1-2-dielaidoylphosphatidylethanolamine and Osteosarcoma

We recently have developed a unique cytoplasmic transient gene expression system based on cotransfection of target cells with bacteriophage T7 RNA polymerase (RNAP) and plasmid DNA vectors containing a T7 autogene. Because this T7 system is self-initiating, self-maintaining, and requires no cellular factors for transcription, it is therefore likely to function in any mammalian cell with any gene both in vitro and, more importantly, in vivo. In this study we demonstrate that the T7 DNA vector and T7 RNAP could be efficiently codelivered to cultured cells by lipofection. Different target genes were expressed by the T7 system in a wide variety of mammalian cells including several tumor cell lines. Gene expression could be detected in more than 30% of the cells of some tumor cell lines transiently transfected by the T7 vector. Average activity of the reporter enzyme (luciferase) expressed by a transfected cell was relatively constant regardless of the cell line used. When a T7-luciferase vector was directly injected into various tissues of mice without the use of liposomes, luciferase activity could be found in the injected liver, muscle, brain and tail connective tissues. The luciferase levels expressed by the T7 system were found to be up to 200-fold higher, depending upon the injected tissues, than levels achieved with a traditional nuclear gene expression vector. Direct tumor injection with a T7-beta-galactosidase (beta-gal) construct resulted in beta-gal gene expression in tumor cells near the injection sites. In addition, direct injection of the T7 system in mice did not generate detectable quantities of antibodies against the T7 RNAP. These results suggest that this gene expression system may be useful in many different medical applications such as cancer gene therapies and DNA vaccination, where transient but rapid and efficient gene expression is required.

Topics: Animals; Antibodies, Viral; Bacteriophage T7; beta-Galactosidase; Cation Exchange Resins; CHO Cells; Cricetinae; Cytoplasm; DNA-Directed RNA Polymerases; Fibrosarcoma; Gene Expression; Genes, Reporter; Genes, Viral; Genetic Therapy; Genetic Vectors; Glioma; Humans; L Cells; Lipids; Liver; Luciferases; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Osteosarcoma; Phosphatidylethanolamines; Plasmids; Promoter Regions, Genetic; Rats; Transfection; Tumor Cells, Cultured; Viral Proteins

A 37-mer hammerhead ribozyme has been designed to efficiently cleave the 1.4 kb mRNA of the urokinase plasminogen activator receptor (uPAR). Under in vitro conditions, the chemically synthesized ribozyme cleaved uPAR mRNA and inhibited its translation in a concentration-dependent fashion. The ribozymes were 5'-[35S]thiophosphorylated and used as a model to analyze conditions for RNA delivery in a cultured human osteosarcoma cell system. Ribozymes degraded immediately in cell-conditioned medium but ribozymes complexed with lipofectin were protected from RNases for up to 22 h. Lipofectin rapidly transported ribozyme into the cell, where it accumulated almost exclusively in the cytoplasm. Thus, lipofectin dramatically enhances stability and cytoplasmic delivery of ribozymes, potentially enabling targeting of mRNA in vivo.

Topics: Base Sequence; Cell Nucleus; Cytoplasm; Drug Delivery Systems; Humans; Molecular Sequence Data; Nucleic Acid Conformation; Osteosarcoma; Phosphatidylethanolamines; Polyribonucleotides; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Catalytic; RNA, Messenger; Tumor Cells, Cultured