Systemic gene therapy: biodistribution and long-term expression of a transgene in mice.

Abstract

We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) Systemic gene therapy by direct delivery of plasmid DNA coupled to synthetic carriers is appealing because of its simplicity, low toxicity, and potential for multiorgan targeting. Although the efficiency of in vitro transfection of DNA plasmids is limited when compared to delivery by viral vectors, recent advances, especially in liposomal delivery, have demonstrated that nonviral gene transfer offers exciting potential (1, 2). Recent attempts to deliver genetic material have provided an impetus to liposome technology (3-6), in particular formation of complexes of DNA with cationic liposomes (7-11). A few therapeutic clinical trial protocols using local administration of these complexes are ongoing but data on systemic administration are still poorly documented (12,13). In this study, we describe a liposomal formulation for gene delivery called DLS, which could be suitable for systemic administration. To obtain long-term expression from a nonintegrated transgene, episomal self-replicating plasmid vectors were constructed and tested. Using the luciferase gene (luc) as a reporter gene (14), we detected transgene expression in various mouse tissues using measurements of luciferase activity, PCR analysis, and immunohistochemistry. Our results demonstrated that a single injection of an episomal DNA vector using the DLS liposome delivery system yielded widespread and long-lasting transgene expression.MATERIALS AND METHODS Materials. Dioctadecylamidoglycylspermidine (7) (15) were deleted to remove expression of these potentially immunogenic proteins. The resultant episomal/reporter expression vector was termed pBKd2RSV-luc. In addition, a second series of pBKdl and pBKd2 vectors were made in which luc expression was placed under the control of the enhancer/promoter sequences from the immediate-early gene of the human cytomegalovirus (CMV) and the polyadenylylation signal and transcriptional termination sequences from the bovine growth hormone gene. The resultant episomal/reporter expression vectors were termed pBKdlCMV-Iuc and pBKd2CMV-Iuc, respectively. The details of these constructs will be reported elsewhere (L.C.M., unpublished data). The nonepisomal pRSV-luc containing luc under control of the RSV long terminal repeat was constructed as reported (14). pCMVintlux plasmid encodes luc under the control of human CMV immediate-early promoter with intron A (16) (generously provided by M. Mansthorpe, Vical, CA). Preparation of the DLS Liposomes. Liposomes were formed by mixing 1 mg of dioctadecylamidoglycylspermidine and 1 mg of dioleoyl phosphatidylethanolamine. After thorough stirring, the mixture was evaporated to dryness in a round-bottomed borosilicate tube using a rotary Vortex evaporator under vacuum. Then the dry lipid film was hydrated with a maximum volume (60 ,ul per mg of lipid) of a solution containing 160 ,ug of plasmid DNA and was slightly Vortex mixed. After in...

Comparison between intratracheal and intravenous administration of liposome–DNA complexes for cystic fibrosis lung gene therapy

“…Balasubramaniam et al 28 show that free DNA transfects respiratory tissue more efficiently than DC-choesterol-DNA or DOTAP-DNA, which are cationic lipids in clinical use, indicating the poor transfection efficiency of those lipids. Other studies 7,13,17,19,29 do not include free DNA controls. Recently, two reports 10,11 describe a drastic improvement in transfection efficiency after intranasal administration of optimized liposome-DNA formulations.…”

“…12 Systemic intravenous infusions of liposome-DNA complexes have previously been shown to transfect lung tissue. 13,14 This mode of administration avoids the mucus barrier in the CF lung, but will also transfect other organs, leading to loss of DNA-liposome material and possibly toxic side-effects. A side by side comparison using the same liposomal vector, will be necessary to determine which route of administration proves to be more effective for gene therapy of CF and other lung diseases.…”

Stabilized plasmid-lipid particles: construction and characterization

“…In the case of nonviral systems such as plasmid DNA-cationic lipid complexes (lipoplexes), the large size and positively charged character of these aggregates also result in rapid clearance, and the highest expression levels are again observed in first-pass organs, particularly the lungs. [1][2][3][4] Plasmid DNA-cationic lipid complexes can also result in toxic side-effects both in vitro 5 and in vivo. 6 The need for a gene delivery system for treatment of systemic disease is obvious.…”

Co-polymer of histidine and lysine markedly enhances transfection efficiency of liposomes

“…Cationic liposomes are the most commonly used nonviral delivery system for delivery of plasmids into cells in vitro [1][2][3] and in vivo. [4][5][6] A significant problem associated with cationic liposomes is their low in vivo transfection efficiency, 7,8 possibly due to inactivation of the liposomes by serum. 9,10 Despite this limitation, cationic liposome carriers have several major advantages such as relative ease of large-scale production and minimal toxicity.…”