Altering the Tropism of Retroviral Vectors For In Vivo Gene Therapy: Pseudotyped Virus Targeting by Ligand-Receptor Interactions: A Dissertation

Abstract

A potential approach to in vivo gene therapy is to target retrovirus to specific receptors through a ligand-receptor interaction. Previous studies have placed a ligand at or close to the N-terminus of the ecotropic Moloney murine leukemia virus envelope and require co-expression of a wild type envelope on the pseudotyped virus for successful transduction of human cells. In this study, over forty chimeric envelopes were generated, which have single or multiple insertions of a 13 or 21 amino acid RGD containing sequence, flanked by cysteine residues, that target the cellular integrin receptors (Chapter III). Virus displaying only the chimeric envelopes was generated from packaging cell lines that express the gag and pol genes. Many of the mutant envelopes demonstrated the formation of syncytia when they were transfected into the XC indicator cell line, which is frequently used to determine envelope binding and fusion capabilities. Pseudotyped virus for several of the chimeric envelopes, transduced both NIH 3T3 mouse fibroblasts and human A375 melanoma cells. Ligands placed in the N-terminal region, within the VRA variable domain, and close to the N-terminus of the proline-rich region (PRR), demonstrated transduction into human melanoma cells. Ligands placed within the PRR and the C-terminus of the envelope did not demonstrate transduction into melanoma cells, although host cell transduction was demonstrated. Pseudotyped virus expressing an RGE containing target sequence, replacing the RGD sequence, had significantly lower transduction efficiency of melanoma cells. These data indicate that the MLV envelope tropism can be altered by insertion of short ligands at various locations throughout the envelope. These initial results were promising and helped to define regions within the envelope that could accommodate the insertion of small targeting ligands, that could redirect the tropism of pseudo typed virus to human cells. In the second part of this study, the focus shifted to targeting receptors that were expressed on specific cells, such as carcinoma cells. We inserted short ligands, flanked with cysteines, into the envelope to generate numerous targeting constructs that bind to receptors over-expressed on a variety of carcinoma cells. These pseudotyped retroviral vectors were generated by packaging cell lines that express only the viral Gag and Pol genes, with no wild-type envelope present. Select chimeric envelopes that express the 21 amino acid bombesin (BN)/gastrin releasing protein (GRP) binding sequence successfully transduced human melanoma cells, breast cancer cells, and cells that express the cloned GRP receptor gene. Nine additional chimeric envelopes were generated, that express a modified 56 amino acid heregulin sequence (HRG), that targets c-rbB-3 (Her-3) and c-erbB-4 (Her-4) receptors on breast carcinoma cells. Pseudotyped virus expressing only the BN/GRP mutant envelopes, transduced NIH 3T3 host cells, and two human carcinoma cell lines; A375 melanoma and MDA-MB-231 breast cells. The HRG chimeric envelopes demonstrated transduction of NIH 3T3 cells and human MDA-MB-453 breast carcinoma cells. Finally, a pseudotyped virus that expressed the chimeric BN/GRP envelopes and packaged the thymidine kinase gene, transduced melenoma and breast carcinoma cells and demonstrated ganciclovir cytotoxicity. Collectively, these data indicate that ligands of various sizes can be used to target pseudotyped virus to a variety of human cancer cells and transfer genes of interest. These findings may expand the feasibility and potential scope of gene therapy.