Manipulation of the Moloney Murine Leukemia Virus Envelope Protein in an Effort to Develop Directly and Indirectly Targeted Retroviral Vectors for Use in Human Gene Therapy

Highly effective, targeted therapies against cancer would revolutionize the way people recover from this devastating illness. Gone would be the lingering side effects of the current non-specific treatments and in their place would be faster recovery times, better quality of life both during and after treatment, and less ambiguity about whether or not treatment was effective. This concept will elude modern medicine until treatments can be tailored to the patient\u27s individual and unique disease. This concept of a transient, targeted, and tailored vehicle aimed at cancer cells lends itself to the use of replication deficient retroviral gene therapy vectors with interchangeable receptor binding sites. These vectors may be used separately or in combination with each other to ensure maximum delivery of a suicide gene only to cancerous cells involved with the primary tumor as well as those equally dangerous metastatic cells. Recently, a prototype retroviral vector incorporating the short peptide Somatostatin in place of its natural receptor binding site has been developed. The design of this chimeric envelope protein has the potential to make the insertion of varied receptor binding sites a simple and efficient process. The goal of my studies was to learn more about the potential of this design by taking steps towards challenging its ligand capacity, both in size and secondary structure, and improving its interaction with the target cell. My studies indicate that the peptide ligand Stromal Derived Factor-1α (lysine 22 through lysine 89) is not a good candidate for use with this chimeric design. They also show that only certain modifications within the Moloney Murine Leukemia Virus heparin binding motif can be tolerated. Supplemental studies point out that (1) the removal of an MluI site at the CMV promoter N-terminus may enhance promoter function, (2) TransIT (Mirus) is an effective reagent for transfection of DNA into the MDA-MB-231 breast cancer cell line, and (3) a eukaryotic α-complementation assay is not sensitive enough to detect cell-cell fusion at levels below that of the wild type MoMLV envelope with its natural receptor. While much investigation remains to be done, these observations will help pave the way to the development of effective retroviral vectors for cancer therapy