Development and Delivery of Improved Dna Vectors For Gene Therapy and Dna Vaccines

The purpose of this research is to develop an E. coli host strain that, upon induction, will remove the plasmid elements needed for replication and antibiotic resistance in the bacterial host. A DNA vaccine against Brucella abortus will be developed for proof-of-concept, but the technology has broad application to any DNA vaccine and gene therapy. Two goals will be addressed: 1) an E. coli host strain and vector system capable of removing plasmid replication elements in vitro to create pHalo-based vectors, and 2) development of transfection reagents capable of targeting cell receptors for more efficient, cell-specific transfection. Completion of the goals provides proof-of-concept for targeting a specific cell receptor with a ligand-transfection reagent to deliver pHalo devoid of E. coli replication elements. A review of the literature reveals no technology on the market similar to what is being developed for either goal. Combined, these technologies provide a safer, more efficient
DNA transfer technology. A DNA vaccine against the human and livestock pathogen B. abortus is being designed for the purpose of proving the concept and potential international impact; due to the amount of research conducted in Dr. Elzer's laboratory, the tools are available to evaluate antigenic proteins produced in cell culture and study vaccine efficacy in an animal model. The proposed Anaplasma marginale vaccine will have direct impact on cattle operations in Louisiana. By providing an efficacious vaccine to eliminate the need for antibiotic treatment, farmers will have a tool to produce healthier cattle and eliminate the possibility of antibiotic resistant bacteria.An E. coli host strain and vector system will be completed that is capable of normal plasmid replication until late log phase. In late log phase, an inducible promoter will express a recombinase system that removes the antibiotic selection marker and the origin of replication, leaving only the gene-of-interest (GOI)
in a "mini-halo" plasmid. The Food and Drug Administration (FDA) is developing policies on DNA vaccines and gene therapy products with a significant push towards "zero tolerance" on the inclusion of antibiotic selection markers and replicative elements. The concern is the horizontal gene transfer (HGT) of antibiotic resistance to other bacteria; and current methods to remove these elements are too costly, which can drive costs beyond what is affordable in the veterinary industry and developing countries. Second, two DNA transfection reagents will be developed containing receptor-specific ligands to increase plasmid delivery to a target cell. The expected outcome is twofold: 1) DNA uptake by non-target cells will be minimized; and 2) transfection efficiency will be increased. These transfection reagents will function in a similar manner to viral delivery vectors, targeting specific cell receptors with similar transfection efficiencies as viral vectors, yet retaining the non-immune
stimulating feature of lipid-based transfection reagents.The specific objectives are:Objective 1.Complete the recombinase gene construct and incorporate into the E. coli chromosome by homologous recombinationClone the recombinase recognition sequences into a plasmid vector pHaloEstablish a growth curve for optimal timing of induction and recombinase expressionAnalyze pHalo production efficiencyGoal 1 will result in an E. coli based system capable of removing plasmid replication elements in situ and allow normal plasmid purification procedures.Objective 2.Attach peptide ligands to transfecting reagents with extensive transfection use in the literatureCompare peptide-bearing transfection reagents to those without peptide ligands to determine transfection efficiency with a reporter gene Goal 2 will provide a mechanism to deliver plasmid DNA or other nucleic acids to specific cells bearing the target receptor.Objective 3.Using a combination of computer modeling software, published
research on Brucella and Anaplasma antigenic proteins, and collaborator knowledge, multi-epitope DNA vaccine(s) will be developed.Expression of the DNA vaccines will be tested in cell culture