Progress toward a combined bacterial and viral gene delivery system for mammalian cells
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Gene therapy requires efficient delivery and expression of genes in specific target cells, but most current gene therapy vectors fail to accomplish this. The goal of these experiments was development of a gene therapy system for breast cancer. The combination of viral and bacterial vectors would create one gene therapy system that achieves these goals and consists of (i) highly infectious, attenuated bacteria containing a retroviral vector that would be efficiently delivered to lymphoid cells in the gut and (ii) a mouse mammary tumor virus (MMTV)-based vector capable of trafficking from lymphocytes to mammary epithelial cells where target gene expression can occur. In initial studies, development of a bacterial delivery system that traffics through M cells and targets lymphocytes was attempted using Shigella, Yersinia, and Escherichia coli. A Shigella asd mutant was unable to efficiently deliver genes to lymphocytes, while gene delivery by the Yersinia type III secretion system caused toxicity to eukaryotic cells. E. coli expressing invasin, which binds b1 integrins on lymphocytes, was still unable to specifically target lymphoid cells. In attempts to develop a retroviral gene therapy vector, an MMTV hybrid provirus was tagged with a green fluorescent protein (GFP) to follow MMTV trafficking in the mouse. However, GFP insertion into the provirus disrupted either cis- or trans-acting elements necessary for replication. To facilitate the mapping of these elements in the MMTV genome, transposon-based mutagenesis was performed, and three MMTV mutants were identified with insertions in the env gene. These mutants had defective cytoplasmic export of unspliced gag mRNA and, thus, were unable to synthesize Gag. In coinfection experiments with MMTV env mutants, wild-type MMTV complemented the defect in unspliced mRNA export. A novel, doubly spliced, mRNA was identified from MMTVinfected cells, and predicted protein motifs showed similarities to other retroviral trans-acting proteins involved in unspliced mRNA export. These results suggested that MMTV expresses a trans-acting protein that allows nuclear export of intron-containing RNA and should be reclassified as a complex retrovirus.