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The problem of assessing in vivo activity of gene delivery systems is complex. The reporter gene must be carefully chosen depending on the application. Plasmids with strong promoters, enhancers and other elements that optimize transcription and translation should be employed, such as the CMVint and pCIS-CAT constructs. Formulation aspects of cationic lipid-DNA complexes are being studied in several laboratories, and the physical properties and molecular organization of the complexes are being elucidated. Likewise, studies on the mechanism of DNA delivery with cationic lipids are accumulating which support the basic concept that the complexes fuse with biological membranes leading to the entry of intact DNA into the cytoplasm. Naked plasmid DNA administered by various routes is expressed at significant levels in vivo. This observation is not restricted to skeletal and heart muscle, but has been observed in lung, dermis, and in undefined tissues following intravenous administration. Most of the widely available cationic lipids, including Lipofectin, Lipofectamine and DC-cholesterol have a very poor ability to enhance DNA expression above the baseline naked DNA level, at least in lung. In this report we have revealed a novel cationic lipid, DLRIE, which can significantly enhance CAT expression in mouse lung by 25-fold above the naked DNA level. Other compounds are currently being evaluated which can enhance the naked DNA expression even higher. Plasmid vector improvements have led to further increase in in vivo lung expression, so that the net improvement is > 5,000-fold. Results of this nature are advancing the pharmaceutical gene therapy opportunities for synthetic cationic lipid based gene delivery systems.

A stable single vial lipoplex formulation has been of a T-connector. Homogenous cationic liposome prepdeveloped that can be stored frozen without losing either arations were prepared by extrusion in two different size biological activity or physical stability. This formulation was ranges of either 400 or 100 nm. Extruded liposomes proidentified by systematically controlling several formulation duced more monodisperse and physically stable lipoplex variables and without introducing either stabilizers or surformulations than unextruded liposomes, but the formufactants. Analytical assays were used to unambiguously lations prepared with 100 nm liposomes were less active characterize the formulations. The critical formulation parain in vitro transfection assays than either the 400 nm or meters were: (1) the size of the cationic liposomes; (2) the unextruded liposomes. Low ionic strength and 5% sorbitol rate and method of DNA and cationic liposome mixing; and were required for the lipoplex formulations to survive freez-(3) the ionic strength of the suspending vehicle. The mixing ing and thawing. A frozen lipoplex formulation stored for conditions were precisely controlled by using a novel, spemore than a year maintained its biological activity. These cially designed continuous flow pumping system in which results have broad implications for the pharmaceutical the DNA and liposome solutions were mixed at the junction development of lipoplex formulations for gene delivery.

Cytofectins are positively charged lipophilic molecules that readily form complexes with DNA and other anionic polynucleotides. Normally, cytofectins are combined with an activity-augmenting phospholipid such as dioleoylphosphatidylethanolamine (DOPE), and a film of dried, mixed lipid is prepared and hydrated to form cationic liposomes. The liposome solution is then mixed with a plasmid DNA solution to afford cytofectin-DNA complexes which, when presented to living cells, are internalized and the transgene is expressed. One of the most potent cytofectins, dimyristoyl Rosenthal inhibitor ether (DMRIE), is presently being used to deliver transcriptionally active DNA into human tumor tissues. Here we report the remarkable consequences of replacing the alcohol moiety of DMRIE with a primary amine. The resulting cytofectin, called beta-aminoethyl-DMRIE (betaAE-DMRIE), promoted high level transfection over a broad range of DNA and cationic lipid concentrations. A comparison of in vitro transfection activity between DMRIE and betaAE-DMRIE in 10 cell types revealed that betaAE-DMRIE was more active than DMRIE, and that betaAE-DMRIE, unlike DMRIE, was maximally effective in the absence of colipid. The consequences of the alcohol-to-amine conversion on the structure of the cytofectin/DNA complex was also examined by Atomic Force Microscopy. Strikingly dissimilar images were found for plasmid DNA alone and for the plasmid complexes of betaAE-DMRIE and DMRIE/DOPE.