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992. Cytoreductive Cancer Gene Therapy Using Parvoviral Replicon Vectors
NAKED DNA: APPLICATIONS 991. Application of Lysosome Targeting Sequence in DNA Constructs Encoding Mite Allergen Der p 1 Taoqi Huangfu,1 Lay Hong Lim,1 Kaw Yan Chua.1 Department of Paediatrics, National University of Singapore, Singapore, Singapore, Singapore. 1
Mite allergy related asthma is highly prevalent worldwide. Der p 1 is a major allergen from house dust mite Dermatophagoides pteronyssinus, which reacts with IgE in more than 70% allergic sera, and therefore is a good candidate for DNA vaccine development. Previous study in our lab has demonstrated that intramuscular injection of plasmid DNA containing the Der p 1 gene in mice induced specific Th1 skewed immune response, with a significant amount of Der p 1 specific IgG. However, the magnitude of the immune response was too low to achieve satisfactory therapeutic and preventive efficacies, thus our main focus is to explore the various strategies to optimize the magnitude of the antigen-specific immune response. Codon optimization has been reported to enhance the expression of foreign genes by the mammalian transcriptional and translational machinery, and the lysosome targeting sequence from LAMP has been shown to direct the encoded protein to the MHC II compartment, and enhance the activation of CD4+ T cells. The aim of this study is to test the feasibility of using the LAMP lysosome targeting sequence and the codon optimization strategy to improve the efficacy of DNA vaccination. Codon optimization was done by changing the codon usage of Der p 1 cDNA sequence according to highly expressed human genes. The humanized gene was then linked to the LAMP lysosome targeting sequence, and the whole cassette was subsequently inserted into the multiple cloning site of pVAX1, which was used for DNA immunization. Following intramuscular injection of naked plasmid DNA with electroporation in BALB/c mice, antibody kinetics was followed for 70 days, and T cell cytokine profiles will be evaluated by cytokine ELISA and intracellular cytokine staining. Current results indicated that the humoral response was significantly enhanced by codon optimization possibly due to the higher level of protein expression. The efficacy of LAMP lysosome targeting sequence is still under evaluation and will be presented in the meeting. The information obtained will facilitate the design of Der p1 vaccines for allergic diseases.
992. Cytoreductive Cancer Gene Therapy Using Parvoviral Replicon Vectors Boris R. A. Blechacz,1 Jean Rommelaere,2 Jan J. Cornelis,2 Christiane Dinsart,2 Annick Brandenburger,3 Stephen J. Russell.1 1 Molecular Medicine Program, Mayo Foundation, Rochester, MN, United States; 2Applied Tumor Virology Program, German Cancer Research Center, Heidelberg, Germany; 3IRIBHN-IBMM, Universite Libre de Bruxelles, Gosselies, Belgium. Background: Cytoreductive gene therapy is an appealing approach to the treatment of human malignancies but significant barriers exist. In particular, the use of viruses as gene transfer vectors is limited by their immunogenic potential and existing nonviral systems are far less efficient than viral systems. We developed replicon vectors based on the autonomous parvovirus Minute Virus of Mice (MVMp) which is known to be tumorsuppressive and tumorcellspecific. As therapeutic transgenes we used the human sodium iodide symporter gene (hNIS) and the Gibbon ape leukaemia virus fusogenic membrane glycoprotein gene (Galv-FMG). Both of them have already proved their strong antitumoral potential in different tumor-models. The rational behind these replicon vectors is to take advantage of the tumorcell-specific cytotoxicity of parvoviruses and avoiding the disadvantages of their immunogenicity at the same time. By combining these parvoviral replicons with Galv-FMG and hNIS as therapeutic transgenes we expect high level expression of these highpotency therapeutic transgenes with major bystander killing potential. S382
Methods: Engineering of recombinant parvoviral replicon vectors was performed by replacing the capsid-coding region of the MVMgenome by transgene cDNA. For examination of size limitation in terms of gene replication and expression a polylinker was cloned between element A/B and the right palindrome; stuffer DNA of different size was cloned into this position. For functional testing of the parvoviral constructs 293-T cells were used. As an in vitro tumor model we used the human Hepatoma cell lines HUH-7, Hep3B and Hep-G2. Transfection was performed by using the lipotransfection method. For analysis of excision and parvoviral DNA-replication Southern blot analysis of Hirt extracts was performed. Transgene expression was analyzed in dependence of the transgene: semi-quantitative optical analysis, luciferase assay or CEA-level quantification. Results: MVM replicon vectors containing the marker genes CEA, luciferase and GFP as well as the therapeutic transgenes Galv-FMG and hNIS have been cloned. Excision and replication of parvoviral DNA has been shown as early as 24 h after transfection. High-level transgene expression in different cell lines was achieved. Parvoviral replicons containing Galv-FMG as therapeutic transgene were shown to have a very strong cytotoxic potential leading to an almost complete destruction of cell layers. Altogether these results show that the use of parvoviral replicons is a very promising approach to the gene therapy of cancer.
993. Thermosensitive Hydrogel Mediated Gene Transfer for Diabetic Wound Healing Pui-yan R. Lee,1 Zhenhua Li,2 Leaf Huang.2 Department of Bioengineering, School of Engineering, University of Pittsburgh, Pittsburgh, PA, United States; 2Center for Pharmogenetics, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States. 1
Of late, there have been a lot of interests in the role of TGF-b1 gene in impaired wound healing by using various delivery methods. In present study, we investigated the feasibility of a thermosensitive hydrogel made of a triblock copolymer, PEG-PLGA-PEG, as a vector of naked DNA delivery in genetically diabetic mouse (C57BKS.Cg-m +/+ Leprdb female mice) model. The hydrogel itself is a simple and good wound dressing. When the hydrogel formulated with TGF-b1 gene (naked DNA) applied on the wound, we observed a further enhanced healing in the mouse model as a result of significant therapeutic effects of TGF-b1 gene. A markedly enhanced wound closing was observed in the early phase of healing (day 0-day5). A complete closure was observed on day 9 as compared with day 1114 of the wounds with hydrogel alone treatment and no treatment. We have found an enhanced re-epithelization, cell proliferation as well as a more mature collagen deposition at day 5 as compared with hydrogel alone treatment and no treatment. The data suggest that this novel gene delivery system promotes healing as a result of effective transfer of naked DNA in additional to its wound dressing effect, and has potential in treating clinically diabetic wound healing.
994. Transfection of Transitional Cell Carcinoma by Extracorporeal Acoustcal Energy: A New Method for Gene Therapy of Localized Bladder Tumors? Axel Schaaf, Sreedhar Sagi, Michael Siegsmund, Peter Alken, Maurice S. Michel. 1 Urological Department, University Hospital Mannheim, Mannheim, Germany. INTRODUCTION AND OBJECTIVES: Uptake of naked functional DNA into carcinoma cells can be achieved in vitro by a number of physical methods. However, for most of these techniques possibilities for therapeutic in vivo applications - especially to bladder tumors - are often limited. The Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
Mite allergy related asthma is highly prevalent worldwide. Der p 1 is a major allergen from house dust mite Dermatophagoides pteronyssinus, which reacts with IgE in more than 70% allergic sera, and therefore is a good candidate for DNA vaccine development. Previous study in our lab has demonstrated that intramuscular injection of plasmid DNA containing the Der p 1 gene in mice induced specific Th1 skewed immune response, with a significant amount of Der p 1 specific IgG. However, the magnitude of the immune response was too low to achieve satisfactory therapeutic and preventive efficacies, thus our main focus is to explore the various strategies to optimize the magnitude of the antigen-specific immune response. Codon optimization has been reported to enhance the expression of foreign genes by the mammalian transcriptional and translational machinery, and the lysosome targeting sequence from LAMP has been shown to direct the encoded protein to the MHC II compartment, and enhance the activation of CD4+ T cells. The aim of this study is to test the feasibility of using the LAMP lysosome targeting sequence and the codon optimization strategy to improve the efficacy of DNA vaccination. Codon optimization was done by changing the codon usage of Der p 1 cDNA sequence according to highly expressed human genes. The humanized gene was then linked to the LAMP lysosome targeting sequence, and the whole cassette was subsequently inserted into the multiple cloning site of pVAX1, which was used for DNA immunization. Following intramuscular injection of naked plasmid DNA with electroporation in BALB/c mice, antibody kinetics was followed for 70 days, and T cell cytokine profiles will be evaluated by cytokine ELISA and intracellular cytokine staining. Current results indicated that the humoral response was significantly enhanced by codon optimization possibly due to the higher level of protein expression. The efficacy of LAMP lysosome targeting sequence is still under evaluation and will be presented in the meeting. The information obtained will facilitate the design of Der p1 vaccines for allergic diseases.
992. Cytoreductive Cancer Gene Therapy Using Parvoviral Replicon Vectors Boris R. A. Blechacz,1 Jean Rommelaere,2 Jan J. Cornelis,2 Christiane Dinsart,2 Annick Brandenburger,3 Stephen J. Russell.1 1 Molecular Medicine Program, Mayo Foundation, Rochester, MN, United States; 2Applied Tumor Virology Program, German Cancer Research Center, Heidelberg, Germany; 3IRIBHN-IBMM, Universite Libre de Bruxelles, Gosselies, Belgium. Background: Cytoreductive gene therapy is an appealing approach to the treatment of human malignancies but significant barriers exist. In particular, the use of viruses as gene transfer vectors is limited by their immunogenic potential and existing nonviral systems are far less efficient than viral systems. We developed replicon vectors based on the autonomous parvovirus Minute Virus of Mice (MVMp) which is known to be tumorsuppressive and tumorcellspecific. As therapeutic transgenes we used the human sodium iodide symporter gene (hNIS) and the Gibbon ape leukaemia virus fusogenic membrane glycoprotein gene (Galv-FMG). Both of them have already proved their strong antitumoral potential in different tumor-models. The rational behind these replicon vectors is to take advantage of the tumorcell-specific cytotoxicity of parvoviruses and avoiding the disadvantages of their immunogenicity at the same time. By combining these parvoviral replicons with Galv-FMG and hNIS as therapeutic transgenes we expect high level expression of these highpotency therapeutic transgenes with major bystander killing potential. S382
Methods: Engineering of recombinant parvoviral replicon vectors was performed by replacing the capsid-coding region of the MVMgenome by transgene cDNA. For examination of size limitation in terms of gene replication and expression a polylinker was cloned between element A/B and the right palindrome; stuffer DNA of different size was cloned into this position. For functional testing of the parvoviral constructs 293-T cells were used. As an in vitro tumor model we used the human Hepatoma cell lines HUH-7, Hep3B and Hep-G2. Transfection was performed by using the lipotransfection method. For analysis of excision and parvoviral DNA-replication Southern blot analysis of Hirt extracts was performed. Transgene expression was analyzed in dependence of the transgene: semi-quantitative optical analysis, luciferase assay or CEA-level quantification. Results: MVM replicon vectors containing the marker genes CEA, luciferase and GFP as well as the therapeutic transgenes Galv-FMG and hNIS have been cloned. Excision and replication of parvoviral DNA has been shown as early as 24 h after transfection. High-level transgene expression in different cell lines was achieved. Parvoviral replicons containing Galv-FMG as therapeutic transgene were shown to have a very strong cytotoxic potential leading to an almost complete destruction of cell layers. Altogether these results show that the use of parvoviral replicons is a very promising approach to the gene therapy of cancer.
993. Thermosensitive Hydrogel Mediated Gene Transfer for Diabetic Wound Healing Pui-yan R. Lee,1 Zhenhua Li,2 Leaf Huang.2 Department of Bioengineering, School of Engineering, University of Pittsburgh, Pittsburgh, PA, United States; 2Center for Pharmogenetics, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States. 1
Of late, there have been a lot of interests in the role of TGF-b1 gene in impaired wound healing by using various delivery methods. In present study, we investigated the feasibility of a thermosensitive hydrogel made of a triblock copolymer, PEG-PLGA-PEG, as a vector of naked DNA delivery in genetically diabetic mouse (C57BKS.Cg-m +/+ Leprdb female mice) model. The hydrogel itself is a simple and good wound dressing. When the hydrogel formulated with TGF-b1 gene (naked DNA) applied on the wound, we observed a further enhanced healing in the mouse model as a result of significant therapeutic effects of TGF-b1 gene. A markedly enhanced wound closing was observed in the early phase of healing (day 0-day5). A complete closure was observed on day 9 as compared with day 1114 of the wounds with hydrogel alone treatment and no treatment. We have found an enhanced re-epithelization, cell proliferation as well as a more mature collagen deposition at day 5 as compared with hydrogel alone treatment and no treatment. The data suggest that this novel gene delivery system promotes healing as a result of effective transfer of naked DNA in additional to its wound dressing effect, and has potential in treating clinically diabetic wound healing.
994. Transfection of Transitional Cell Carcinoma by Extracorporeal Acoustcal Energy: A New Method for Gene Therapy of Localized Bladder Tumors? Axel Schaaf, Sreedhar Sagi, Michael Siegsmund, Peter Alken, Maurice S. Michel. 1 Urological Department, University Hospital Mannheim, Mannheim, Germany. INTRODUCTION AND OBJECTIVES: Uptake of naked functional DNA into carcinoma cells can be achieved in vitro by a number of physical methods. However, for most of these techniques possibilities for therapeutic in vivo applications - especially to bladder tumors - are often limited. The Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy