Methods for Gene Transfer Using DNA-Adenovirus Conjugates

Strategies have been developed to accomplish gene delivery via the receptor-mediated pathway employing molecular conjugate vectors (1-13). As cells possess endogenous pathways for internalization of macromolecules, the utilization of these pathways for the purpose of DNA delivery represents a strategy that potentially allows certain practical advantages. In this regard, these cellular internalization pathways can be highly efficient. For example, internalization of the iron transport protein transferrin can be on the order of thousands of molecules per minute per cell (14,15). These pathways thus represent a potentially efficient physiologic method to transport DNA across the cell membrane of eukaryotic cells. To accomplish gene transfer via receptor-mediated endocytosis, a vehicle must be derived that allows DNA entry into these cellular pathways. For this purpose, molecular conjugate vectors have been derived. These vector agents consist of two linked functional domains: a DNA-binding domain to transport the DNA as part of the vector complex, and a ligand domain to target a cellular receptor that allows entry of the conjugate-DNA complex into a receptor-mediated endocytosis pathway. For incorporating DNA into the complex for gene delivery, binding must be achieved in a nondamaging, reversible manner. For this linkage, an electrostatic association between the binding domain and the nucleic acid is accomplished. To achieve this, the DNA binding domain is comprised of a polycationic amine, such as poly(L)lysine. This can associate with the negatively charged DNA in an electrostatic, noncovalent manner. To achieve entry of the complex through a receptor-mediated pathway, a ligand for the target cell is utilized. The ligand domain is covalently linked to the polylysine to create the molecular conjugate vector. The ligand domain may be a native or synthetic cell surface receptor ligand, an antireceptor antibody, or other agent that allows specific association with target cell membranes.