Development of bacterial delivery systems for the introduction of DNA into eukaryotic cells
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Gene therapy involves the delivery of DNA to cells in order to correct a genetic defect or kill a malignant cell. A major drawback to current approaches involves the targeted delivery to the tissue of interest. This work evaluates two different approaches that both utilize an attenuated bacterium for the delivery of DNA constructs to eukaryotic cells in vitro. The initial approach was to use an invasive bacterium, Shigella flexneri. A mutation in the asd gene was introduced into S. flexneri, rendering the bacterium dependent on diaminopimelic acid (DAPA). Following invasion of the host cell, the absence of DAPA causes the bacterium to lyse and release its content into the cytoplasm of the eukaryotic cell. This mutant was used to demonstrate its ability to deliver eukaryotic reporter plasmids into epithelial cells. In addition, it was shown that S. flexneri can infect various T and B cell lines and deliver the mouse viii mammary tumor virus (MMTV) provirus to these cells. The same mutant was used to deliver a reporter plasmid to murine M cells in situ. DNA delivery to the gut-associated lymphatic tissue was not detected. A second approach was based on the ability of Yersinia spp. to inject effector proteins (Yersinia outer proteins; Yops) via a type III secretion machinery directly into the cytoplasm of a eukaryotic cell. A fusion protein was generated between the secretion signal of one such effector protein, YopE, and a DNA-binding protein, LacI. Secretion of this fusion protein was shown to be dependent on the presence of the type III secretion apparatus. It was demonstrated that the ability to secrete a plasmid containing the LacI binding site (lacO) required the presence of the type III secretion apparatus. Furthermore, the ability of this system to translocate lacO-containing reporter plasmids into epithelial cells was demonstrated in tissue culture experiments. Potential applications for this novel DNA delivery system are discussed.