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Further advances in immuno-gene therapy against human papillomavirus by adeno-associated virus gene transfer into dendritic cells
TUMOR VACCINES Lived leukemic cells transduced with the different transgene showed reduced leukemogenicity but protective effect was reduced when 106 leukemic cells were injected. Vaccines with lethally irradiated IP10 transduced cells were able to cure mice previously injected with 104 leukemic cells. We detected CTL and Natural killer cells (NK) activity with cells isolated from mice vaccinated with IP10 transduced cells against the DA1-3b leukemic cell line. Depletion in vivo showed that the immune response induced by IP10 was NK cells, CD4+ and CD8+ dependent. Real time BCR/ABL PCR showed that IP10 was the only one transgene resulting in real molecular remission with no detection of residual disease. We conclude that IP10 gene is the more potent candidate for gene therapy of acute leukemia that we envisaged.
587. Further Advances in Immuno-Gene Therapy Against Human Papillomavirus by Adeno-Associated Virus Gene Transfer into Dendritic Cells Yong Liu,1 Maurizio Chiriva-Internati,2 Hong You,1 Jawahar L. Mehta,1 Paul L. Hermonat.1 1 Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, AR; 2Microbiology and Immunology, Texas Tech University, Lubbock, TX. Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen-loading of dendritic cells (DC) generates significant and rapid (one stimulation/one week) cytotoxic T lymphocyte (CTL) responses in vitro against viral and self antigens. To further improve this already potent technique we compared coinfection of an AAV/antigen vector, AAV/HPV-E6/Neo, with a rAAV vector carrying one of four cytokines. These included interleukin (IL)-2, interferon (IFN) gamma, granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-7. Previously we have shown that the delivery of the GM-CSF gene into DC resulted in secretion of this transgene cytokine. Furthermore the freshly secreted cytokine had a 100-1000 fold higher biological activity on a per weight basis compared to recombinant commercial exogenously added cytokine. Here, in a head-to-head comparison the delivery of the IL-7 gene appeared superior to IL-2 and IFN-gamma for improving CTL response. In a second comparison the infection of DC by IL-7 was compared to the direct infection of T cells. In a head-to-head comparison the direct delivery/infection of IL-7 into the T cell population generated significantly higher levels of killing of E7positive target cells. These data suggest that the use of cytokine genes can improve CTL response and also suggests that a number of further procedural improvements can be made.
588. A Central Role of RNA in Developing Therapeutic Vaccines for Cancer and Infectious Diseases Eli Gilboa, Sandra Santulli-Marotto, Smita K. Nair, David Boczkowski, Yanbing Zhao. 1 Surgery, Duke University Medical Center, Durham, NC. The dendritic cell (DC) is a newly discovered member of the immune system specialized in presenting antigen to naive T cells, and consequently playing a central role in the induction of T cell, as well as B-cell, immunity. Immunization with DC loaded with tumor antigens could, therefore, represent a powerful method of inducing antitumor immunity. Studies in our laboratory have shown that DC transfected with mRNA encoding tumor antigens are effective inducers of cytotoxic T cells (CTL) and tumor immunity. Tumor RNA can be amplified from microscopic amounts of tumor tissue obtained by microdissection from needle biopsies or fixed tissue sections providing virtually inexhaustible amounts of tumor antigens S222
without the need to identify the relevant antigens in each patient. Phase I/II clinical studies in patients with prostate and renal cancer have established the safety, and feasibility of this approach and despite the advanced nature of the disease, the majority of the patients exhibited specific immunological responses. To enhance the therapeutic impact of this vaccination strategy antisense and siRNA technology are used to induce CD4+ T cell immunity by inhibiting Invariant chain expression in the (mRNA transfected) DC, and CTLA-4 binding RNA aptamers are used to enhance the persistence of vaccine-induced antitumor immunity. Therapeutic vaccination with dendritic cells also offers a powerful method to stimulate T cell immunity in the setting of infectious diseases such as HIV and HCV. However, the propensity of HIV and HCV to generate immunological escape variants could defeat current vaccination strategies, regardless how effective they may be. The mRNA platform, by amplifying viral mRNA from infected patients, provides a real-time approach to vaccinate against the patient’s HIV or HCV quasispecies, and thereby overcome the problem stemming from the emergence of treatment-resistant virus. Founder and consultant to MERIX Bioscience Inc.
589. Therapeutic Efficacy and Cellular Mechanism Mediated by Intratumoral and Intramuscular IL-12 Electrogenetherapy Shulin Li,1 Francesca Mellieon,1 Robin Barret.1 1 Comparative Biomedical Sciences, Louisiana State Unviersity, Baton Rouge, LA. Intratumoral (tumor local) and intramuscular (systemic) immunoelectrogenetherapies are important for treating solid tumor and residual or metastatic tumors, respectively. However, intratumoral injection of IL-12 gene via electroporation is more effective in the inhibition of tumor growth than intramuscular injection, but the underlying mechanism that causes this administration routedependent inhibition of tumor growth is not fully understood. To understand the cellular mechanism by which tumor growth is significantly inhibited by intratumoral but not by intramuscular IL12 electro-gene therapies, induction of T-cell infiltration, CTL activity, inhibition of angiogenesis and transgene expression were determined using northern blot, ELISA, immunohistochemistry, and fluorescence-based CTL analysis, and the therapeutic effect was determined in immunocompetent, immune cell-depleted, and immunodeficient mice. The results indicated that intratumoral electric pulses, when combined with the injection of plasmid DNA, is one of the primary factors that results in the different therapeutic efficacies between intratumoral and intramuscular delivery. Intratumoral injection of control plasmid DNA via electric pulses yields a small tumor volume that is only 37% and 58% of the tumor volumes from mice treated with intramuscular injection of plasmid DNA without and with IL-12 via electroporation, respectively. Inclusion of IL-12 in the intratumoral injection further inhibits the tumor growth and yields a tumor volume that is 6% and 9% of the tumor volume from mice treated with intramuscular injection of control plasmid DNA and IL-12 gene, respectively. CD8 T-cells are the primary effector cells that are associated with this administration route-dependent tumor inhibition because intratumoral, but not intramuscular injection via electric pulses induces CD8+ T-cell infiltration and CTL activity, and because both intratumoral and intramuscular IL-12 electro-gene therapies induce similar levels of antiangiogenesis- and NK cell-mediated antitumor effect. These findings indicate that induction of tumor-specific CD8+ T-cell response is a prerequisite for improving the therapeutic effect mediated by the systemic immuno-electrogenetherapy.
Molecular Therapy Volume 9, Supplement 1, Ma y 2004
Copyright The American Society of Gene Therapy
587. Further Advances in Immuno-Gene Therapy Against Human Papillomavirus by Adeno-Associated Virus Gene Transfer into Dendritic Cells Yong Liu,1 Maurizio Chiriva-Internati,2 Hong You,1 Jawahar L. Mehta,1 Paul L. Hermonat.1 1 Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, AR; 2Microbiology and Immunology, Texas Tech University, Lubbock, TX. Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen-loading of dendritic cells (DC) generates significant and rapid (one stimulation/one week) cytotoxic T lymphocyte (CTL) responses in vitro against viral and self antigens. To further improve this already potent technique we compared coinfection of an AAV/antigen vector, AAV/HPV-E6/Neo, with a rAAV vector carrying one of four cytokines. These included interleukin (IL)-2, interferon (IFN) gamma, granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-7. Previously we have shown that the delivery of the GM-CSF gene into DC resulted in secretion of this transgene cytokine. Furthermore the freshly secreted cytokine had a 100-1000 fold higher biological activity on a per weight basis compared to recombinant commercial exogenously added cytokine. Here, in a head-to-head comparison the delivery of the IL-7 gene appeared superior to IL-2 and IFN-gamma for improving CTL response. In a second comparison the infection of DC by IL-7 was compared to the direct infection of T cells. In a head-to-head comparison the direct delivery/infection of IL-7 into the T cell population generated significantly higher levels of killing of E7positive target cells. These data suggest that the use of cytokine genes can improve CTL response and also suggests that a number of further procedural improvements can be made.
588. A Central Role of RNA in Developing Therapeutic Vaccines for Cancer and Infectious Diseases Eli Gilboa, Sandra Santulli-Marotto, Smita K. Nair, David Boczkowski, Yanbing Zhao. 1 Surgery, Duke University Medical Center, Durham, NC. The dendritic cell (DC) is a newly discovered member of the immune system specialized in presenting antigen to naive T cells, and consequently playing a central role in the induction of T cell, as well as B-cell, immunity. Immunization with DC loaded with tumor antigens could, therefore, represent a powerful method of inducing antitumor immunity. Studies in our laboratory have shown that DC transfected with mRNA encoding tumor antigens are effective inducers of cytotoxic T cells (CTL) and tumor immunity. Tumor RNA can be amplified from microscopic amounts of tumor tissue obtained by microdissection from needle biopsies or fixed tissue sections providing virtually inexhaustible amounts of tumor antigens S222
without the need to identify the relevant antigens in each patient. Phase I/II clinical studies in patients with prostate and renal cancer have established the safety, and feasibility of this approach and despite the advanced nature of the disease, the majority of the patients exhibited specific immunological responses. To enhance the therapeutic impact of this vaccination strategy antisense and siRNA technology are used to induce CD4+ T cell immunity by inhibiting Invariant chain expression in the (mRNA transfected) DC, and CTLA-4 binding RNA aptamers are used to enhance the persistence of vaccine-induced antitumor immunity. Therapeutic vaccination with dendritic cells also offers a powerful method to stimulate T cell immunity in the setting of infectious diseases such as HIV and HCV. However, the propensity of HIV and HCV to generate immunological escape variants could defeat current vaccination strategies, regardless how effective they may be. The mRNA platform, by amplifying viral mRNA from infected patients, provides a real-time approach to vaccinate against the patient’s HIV or HCV quasispecies, and thereby overcome the problem stemming from the emergence of treatment-resistant virus. Founder and consultant to MERIX Bioscience Inc.
589. Therapeutic Efficacy and Cellular Mechanism Mediated by Intratumoral and Intramuscular IL-12 Electrogenetherapy Shulin Li,1 Francesca Mellieon,1 Robin Barret.1 1 Comparative Biomedical Sciences, Louisiana State Unviersity, Baton Rouge, LA. Intratumoral (tumor local) and intramuscular (systemic) immunoelectrogenetherapies are important for treating solid tumor and residual or metastatic tumors, respectively. However, intratumoral injection of IL-12 gene via electroporation is more effective in the inhibition of tumor growth than intramuscular injection, but the underlying mechanism that causes this administration routedependent inhibition of tumor growth is not fully understood. To understand the cellular mechanism by which tumor growth is significantly inhibited by intratumoral but not by intramuscular IL12 electro-gene therapies, induction of T-cell infiltration, CTL activity, inhibition of angiogenesis and transgene expression were determined using northern blot, ELISA, immunohistochemistry, and fluorescence-based CTL analysis, and the therapeutic effect was determined in immunocompetent, immune cell-depleted, and immunodeficient mice. The results indicated that intratumoral electric pulses, when combined with the injection of plasmid DNA, is one of the primary factors that results in the different therapeutic efficacies between intratumoral and intramuscular delivery. Intratumoral injection of control plasmid DNA via electric pulses yields a small tumor volume that is only 37% and 58% of the tumor volumes from mice treated with intramuscular injection of plasmid DNA without and with IL-12 via electroporation, respectively. Inclusion of IL-12 in the intratumoral injection further inhibits the tumor growth and yields a tumor volume that is 6% and 9% of the tumor volume from mice treated with intramuscular injection of control plasmid DNA and IL-12 gene, respectively. CD8 T-cells are the primary effector cells that are associated with this administration route-dependent tumor inhibition because intratumoral, but not intramuscular injection via electric pulses induces CD8+ T-cell infiltration and CTL activity, and because both intratumoral and intramuscular IL-12 electro-gene therapies induce similar levels of antiangiogenesis- and NK cell-mediated antitumor effect. These findings indicate that induction of tumor-specific CD8+ T-cell response is a prerequisite for improving the therapeutic effect mediated by the systemic immuno-electrogenetherapy.
Molecular Therapy Volume 9, Supplement 1, Ma y 2004
Copyright The American Society of Gene Therapy