In addition to tracking the radioactive lipid label we also performed a PCR analysis of tissue samples in order to verify that the gene cargo of liposomes was present in the different organs. Indeed we find more found that both luciferase and EGFP expression plasmids were detected in a semi-quantitative assay in good alignment with the distribution of the radioactive

lipid label (Fig. 6). In our strategy to develop a suicide gene therapy for small cell lung cancer the most promising system to date is the yeast cytosine deaminase (YCD) gene fused with the uracil phosphoribosyl transferase (YUPRT) gene driven from the human INSM1 promoter in combination with administration of 5-fluoro-cytosine (5-FC) prodrug [13]. When we tailvein-injected tumor-bearing nude mice with suicide gene encapsulating SPLPs and administered prodrug intraperitoneally in two preliminary experiments, we could neither observe a significant reduction in tumor size by caliper measurements (Fig. 8) nor an increase in dead tumor cells measured by TUNEL-positive cells in fixed tissue sections. A high apoptotic index of the cancer cells ZVADFMK within the tumor could be masking a subtle effect of the suicide gene therapy treatment ([13], data not shown).

Previous trials with this suicide gene system utilizing intratumoral delivery showed a prompt response in tumor growth already after one or two days [13], hence these results are in alignment with the low efficiency of transgene expression as described above using luciferase and EGFP reporters. Even so, the system constitutes an attractive delivery vehicle that enables tumor targeting after systemic administration without causing adverse retention in non-target organs allowing evaluation of cancer gene therapy strategies within the appropriate tissue of a xenograft tumor model. Obviously the transfection efficiency in target tissue requires augmentation of

the present results with commercially available lipids and optimization of lipids formulated into SPLPs is ongoing [25] and [35]. Furthermore we are aiming to incorporate lipids responsive to local tumor environment, e.g. using pH-sensitive, detachable Tryptophan synthase PEG-moieties [36] or other enzyme activities found in tumor environment [37] and hereby arriving at a transfection efficiency that is useful in a therapeutic setting. A protocol is described for the preparation of SPLPs with encapsulated plasmid DNA for treatment of SCLC using a transcriptionally targeted suicide gene therapy approach. The system was tested for systemic delivery to xenograft tumors in nude mice and showed attractive properties of circulation and tumor accumulation, however without causing effective transfection.