Intracellular gene delivery is dependent on the type of non-viral carrier and defined by the cell surface glycosaminoglycans.

Intracellular limiting steps and molecules involved in internalization and intracellular routing of non-viral gene delivery systems are still poorly understood. In this study, the intracellular kinetics of three different gene delivery systems calcium phosphate precipitates (CaP), polyethyleneimine (PEI) and N-[1-(2,3-dioleyl)propyl]-N,N,N-trimethylammonium chloride (DOTAP)) were quantified at cellular, nuclear, transcriptional and translational levels by using qRT-PCR. Additionally, a role of cell surface glycosaminoglycans (GAGs) was evaluated by performing the aforementioned studies in cells devoid of GAGs (pgsB-618) and cells lacking heparan sulphate (HS). The obtained data showed that the intracellular kinetics was dependent on the type of gene carrier and the weakest intracellular step varied between the carriers; rapid elimination of cell-associated pDNA in CaP, nuclear uptake in DOTAP and transcriptional and translational events in PEI mediated transfections. Overall, neither the amount of cell- nor nuclear associated pDNA correlated with transgene expression but the mRNA expression of the transgene correlated well with the expression at protein level. The nuclear uptake of pDNA in all cases was rapid and efficient thus indicating that the post-nuclear processes including transcription and translation steps have a critical role in defining the efficiency of non-viral gene delivery systems. Our study demonstrated that cell-surface GAGs are not essential for cell surface binding and internalization of gene delivery complexes, but they are able to define the intracellular routing of the complexes by leading them to pathways with high pDNA elimination.

Endothelium-specific overexpression of human vascular endothelial growth factor-D in mice leads to increased tumor frequency and a reduced lifespan.

BACKGROUND: Vascular endothelial growth factors (VEGFs) are central mediators in vascular development and lymphangiogenesis. VEGF-D contributes to the growth and formation of blood and lymphatic vessels, although its biological role is still somewhat unclear. METHODS: Transgenic mice, which express the mature form of human VEGF-D under endothelium-specific Tie1 promoter, were produced by the lentiviral perivitelline-injection method. The mice were followed up to generation F(5) and the effect of the transgene was analyzed. RESULTS: Transgenic mice had a high expression of human (h)VEGF-D in the endothelium in several tissues, such as kidney, liver, lung and spleen. However, transgenic mice developed tumors in lungs, kidneys, liver, mammary glands and lymph nodes upon aging and their mortality was also increased as a result of other pathological conditions. Hind limb ischemia was surgically induced in these mice and they were analyzed 1, 2 and 3 weeks after the ischemia operation. No significant differences were found in hVEGF-D mRNA expression, the number of capillaries or tissue repair between ischemic transgenic mice and transgene negative littermates. CONCLUSIONS: It is concluded that targeted unregulated long-term expression of hVEGF-D in endothelium may not be useful and reduces the life span of transgenic mice.