Kouichi Ohno

Cell-specific targeting of a thymidine kinase/ganciclovir gene therapy system using a recombinant Sindbis virus vector.

Transfer of the herpes simplex virus type I thymidine kinase (HSV-TK) gene into tumor cells using virus-based vectors in conjunction with ganciclovir (GCV) exposure provides a potential gene therapy strategy for the treatment of cancer. The possibility of using a novel targetable Sindbis virus expression vector containing the HSV-TK gene was examined. Baby hamster kidney (BHK) cells and several human tumor cells infected with a Sindbis virus containing the HSV-TK gene showed strong expression of HSV-TK protein. Cells transduced with the HSV-TK gene exhibited increased TK activity, ranging from 3- to 20-fold over an average baseline level. The human HeLa-CD4+ cells infected with recombinant Sindbis virus containing the HSV-TK gene were sensitive to low concentrations of GCV (0.1-1 microg/ml) and the 50% growth inhibitory concentration (IC50) was 0.6 microg/ml. We also demonstrated applications of cell type-specific Sindbis virus-mediated antigen-antibody targeting of the HSV-TK/GCV system in vitro. Sindbis virus containing the HSV-TK gene packaged in a helper virus displaying the IgG-binding domain of protein A on its envelope could infect various tumor cell lines in the presence of specific antibodies that recognize antigens on their surfaces. HSV-TK-transduced tumor cell lines exhibited sensitivity to GCV. Our data suggest the potential for targeted gene therapy of the HSV-TK/GCV system using a cell type-specific recombinant Sindbis virus vector-antibody system.

Specific Cell Targeting for Delivery of Toxins into Small-Cell Lung Cancer Using a Streptavidin Fusion Protein Complex

New modalities of treatment for small-cell lung cancer (SCLC) are needed, because the majority of patients continue to die of disseminated disease despite an initial response to conventional chemotherapy. Abnormal surface expression of the neural-cell adhesion molecule (NCAM) has been noted to be highly associated with SCLC. We examined the ability and efficiency of a streptavidin-Protein A (ST-PA) fusion protein complexed with an anti-NCAM monoclonal antibody (Mab) to transfer biotinylated beta-galactosidase into human SCLC cell lines NCI-H69, NCI-H526, and NCI-H446. When the surface molecule NCAM was targeted with this system, more than 99% of the targeted cells internalized and exhibited beta-galactosidase activity. In addition, we evaluated cytotoxic activity against SCLC lines NCI-H69 and NCI-H526 by efficient delivery of biotinylated glucose oxidase using the same ST-PA/anti-NCAM Mab complex. Cytotoxicity of the transduced cells (SCLC) was 10-fold and 100-fold greater, respectively, than the glucose oxidase control. This system could be widely applied for specific therapy of cancer cells by targeting unique surface molecules (antigens) using the corresponding Mab/ST-PA complex to transfer a variety of effector molecules; e.g., immunotoxic compounds, into target cells with a high degree of efficiency and specificity.

Infection of human cells by murine ecotropic viruses: retroviral vectors carrying the hygromycin resistance-encoding gene

The construction of a new retroviral vector, pSKV, is described. This vector carries two unique cloning sites, located between two Moloney leukemia virus-derived LTR, into which genes of interest may be introduced. The gene encoding hygromycin resistance (HyR) was subsequently introduced into one of the two sites, producing a second vector (pSKV/HyR) containing a unique SfiI site for the introduction of cDNA clones under the control of the cytomegalovirus (CMV) promoter (P-CMV). The cDNA (mH13), encoding a protein that has been shown to serve as a murine ecotropic retroviral receptor in transient assays, was cloned into the SfiI site (pSKV/HyR/mH13). Both constructs can be packaged into retroviral particles following transfection into an appropriate packaging cell line. Stable transfectants of the human glioblastoma cell line (U118MG) carrying each of these two constructs were generated by transfection and subsequent Hy selection. Clones expressing both the selectable marker and the mH13 gene, but not those expressing only the selectable marker, are shown to be susceptible to infection with murine ecotropic retroviral particles. These cells (HyR and mH13 positive) were then exposed to CRE/Xtk culture supernatant, a packaging cell line producing ecotropic retroviral particles carrying the HSV-TK (Herpes simplex virus-thymidine kinase) and neoR (neomycin-resistance) genes. Selection was in the presence of G418. In vitro growth of the U118MG/HyR/mH13/TK cells, but not that of the U118MG/HyR/mH13 cells, was inhibited by ganciclovir (GCV), indicating the successful transfer of HSV-TK by infection of human cells with murine retroviruses via the mH13 product.