Year: 2004
Type: dissertation
Abstract: Gene therapy requires efficient delivery and expression of genes in \nspecific target cells, but most current gene therapy vectors fail to accomplish this. \nThe goal of these experiments was development of a gene therapy system for \nbreast cancer. The combination of viral and bacterial vectors would create one \ngene therapy system that achieves these goals and consists of (i) highly infectious, \nattenuated bacteria containing a retroviral vector that would be efficiently \ndelivered to lymphoid cells in the gut and (ii) a mouse mammary tumor virus \n(MMTV)-based vector capable of trafficking from lymphocytes to mammary \nepithelial cells where target gene expression can occur. \nIn initial studies, development of a bacterial delivery system that traffics \nthrough M cells and targets lymphocytes was attempted using Shigella, Yersinia, \nand Escherichia coli. A Shigella asd mutant was unable to efficiently deliver \ngenes to lymphocytes, while gene delivery by the Yersinia type III secretion \nsystem caused toxicity to eukaryotic cells. E. coli expressing invasin, which \nbinds b1 integrins on lymphocytes, was still unable to specifically target \nlymphoid cells. \nIn attempts to develop a retroviral gene therapy vector, an MMTV hybrid \nprovirus was tagged with a green fluorescent protein (GFP) to follow MMTV \ntrafficking in the mouse. However, GFP insertion into the provirus disrupted \neither cis- or trans-acting elements necessary for replication. To facilitate the \nmapping of these elements in the MMTV genome, transposon-based mutagenesis \nwas performed, and three MMTV mutants were identified with insertions in the \nenv gene. These mutants had defective cytoplasmic export of unspliced gag \nmRNA and, thus, were unable to synthesize Gag. In coinfection experiments with \nMMTV env mutants, wild-type MMTV complemented the defect in unspliced \nmRNA export. A novel, doubly spliced, mRNA was identified from MMTVinfected \ncells, and predicted protein motifs showed similarities to other retroviral \ntrans-acting proteins involved in unspliced mRNA export. These results \nsuggested that MMTV expresses a trans-acting protein that allows nuclear export \nof intron-containing RNA and should be reclassified as a complex retrovirus.
Author: Melissa Simper
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1903835676