Escherichia coli as a vector for gene delivery to mammalian macrophage cells.

摘要

Bacterial vectors offer a biological route to gene and protein delivery to antigen-presenting cells (APCs). Primarily in the context of immune stimulation against infectious disease or cancer, the goal of bacterially mediated delivery is to overcome the hurdles to effective macromolecule delivery.; This project has been focused on Escherichia coli ( E. coli) as a gene delivery device with both innate and acquirable (or engineered) biological features to facilitate delivery to APCs. E. coli strain BL21(DE3) was tested as a delivery vector for gene transfer to a murine P388D1 macrophage cell line using a 96-well high throughput assay. Five recombinant strains of E. coli were compared to quantitatively identify the effect heterologously introduced gene products and associated gene expression parameters had on final gene delivery. Recombinant lysteriolysin O (LLO) was expressed from plasmid and chromosomal locations under the control of constitutive Tet or inducible T7 promoters. The E. coli vectors delivered the luciferase reporter gene and delivery success was assessed by recording luciferase luminescence activity within the macrophage cell line. Each E. coli strain carrying the luciferase reporter plasmid exhibited gene delivery as measured by luciferase expression and concomitant luminescence with strain BL21(DE3) harboring a chromosomal copy of a T7-driven LLO showing the greatest relative measure of gene delivery. In addition, the LLO gene delivery vector BL21(DE3) with a chromosomal T7 promoter proved as effective with comparable results to gene delivery using 15% PolyC-PLGA microspheres, affirming the efficiency of E. coli vectors as an alternate for gene delivery to macrophages, especially at higher dosages.; With biological vectors, the use of recombinant DNA technology allows for biological engineering to take place for improved bacterial-mediated gene delivery and such tools provide a level of sophistication and uniqueness when comparing the methods to improve delivery between biological and non-biological delivery systems. In the future, large-scale manufacture, storage, and distribution must be considered if the bacterial delivery devices are ever to be used in a global market.