- Email: [email protected]
185. Identification of Oncolytic Virus-Sensitizing Genes by an siRNA Based Screen
Cancer - Viral Vector Cancer Gene Therapy 183.
Abstract Withdrawn
Approximately 60% of CD19+ ALL cytotoxicity was maintained by mRNA-modified NK cells 3-4 d after transfection at an E:T ratio of 1:1. The method described here should greatly facilitate effective NK cell therapy of CD19+ leukemia and lymphoma, and has potential for a wide range of clinical applications in NK cell therapy of cancer.
Cancer – Viral Vector Cancer Gene Therapy 185. Identification of Oncolytic Virus-Sensitizing Genes by an siRNA Based Screen
Jenny Puhlmann,1 Florian Puehler,1 Petra Boukamp,2 Dominik Mumberg,1 Rudolf Beier.1 1 TRG Oncology, Bayer Schering Pharma AG, Berlin, Germany; 2 Division of Genetics of Skin Carcinogenesis, DKFZ, Heidelberg, Germany.
184. A Clinical-Grade Method to Genetically Modify Natural Killer Cells for Leukemia and Lymphoma Therapy
Linhong Li,1 Linda N. Liu,1 Stephanie Feller,1 Cornell Allen,1 Rama Shivakumar,1 Hiroyuki Fujisaki,2 Dario Campana,2 Joseph Fratantoni,1 Madhusudan Peshwa.1 1 MaxCyte Inc., Gaithersburg, MD; 2Oncology and Pathology, St. Jude Children’s Research Hospital, Memphis, TN. The infusion of allogeneic natural killer (NK) cells has been shown to induce clinical responses in patients with acute myeloblastic leukemia and is an attractive new tool to enhance the effects of chemotherapy and hematopoietic stem cell transplantation in patients with hematologic malignancies. NK cells have poor innate cytotoxicity against certain leukemic cell types, e.g., those of acute lymphoblastic leukemia (ALL). However, they can be rendered extremely effective by enforcing the expression of chimeric signaling receptors that recognize CD19, a surface molecule widely expressed in B-lineage ALL and B-cell non-Hodgkin lymphoma (Imai et al. Blood 2005). We here report an electrotransfection and cGMP compliant method designed for clinical-grade genetic modification of NK cells with mRNA. In preliminary experiments, we expanded NK from the peripheral blood of healthy donors using the method described by Imai et al., and then transfected them with various molecules, including FITC-dextran (500kD), siRNA and eGFPencoded mRNA. Efficiencies of transfection were approximately 80% and cell viabilities after transfection were 80%-90%. Then, we prepared mRNA encoding a chimeric receptor consisting of an anti-CD19 scFV fused with the signaling domains of CD3zeta and 4-1BB. Expression of the chimeric receptor was detected among 56% ± 13% (n=26) of expanded NK cells after transfection. Receptor expression progressively declined but was still detectable 4 days post transfection. Expression of the anti-CD19 chimeric receptor dramatically enhanced specific lysis of CD19+ ALL cell line(s) and the CD19+ primary allogeneic B-chronic lymphocytic leukemia (CLL) cells. At an effector : target (E:T) ratio of 1:1, expanded NK cells transfected 1 day earlier with the anti-CD19 receptor lysed 95% ± 2% of CD19+ ALL cells whereas cytotoxicity of non transfected cells was 14% ± 7%; cytotoxicity against primary CLL cells reached 81% ± 2% versus 28% ± 6% (2 CLL donors; 3 experiments). The lysis of CD19+ ALL cells by transfected NK cells decreased with time after transfection. Significant lysis of CD19+ ALL cells was observed as early as 3 hours and as late as 4 days subsequent to transfection. Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Newcastle disease virus (NDV) is an avian paramyxovirus with a negative stranded RNA genome. The virus has a natural preference for replication in tumor cells but not in normal cells. Therefore, NDV is under current investigations for oncolytic therapy. A defective antiviral response in tumor cells is assumed to be responsible for tumor-selective viral replication. However, the precise link between tumorigenesis and sensitivity to oncolytic viruses is not fully understood and needs to be analysed in more detail. To address this issue, a human cell line model has been set up based on a nontumorigenic keratinocyte cell line (HaCaT) and a ras-transformed and in vivo tumor-passaged metastatic clone (RT3). In contrast to the non-transformed HaCaT cells, the tumorigenic cell line RT3 is susceptible to NDV but the virus induces no significant cytopathic effect. A RT3-derived subclone (RT3 K1), selected by enhanced growth in soft agar, features an increased tumor growth potential in nude mice and a higher NDV susceptibility compared to the parental cell line RT3. This elevated susceptibility corresponds to an enhanced NDV-titer production and a NDV-mediated cell death even apparent at low multiplicities of infection (MOI) in contrast to RT3 cells. To identify tumor specific factors essential for efficient NDV infection and replication, the virus sensitive RT3 K1 cells were transfected with siRNA libraries that silence approximately 300 genes in total (containing 185 genes involved in membrane trafficking and 95 tyrosine kinases). Following siRNA mediated knock-down the cells were infected with a luciferase expressing NDV. Thus, an increase or decrease of replication efficacy of the virus can be detected by a luciferase based reporter gene assay. Several candidate genes influencing the viral infection and replication cycle were identified and selected for hit-validation in secondary assays, whereas the non-transformed HaCaT cell line is included as a control. These virus-sensitizing or virus-repressing genes can hint towards cellular pathways important for the life-cycle of the virus. As the processes of viral susceptibility and tumorigenic transformation are postulated to be correlated, the identified genes or pathways may not only explain the differential viral sensitivity of tumor and normal cells but also give insights into the process of transformation.
S69
Abstract Withdrawn
Approximately 60% of CD19+ ALL cytotoxicity was maintained by mRNA-modified NK cells 3-4 d after transfection at an E:T ratio of 1:1. The method described here should greatly facilitate effective NK cell therapy of CD19+ leukemia and lymphoma, and has potential for a wide range of clinical applications in NK cell therapy of cancer.
Cancer – Viral Vector Cancer Gene Therapy 185. Identification of Oncolytic Virus-Sensitizing Genes by an siRNA Based Screen
Jenny Puhlmann,1 Florian Puehler,1 Petra Boukamp,2 Dominik Mumberg,1 Rudolf Beier.1 1 TRG Oncology, Bayer Schering Pharma AG, Berlin, Germany; 2 Division of Genetics of Skin Carcinogenesis, DKFZ, Heidelberg, Germany.
184. A Clinical-Grade Method to Genetically Modify Natural Killer Cells for Leukemia and Lymphoma Therapy
Linhong Li,1 Linda N. Liu,1 Stephanie Feller,1 Cornell Allen,1 Rama Shivakumar,1 Hiroyuki Fujisaki,2 Dario Campana,2 Joseph Fratantoni,1 Madhusudan Peshwa.1 1 MaxCyte Inc., Gaithersburg, MD; 2Oncology and Pathology, St. Jude Children’s Research Hospital, Memphis, TN. The infusion of allogeneic natural killer (NK) cells has been shown to induce clinical responses in patients with acute myeloblastic leukemia and is an attractive new tool to enhance the effects of chemotherapy and hematopoietic stem cell transplantation in patients with hematologic malignancies. NK cells have poor innate cytotoxicity against certain leukemic cell types, e.g., those of acute lymphoblastic leukemia (ALL). However, they can be rendered extremely effective by enforcing the expression of chimeric signaling receptors that recognize CD19, a surface molecule widely expressed in B-lineage ALL and B-cell non-Hodgkin lymphoma (Imai et al. Blood 2005). We here report an electrotransfection and cGMP compliant method designed for clinical-grade genetic modification of NK cells with mRNA. In preliminary experiments, we expanded NK from the peripheral blood of healthy donors using the method described by Imai et al., and then transfected them with various molecules, including FITC-dextran (500kD), siRNA and eGFPencoded mRNA. Efficiencies of transfection were approximately 80% and cell viabilities after transfection were 80%-90%. Then, we prepared mRNA encoding a chimeric receptor consisting of an anti-CD19 scFV fused with the signaling domains of CD3zeta and 4-1BB. Expression of the chimeric receptor was detected among 56% ± 13% (n=26) of expanded NK cells after transfection. Receptor expression progressively declined but was still detectable 4 days post transfection. Expression of the anti-CD19 chimeric receptor dramatically enhanced specific lysis of CD19+ ALL cell line(s) and the CD19+ primary allogeneic B-chronic lymphocytic leukemia (CLL) cells. At an effector : target (E:T) ratio of 1:1, expanded NK cells transfected 1 day earlier with the anti-CD19 receptor lysed 95% ± 2% of CD19+ ALL cells whereas cytotoxicity of non transfected cells was 14% ± 7%; cytotoxicity against primary CLL cells reached 81% ± 2% versus 28% ± 6% (2 CLL donors; 3 experiments). The lysis of CD19+ ALL cells by transfected NK cells decreased with time after transfection. Significant lysis of CD19+ ALL cells was observed as early as 3 hours and as late as 4 days subsequent to transfection. Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Newcastle disease virus (NDV) is an avian paramyxovirus with a negative stranded RNA genome. The virus has a natural preference for replication in tumor cells but not in normal cells. Therefore, NDV is under current investigations for oncolytic therapy. A defective antiviral response in tumor cells is assumed to be responsible for tumor-selective viral replication. However, the precise link between tumorigenesis and sensitivity to oncolytic viruses is not fully understood and needs to be analysed in more detail. To address this issue, a human cell line model has been set up based on a nontumorigenic keratinocyte cell line (HaCaT) and a ras-transformed and in vivo tumor-passaged metastatic clone (RT3). In contrast to the non-transformed HaCaT cells, the tumorigenic cell line RT3 is susceptible to NDV but the virus induces no significant cytopathic effect. A RT3-derived subclone (RT3 K1), selected by enhanced growth in soft agar, features an increased tumor growth potential in nude mice and a higher NDV susceptibility compared to the parental cell line RT3. This elevated susceptibility corresponds to an enhanced NDV-titer production and a NDV-mediated cell death even apparent at low multiplicities of infection (MOI) in contrast to RT3 cells. To identify tumor specific factors essential for efficient NDV infection and replication, the virus sensitive RT3 K1 cells were transfected with siRNA libraries that silence approximately 300 genes in total (containing 185 genes involved in membrane trafficking and 95 tyrosine kinases). Following siRNA mediated knock-down the cells were infected with a luciferase expressing NDV. Thus, an increase or decrease of replication efficacy of the virus can be detected by a luciferase based reporter gene assay. Several candidate genes influencing the viral infection and replication cycle were identified and selected for hit-validation in secondary assays, whereas the non-transformed HaCaT cell line is included as a control. These virus-sensitizing or virus-repressing genes can hint towards cellular pathways important for the life-cycle of the virus. As the processes of viral susceptibility and tumorigenic transformation are postulated to be correlated, the identified genes or pathways may not only explain the differential viral sensitivity of tumor and normal cells but also give insights into the process of transformation.
S69