtration. The effect of tissue culture medium on trapped volume was also investigated. A lipid-mixing assay was employed to further characterize the aggregation events that influence trap volume. The trapped volume (Vt) of neutral control liposomes was 1.1 +/- 0.04 microL/mumol, which was not affected by the addition of DNA. For cationic liposomes in the absence of DNA the Vt was 1.45 +/- 0.46 and 1.54 +/- 0.08 microL/mumol, as measured by the filtration and dialysis methods, respectively. After addition of DNA, the residual trapped volume (RVt) decreased to 0.43 +/- 0.1 microL/mumol and 0.47 +/- 0.05 microL/mumol, as determined by each method, respectively. RVt increased as the ratio of cationic lipid to DNA (nmol of lipid/mg of DNA) was increased above 10, a ratio that corresponds to a charge ratio (positively charged lipids to negatively charged phosphate groups) of 1.62. Aggregation and lipid-mixing were greatest at charge ratios coinciding with the lowest trapped volume. In the presence of tissue culture medium, the Vt of cationic liposomes but not neutral liposomes was reduced, suggesting that the salts have a direct effect on cationic liposomes in the absence of DNA. The RVt of both neutral and cationic liposomes in the presence of DNA, however, was not different from that of the liposomes in the absence of DNA. These results suggest that a significant trapped volume is retained by cationic liposomes after binding to plasmid DNA. This is an important finding with regard to the potential use of DNA/liposome complexes in the codelivery of other bioactive molecules at the time of cell transfection.

Plasmid DNA is protected against ultrasonic cavitation-induced damage when complexed to cationic liposomes.

Cationic liposomes bound to plasmid DNA are currently used for in vitro and in vivo gene therapy applications, but such complexes readily form large, heterogeneous aggregates that are not appropriate for pharmaceutical development. More importantly, size heterogeneity makes studies focused on optimizing gene transfer to cells difficult to conduct or understand. For this reason we have evaluated the effect of microprobe sonication on these complexes in an effort to achieve process-controlled size homogeneity. Complexes were prepared using a 7.2 kb reporter plasmid and the following liposomal lipid combinations: DDAB/DOPE (50:50 mol %), DDAB/DOPE/PEG-PE (50:45:5 mol %), DDAB/EPC (50:50 mol %), DDAB/EPC/PEG-PE (50:45:5, 50:40:10, 50:35:15 mol %), DODAC/DOPE (50:50 mol %), and DODAC/EPC (50:50 mol %) (DDAB, dimethyldioctadecylammonium bromide; DOPE, dioleoylphosphatidylethanolamine; PEG-PE, monomethoxypolyethylene glycol2000 succinate- distearoylphosphatidylethanolamine; EPC, egg phosphatidylcholine; DODAC, dioleoyldimethylammonium chloride). The influence of complex composition and lipid:DNA ratio was evaluated. Particle size was determined before and after complexation and again after sonication using the quasi-elastic light scattering technique. DNA integrity was assessed via agarose gel electrophoresis. Finally, gene transfection was evaluated using CHO cells that were transfected in vitro with sonicated and unsonicated complexes. It is established in this study that size reduction can occur, but this is dependent on cationic and neutral lipid composition and, in some cases, lipid:DNA ratio. Surprisingly, the process of sonication leaves a significant percentage of the plasmid DNA intact and capable of in vitro transfection. This study shows that plasmid DNA can be protected from damage due to sonication by liposome complex formation. This may indicate that more common pharmaceutical methods for size reduction which subject particles to mechanical stress may be applicable in preparation of liposome/DNA formulations for in vivo application.