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459. Regulatory T Cell Specific Gene Expression through a Novel Adenovirus Encoding the FOXP3 Promoter
Adenovirus Vectors: Cancer Therapy 458. Deletion of the E1B19K Gene of Oncolytic Adenoviruses Does Not Interfere with the Expression of Transgenes, but Decreases Lytic Activity in Some Tumor Cells
Stanimira Rohmer,1,2 Andrea Hesse,2 Christina Quirin,1,2 Dirk M. Nettelbeck.1,2 1 Helmholtz-University Group Oncolytic Adenoviruses, German Cancer Research Center and Heidelberg Univerity Hospital, Heidelberg, Germany; 2Department of Dermatology, University Hospital Erlangen, Erlangen, Germany.
Oncolytic adenoviruses are promising new anticancer agents for specific replication in/lysis of tumor cells and subsequent spread in the tumor. Several clinical trials with oncolytic adenoviruses have demonstrated proof of principle and a favourable safety profile, but intratumoral spread and therapeutic efficacy were limited. Recent strategies to address this drawback have been the development of virus mutants with enhanced lytic activity and of so-called “armed” oncolytic adenoviruses by incorporation of therapeutic genes into the viral genome. We previously established strategies for coexpression of transgenes from the late viral transcription unit and for efficient viral cell entry by modifying the virus capsid. Here we investigated in a panel of tumor cell lines and primary tumor cells how the deletion of the anti-apoptotic early viral E1B19K gene affects both oncolytic potency and transgene expression of these viruses. Our results demonstrate that the deletion of the E1B19K gene results in a remarkable increase in oncolytic activity in some tumor cells, but in a drastic reduction of lytic activity in others. Cells infected with the mutant viruses showed clear signs of apoptosis. In contrast, cells infected with E1B19K wild-type viruses showed either no or delayed apoptosis induction. The reason for differences in viral cell lysis by E1B19K mutant viruses between cell types is currently under investigation, data will be presented. This work should help to understand the basis for suboptimal adenovirus replication and spread in tumor cells. Finally, in reporter gene assays we revealed that the deletion of the E1B19K gene, and thus induction of apoptosis during virus replication, does not interfere with the expression of transgenes inserted into the late viral transcription unit of oncolytic adenoviruses. Taken together, the deletion of the E1B19K gene is a promising strategy to increase the potency of armed oncolytic adenoviruses, however, its feasibility needs to be assessed individually for each tumor target.
459. Regulatory T Cell Specific Gene Expression through a Novel Adenovirus Encoding the FOXP3 Promoter
Matthew S. Beatty,1,2 Joel N. Glasgow,1 Tyler J. Curiel,3 David T. Curiel.1 1 Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 2Division of Molecular and Cellular Pathology, Department of Pathology, University of Alabama at Birmingham, Birmingham, AL; 3Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio, TX.
The use of immunotherapy holds much promise in the treatment of cancer in conjunction with established therapies. Most immunotherapy trials focus on eliciting a cellular immune response against tumor using vaccines, cytokines or adoptive cell transfer. These strategies generally fail as tumors actively down regulate immune response through regulatory T cell (Treg) suppression and other suppressive mechanisms. Targeting Tregs for depletion is a novel strategy tested in both animal models and clinical trials with promising results. However, no strategy yet tested for Treg depletion is specific. Development of novel methods for targeting the specific S174
Treg subset while not depleting other immune cell populations is essential to maximize therapeutic value. Current strategies for Treg depletion rely on targeting cellular markers such as CD25, but these molecules are also expressed on activated effector cells. The discovery of the forkhead transcription factor 3 (FOXP3), which regulates Treg differentiation, finally allowed for specific identification. Given that FOXP3 is relatively specific to Tregs, we hypothesize that an adenovirus (Ad) using the FOXP3 promoter to drive gene expression will allow for greater specificity in targeting Tregs than current therapies. To test this hypothesis, we genetically engineered recombinant Ad encoding varying lengths of the human FOXP3 promoter. We developed four lengths of the promoter, varying in size from -1657 base pairs from the transcriptional start site to -307 base pairs, that drive expression of the luciferase gene. These vectors are currently being characterized in CD4+CD25+ Tregs and CD4+CD25- T cells isolated from hCAR expressing mice. The development of novel immunotherapy strategies for cancer therapy, such as Treg depletion, shows great potential. With current Treg depletion strategies, however, problems arise in that other T cell populations required for tumor rejection are also targeted. An Ad vector using the Treg specific FOXP3 promoter will greatly enhance targeting specificity and allow for more accurate depletion of Tregs while leaving other immune cell populations intact.
460. Replication-Competent Adenovirus Containing a Therapeutic Gene in E4orf1-4
Mi-Hyang Lee,1 In-Hoo Kim,1 Jin-Sook Jeong,2 Sang-Young Han,2 Hae-Hyun Seo,1 Sang-Jin Lee.1 1 Genitourinary Cancer Branch, National Cancer Center, Goyang, Korea; 2Pathology, Dong-A University, Pusan, Korea; 3Internal Medicine, Dong-A University, Pusan, Korea. Background: Therapeutic transgene expression from oncolytic adenoviruses is an attractive approach to enhancing the effectiveness of these agents as cancer therapeutics. Few places are available for transgene insertion, since the genomic packaging capacity is constrained inside oncolytic adenovirus. In this study, we tested whether the early E4 region could be utilized for transgene expression via alternative splicing without damaging the replication capability. Method: We examined which region in E4 could be necessary for virus and replication in several cancer cells and then constructed the recombinant adenovirus by; 1) replacing E4 promoter by the prostatespecific enhancer element (PSES), 2) inserting mRFP inside E4 deletion region, 3) sub-cloning EGFP controlled by cytomegalovirus promoter (CMV). Furthermore, we constructed replication-competent adenovirus replacing E4orf1-4 fragment by thymidine kinase gene as a therapeutic adenovirus. Results: When we examined the replication capability of both E4Dorf1-4 and E4Dof1-3, E4orf 1-4 was not so essential for adenovirus replication so that it could be used for an insertion site for transgene and then expression inside target cells. Using mRFP and GFP as reporter genes, we tested mRFP for alternative splicing and GFP for virus replication. The number and intensity of EGFP and mRFP gene products increased in PSESpositive prostate cancer cells, implicating that mRFP gene and E4 genes other than orf1-4 were synthesized from one transcript via alternative splicing as the recombinant adenovirus replicated. RTPCR analysis confirmed the mRFP and E4orf6, E434kDa, E4orf6/7 expression via alternative splicing and replication assay did the virus replication capability. Next, we replaced E4orf1-4 with therapeutic gene, thymidine kinase and evaluated its replication and killing activity. This therapeutic adenovirus effectively lyzed prostate cancer cells and synergistically in the presence of gancyclovir, which was substrate of thymidine kinase. Conclusion: We discovered another insertion site for therapeutic transgene in the adenoviral genome and developed therapeutic adenovirus targeting prostate cancer cells. Furthermore, these findings will enable us to construct therapeutic Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Stanimira Rohmer,1,2 Andrea Hesse,2 Christina Quirin,1,2 Dirk M. Nettelbeck.1,2 1 Helmholtz-University Group Oncolytic Adenoviruses, German Cancer Research Center and Heidelberg Univerity Hospital, Heidelberg, Germany; 2Department of Dermatology, University Hospital Erlangen, Erlangen, Germany.
Oncolytic adenoviruses are promising new anticancer agents for specific replication in/lysis of tumor cells and subsequent spread in the tumor. Several clinical trials with oncolytic adenoviruses have demonstrated proof of principle and a favourable safety profile, but intratumoral spread and therapeutic efficacy were limited. Recent strategies to address this drawback have been the development of virus mutants with enhanced lytic activity and of so-called “armed” oncolytic adenoviruses by incorporation of therapeutic genes into the viral genome. We previously established strategies for coexpression of transgenes from the late viral transcription unit and for efficient viral cell entry by modifying the virus capsid. Here we investigated in a panel of tumor cell lines and primary tumor cells how the deletion of the anti-apoptotic early viral E1B19K gene affects both oncolytic potency and transgene expression of these viruses. Our results demonstrate that the deletion of the E1B19K gene results in a remarkable increase in oncolytic activity in some tumor cells, but in a drastic reduction of lytic activity in others. Cells infected with the mutant viruses showed clear signs of apoptosis. In contrast, cells infected with E1B19K wild-type viruses showed either no or delayed apoptosis induction. The reason for differences in viral cell lysis by E1B19K mutant viruses between cell types is currently under investigation, data will be presented. This work should help to understand the basis for suboptimal adenovirus replication and spread in tumor cells. Finally, in reporter gene assays we revealed that the deletion of the E1B19K gene, and thus induction of apoptosis during virus replication, does not interfere with the expression of transgenes inserted into the late viral transcription unit of oncolytic adenoviruses. Taken together, the deletion of the E1B19K gene is a promising strategy to increase the potency of armed oncolytic adenoviruses, however, its feasibility needs to be assessed individually for each tumor target.
459. Regulatory T Cell Specific Gene Expression through a Novel Adenovirus Encoding the FOXP3 Promoter
Matthew S. Beatty,1,2 Joel N. Glasgow,1 Tyler J. Curiel,3 David T. Curiel.1 1 Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 2Division of Molecular and Cellular Pathology, Department of Pathology, University of Alabama at Birmingham, Birmingham, AL; 3Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio, TX.
The use of immunotherapy holds much promise in the treatment of cancer in conjunction with established therapies. Most immunotherapy trials focus on eliciting a cellular immune response against tumor using vaccines, cytokines or adoptive cell transfer. These strategies generally fail as tumors actively down regulate immune response through regulatory T cell (Treg) suppression and other suppressive mechanisms. Targeting Tregs for depletion is a novel strategy tested in both animal models and clinical trials with promising results. However, no strategy yet tested for Treg depletion is specific. Development of novel methods for targeting the specific S174
Treg subset while not depleting other immune cell populations is essential to maximize therapeutic value. Current strategies for Treg depletion rely on targeting cellular markers such as CD25, but these molecules are also expressed on activated effector cells. The discovery of the forkhead transcription factor 3 (FOXP3), which regulates Treg differentiation, finally allowed for specific identification. Given that FOXP3 is relatively specific to Tregs, we hypothesize that an adenovirus (Ad) using the FOXP3 promoter to drive gene expression will allow for greater specificity in targeting Tregs than current therapies. To test this hypothesis, we genetically engineered recombinant Ad encoding varying lengths of the human FOXP3 promoter. We developed four lengths of the promoter, varying in size from -1657 base pairs from the transcriptional start site to -307 base pairs, that drive expression of the luciferase gene. These vectors are currently being characterized in CD4+CD25+ Tregs and CD4+CD25- T cells isolated from hCAR expressing mice. The development of novel immunotherapy strategies for cancer therapy, such as Treg depletion, shows great potential. With current Treg depletion strategies, however, problems arise in that other T cell populations required for tumor rejection are also targeted. An Ad vector using the Treg specific FOXP3 promoter will greatly enhance targeting specificity and allow for more accurate depletion of Tregs while leaving other immune cell populations intact.
460. Replication-Competent Adenovirus Containing a Therapeutic Gene in E4orf1-4
Mi-Hyang Lee,1 In-Hoo Kim,1 Jin-Sook Jeong,2 Sang-Young Han,2 Hae-Hyun Seo,1 Sang-Jin Lee.1 1 Genitourinary Cancer Branch, National Cancer Center, Goyang, Korea; 2Pathology, Dong-A University, Pusan, Korea; 3Internal Medicine, Dong-A University, Pusan, Korea. Background: Therapeutic transgene expression from oncolytic adenoviruses is an attractive approach to enhancing the effectiveness of these agents as cancer therapeutics. Few places are available for transgene insertion, since the genomic packaging capacity is constrained inside oncolytic adenovirus. In this study, we tested whether the early E4 region could be utilized for transgene expression via alternative splicing without damaging the replication capability. Method: We examined which region in E4 could be necessary for virus and replication in several cancer cells and then constructed the recombinant adenovirus by; 1) replacing E4 promoter by the prostatespecific enhancer element (PSES), 2) inserting mRFP inside E4 deletion region, 3) sub-cloning EGFP controlled by cytomegalovirus promoter (CMV). Furthermore, we constructed replication-competent adenovirus replacing E4orf1-4 fragment by thymidine kinase gene as a therapeutic adenovirus. Results: When we examined the replication capability of both E4Dorf1-4 and E4Dof1-3, E4orf 1-4 was not so essential for adenovirus replication so that it could be used for an insertion site for transgene and then expression inside target cells. Using mRFP and GFP as reporter genes, we tested mRFP for alternative splicing and GFP for virus replication. The number and intensity of EGFP and mRFP gene products increased in PSESpositive prostate cancer cells, implicating that mRFP gene and E4 genes other than orf1-4 were synthesized from one transcript via alternative splicing as the recombinant adenovirus replicated. RTPCR analysis confirmed the mRFP and E4orf6, E434kDa, E4orf6/7 expression via alternative splicing and replication assay did the virus replication capability. Next, we replaced E4orf1-4 with therapeutic gene, thymidine kinase and evaluated its replication and killing activity. This therapeutic adenovirus effectively lyzed prostate cancer cells and synergistically in the presence of gancyclovir, which was substrate of thymidine kinase. Conclusion: We discovered another insertion site for therapeutic transgene in the adenoviral genome and developed therapeutic adenovirus targeting prostate cancer cells. Furthermore, these findings will enable us to construct therapeutic Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy