PubMed2002
The ability of a systemically administered gene therapy vector to exhibit extended circulation lifetimes, accumulate at a distal tumor site, and enable transgene expression is unique to SPLP. The flexibility and low toxicity of SPLP as a platform technology for systemic gene therapy allows for further optimization of tumor transfection properties following systemic administration. For example, the PEG coating of SPLP is necessary to engender the long circulation lifetimes required to achieve tumor delivery. However, PEG coatings have also been shown to inhibit cell association and uptake required for transfection. The dissociation rate of the PEG coating from SPLP can be modulated by varying the acyl chain length of the ceramide anchor, suggesting the possibility of developing PEG-Cer molecules that remain associated with SPLP long enough to promote tumor delivery, but which dissociate quickly enough to allow transfection. Alternatively, improvements may be expected from inclusion of cell-specific targeting ligands in SPLP to promote cell association and uptake. Finally, the nontoxic properties of SPLP allow the possibility of higher doses. A dose of 100 micrograms plasmid DNA per mouse corresponds to a dose of approximately 5 mg plasmid DNA per kg body weight. This compares well to small molecules used for cancer therapy, which typically are used at dose levels of 10 to 50 mg per kg body weight. In summary, SPLP consist of plasmid encapsulated in a lipid vesicle that, in contrast to naked plasmid or complexes, exhibit extended circulation lifetimes following intravenous injection, resulting in accumulation and transgene expression at a distal tumor site in a murine model. The pharmacokinetics, biodistribution, and tumor transfection properties of SPLP are highly sensitive to the nature of the ceramide anchor employed to attach the PEG to the SPLP surface. The SPLP-CerC20 system in which the PEG-Cer does not readily dissociate exhibits good serum stability, long circulation lifetimes, and high levels of tumor accumulation and mediates marker gene expression at the tumor site. The flexibility of the SPLP system offers the potential of further optimization to achieve therapeutically effective levels of gene transfer and clearly has considerable potential as a nontoxic systemic gene therapy vehicle with general applicability. These features of SPLP contrast favorably with previous plasmid encapsulation procedures. Plasmid DNA has been encapsulated by a variety of methods, including reverse phase evaporation, ether injection, detergent dialysis in the absence of PEG stabilization, lipid hydration and dehydration-rehydration techniques, and sonication, among others. The characteristics of these protocols are summarized in Table I. None of these procedures yields small, serum-stable particles at high plasmid concentrations and plasmid-to-lipid ratios in combination with high plasmid-encapsulation efficiencies. Trapping efficiencies comparable with the SPLP procedure can be achieved employing methods relying on sonication. However, sonication is a harsh technique that can shear nucleic acids. Size ranges of 100 mm diameter or less can be achieved by reverse-phase techniques; however, this requires an extrusion step through filters with 100 nm or smaller pore size which can often lead to significant loss of plasmid. Finally, it may be noted that the plasmid DNA-to-lipid ratios that can be achieved for SPLP are significantly higher than those achievable by any other encapsulation procedure.