A major concern associated with We use of recombinant adenoviral vectors is that viral receptors are found on the surface of many cell types and systemic in vivo delivery of the viral vector could result in uncontrolled and widespread expression of therapeutic molecules in many tissues, To construct a cell-type specific recombinant adenoviral vector a new binding specificity must be added to the virus, and the endogenous binding specificity of We virus must be ablated. In order to introduce a new binding specificity to recombinant adenoviral vectors, We coding sequence of a physiological ligand, the terminal decapeptide of the gastrin releasing peptide (GRP), was placed at the 3' end of the coding sequence of the adenovirus type 5 fiber gene. The resulting fiber-GRP fusion protein was expressed using a T7 vaccinia expression system and has been shown to assemble protein trimers whose quaternary structure is indistinguishable from that of wild-type protein. The fiber-GRP fusion protein was correctly transported to the nucleus of HeLa cells immediately after synthesis. The added GRP ligand in the fiber-GRP fusion protein was accessible to binding by an anti-GRP antibody in both the monomeric and trimeric forms of We chimeric protein. These studies suggest that new cell type specificities for adenovirus binding might be introduced by genetic fusion of peptide ligands on to the carboxyl terminus of the adenovirus fiber protein.

One strategy used for gene therapy of cancer is molecular chemotherapy. This approach is based on selective expression of an encoded toxin in cancer cells to achieve their eradication. One potential advantage of this strategy derives from a phenomenon, termed the bystander effect, whereby only a fraction of cells needs to be transduced to eradicate a tumor population. Despite the theoretical advantages of this phenomenon, it has only been described in a few cellular targets. Therefore, we undertook strategies to develop a molecular chemotherapy approach for ovarian carcinoma utilizing the herpes simplex virus thymidine kinase (HSV-TK) gene. Initially, we established that human ovarian carcinoma cell lines could be transduced at high efficiency with adenoviral vectors encoding reporter genes. We next determined that the human ovarian cell line SKOV3 could exhibit bystander killing by stably transducing it to express HSV-TK and performing cell mixing experiments with varying percentages of HSV-TK-expressing and HSV-TK-nonexpressing cells. Based on these findings, we constructed a recombinant adenovirus encoding HSV-TK and utilized it to induce human ovarian carcinoma cell lines to the sensitizing effects of ganciclovir. In addition, primary cultures of ovarian carcinoma cells were found to be highly transducible with recombinant adenoviral vectors and could be induced to the sensitizing effects of ganciclovir after induction of HSV-TK expression by the adenoviral vector. These studies indicate that molecular chemotherapy using a recombinant adenoviral vector expressing HSV-TK may provide a rational strategy for human ovarian carcinoma.

Gene transfer to eukaryotic cells may be accomplished by capitalizing on endogenous cellular pathways of macromolecular transport. In this regard, molecular conjugate vectors have been developed which deliver DNA via the receptor-mediated endocytosis pathway. An attractive feature of this vector system is the potential to achieve targeted gene delivery based upon flexible incorporation of a targeting ligand. In this review we describe steps that have been taken to optimize this vector system. Specific strategies include the incorporation of mechanisms to achieve conjugate escape from the endosome and the derivation of methods to eliminate sources of nonspecificity. These developments have demonstrated the potential to construct a vector system in which multiple independent components may function in a concerted manner to accomplish targeted high efficiency gene delivery. In their present state of development, molecular conjugate vectors may have many potential applications for in vitro use.

Molecular conjugate vectors may be constructed that accomplish high efficiency gene transfer by the receptor-mediated endocytosis pathway. In order to mediate escape from lysosomal degradation, we have incorporated adenoviruses into the functional design of the conjugate. In doing so, however, we have introduced an additional ligand, which can bind to receptors on the cell surface, undermining the potential for cell specific targeting. To overcome this, we have treated the adenovirus with a monoclonal anti-fiber antibody, which renders the virus incapable of binding to its receptor. The result is a multi-functional molecular conjugate vector, which has preserved its binding specificity while at the same time being capable of preventing lysosomal degradation of endosome-internalized conjugate-DNA complexes. This finding indicates that adenoviral binding is not a prerequisite for adenoviral-mediated endosome disruption.