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1018. Identification of Receptor-Mediated Death Pathway for IL-24
RNA-Based Gene Control & Technical Advances in Gene Regulation 1017. An Enhancer-Less Ubiquitous Chromatin Opening Element (UCOE) Provides Highly Reproducible and Stable Transgene Expression in Haematopoietic Cells Michael Antoniou,1 Fang Zhang,2 Thrasher Adrian.2 1 Medical and Molecular Genetics, King’s College London School of Medicine, London, United Kingdom; 2Molecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom.
Enhancer-mediated insertional mutagenesis and therapeutic transgene silencing within a retroviral vector context are recognised obstacles that hamper future clinical applications. We have therefore assessed the use of a novel enhancer-less ubiquitous chromatin opening element (UCOE) within a SIN lentiviral vector (LV) system. The human HNRPA2B1-CBX3 UCOE (A2UCOE) consists of a methylation-free CpG island spanning closely spaced dual divergently transcribed housekeeping gene promoters (Antoniou M et al. Genomics 82: 269-279, 2003; Williams S et al. BMC Biotechnology, 5: 17, 2005). Employing an ex vivo LV gene transfer approach to mouse bone marrow haematopoietic stem cells (HSC) and engraftment into lethally irradiated recipients, we have found that A2UCOE-EGFP expression is not only at a significantly elevated level relative to the commonly used CMV and SFFV viral promoters but is also highly reproducible, virtually completely resistant to silencing and distributed proportionally between T, B and myeloid cell lineages in peripheral blood cells. Furthermore, an A2UCOE-common cytokine receptor gamma chain gene (A2UCOE-IL2RG) LV restores the IL-2 signalling pathway within human cells deficient in IL2RG in vitro. The A2UCOE-IL2RG LV is also able to completely rescue the SCID-X1 phenotype in a mouse model of this disease in vivo, showing full immuno-reconstitution of T and B cells following engraftment of transduced HSC. The A2UCOE therefore displays highly reliable and stable transcriptional activity from within an LV, overcoming insertion site position effects and giving rise to therapeutically relevant levels of gene expression, which due to the absence of classical enhancer activity should confer a higher safety profile (Zhang F et al., Blood, 110: 1448-1457). Further pre-clinical evaluation studies assessing the genotoxicity, insertional mutagenesis potential of the A2UCOE within lentiviral vectors are underway.
1018. Identification of Receptor-Mediated Death Pathway for IL-24
Mingzhong Zheng,1 Dora Bocangel,1 Rajagopal Ramesh,2 Sunil Chada.1 1 Research Develoopment, Introgen Therapeutics Inc., Houston, TX; 2Department of Thoracic and Cardiovascular Surgery, The University of Texas, MD Anderson Cancer Center, Houston, TX.
IL-24 is a novel cytokine-tumor suppressor in the IL-10 family. Gene transfer of IL-24 results in activation of multiple anti-tumor pathways, including activation of apoptosis; inhibition of metastasis; anti-angiogenesis and induction of anti-tumor immunity. We have previously shown that IL-24 protein can promote tumor cell killing in various tumor cell lines. However, the exact mechanism(s) of IL24-mediated tumor cell killing and the role of the IL-24 receptors in mediating cell killing remain unknown. In the present study we investigated the IL-24-receptor mediated cell death pathway using the non-small cell lung cancer cell line (H1299) that is receptor negative. H1299 cells were stably transfected to express IL-24 receptors, IL20R1/IL-20R2 or IL-22R1/IL-20R2. The receptor positive cell lines isogenic to the parent H1299 cells were labeled H1299/IL-20R1 and H1299/IL-22R1 since the IL-20R2 subunit is common to both receptors. Expression of the receptors in these isogenic cell lines was confirmed by flow cytometry. Treatment with IL-24 protein resulted in dose- and time-dependent cell killing of H1299/IL-20R1 or H1299/ S382
IL-22R1 but not the parental H1299 cells. The specificity of IL-24 binding to its receptors to mediate cell killing was demonstrated by neutralization and blocking studies using anti-IL-24 antibody and anti-receptor antibodies. IL-24-mediated killing in receptorpositive cell lines was abrogated by more than eighty percent upon neutralization of IL-24. Additionally, Western blotting showed STAT3 phosphorylation only in IL-24-treated receptor positive cell lines but not in receptor-negative cells indicating receptor-ligand interaction involves the receptors and contributes to activation of the apoptotic signaling pathway. Furthermore, cell killing was independent of p53 as H1299 cells are null for p53. In summary, our studies demonstrate a novel IL-24-receptor mediated cell death pathway. Therefore IL-24 is the only IL-10 family member with tumor killing capability and is one of the few cytokines which induce apoptosis in tumor cells.
1019. A Versatile Viral Vector Platform Incorporating 2A Peptide Sequences for Multicistronic Gene Expression In Vitro and In Vivo
Charles G. Bailey,1 Cynthia Ng,1 Jessamy C. Tiffen,1 Jeff Holst,1 John E. J. Rasko.1,2 1 Gene and Stem Cell Therapy Program, Centenary Institute, Camperdown, NSW, Australia; 2Sydney Cancer Centre, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
Several viruses, including members of the Picornaviridiae family, use 2A peptides or 2A-like sequences to mediate protein cleavage. The incorporation of a 2A peptide linking sequences between genes encoded in the same open reading frame (ORF) results in near-complete separation and stoichometric production of encoded proteins via a ribosomal skipping mechanism. Cleavage occurs at a highly conserved consensus sequence at the C-terminal end of the 2A peptide encoded within the vector. The consequent retention of 20 amino acids at the carboxy terminus of proteins can be used as a tag for identification and cleavage efficiency determination using a specific anti-2A antibody. We have developed a vector platform for the assembly of multicistronic ORFs to facilitate shuttling into mammalian expression vectors, retroviral and lentiviral vectors. We have integrated autofluorescent and luminescent reporters, mammalian selectable markers and heterologous genes in various combinations into this vector platform for gene transfer experiments in vitro and in vivo. Importantly, selection of cassettes including tandem arrays of reporter genes or markers is tightly linked to the expression of our genes of interest. In an in vivo NOD/SCID tumour model, ACHN renal carcinoma cells and K562 erythroleukemia cells were transduced with lentiviral particles containing eGFP linked by a 2A peptide to firefly luciferase. GFP-positive cells were sorted by FACS, clonally isolated, expanded and then injected subcutaneously into mice. The development of tumours was then tracked in mice following injection of luciferin substrate intravenously and then imaging the anaesthetised mice using the Xenogen IVIS biophotonic imaging system. This ‘toolbox’ of 2A chimeric multicistronic vectors enables analysis and imaging of gene expression in vitro using multi-parametric flow cytometry, confocal microscopy or image streaming technology; and in vivo using bioluminescent or fluorescent imaging.
1020. Optimization of the Slc4a1 (Band 3) in Globin Gene Therapy Vectors
Faith Harrow, Stephanie Battle, Nancy E. Seidel, Amanda P. Cline, David M. Bodine. 1 Genetics and Molecular Biology Branch, Hematopoiesis Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD. Gene therapy holds the promise of a cure for the β globin disorders, sickle cell disease and β-thalassemia. Successful treatment would Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Enhancer-mediated insertional mutagenesis and therapeutic transgene silencing within a retroviral vector context are recognised obstacles that hamper future clinical applications. We have therefore assessed the use of a novel enhancer-less ubiquitous chromatin opening element (UCOE) within a SIN lentiviral vector (LV) system. The human HNRPA2B1-CBX3 UCOE (A2UCOE) consists of a methylation-free CpG island spanning closely spaced dual divergently transcribed housekeeping gene promoters (Antoniou M et al. Genomics 82: 269-279, 2003; Williams S et al. BMC Biotechnology, 5: 17, 2005). Employing an ex vivo LV gene transfer approach to mouse bone marrow haematopoietic stem cells (HSC) and engraftment into lethally irradiated recipients, we have found that A2UCOE-EGFP expression is not only at a significantly elevated level relative to the commonly used CMV and SFFV viral promoters but is also highly reproducible, virtually completely resistant to silencing and distributed proportionally between T, B and myeloid cell lineages in peripheral blood cells. Furthermore, an A2UCOE-common cytokine receptor gamma chain gene (A2UCOE-IL2RG) LV restores the IL-2 signalling pathway within human cells deficient in IL2RG in vitro. The A2UCOE-IL2RG LV is also able to completely rescue the SCID-X1 phenotype in a mouse model of this disease in vivo, showing full immuno-reconstitution of T and B cells following engraftment of transduced HSC. The A2UCOE therefore displays highly reliable and stable transcriptional activity from within an LV, overcoming insertion site position effects and giving rise to therapeutically relevant levels of gene expression, which due to the absence of classical enhancer activity should confer a higher safety profile (Zhang F et al., Blood, 110: 1448-1457). Further pre-clinical evaluation studies assessing the genotoxicity, insertional mutagenesis potential of the A2UCOE within lentiviral vectors are underway.
1018. Identification of Receptor-Mediated Death Pathway for IL-24
Mingzhong Zheng,1 Dora Bocangel,1 Rajagopal Ramesh,2 Sunil Chada.1 1 Research Develoopment, Introgen Therapeutics Inc., Houston, TX; 2Department of Thoracic and Cardiovascular Surgery, The University of Texas, MD Anderson Cancer Center, Houston, TX.
IL-24 is a novel cytokine-tumor suppressor in the IL-10 family. Gene transfer of IL-24 results in activation of multiple anti-tumor pathways, including activation of apoptosis; inhibition of metastasis; anti-angiogenesis and induction of anti-tumor immunity. We have previously shown that IL-24 protein can promote tumor cell killing in various tumor cell lines. However, the exact mechanism(s) of IL24-mediated tumor cell killing and the role of the IL-24 receptors in mediating cell killing remain unknown. In the present study we investigated the IL-24-receptor mediated cell death pathway using the non-small cell lung cancer cell line (H1299) that is receptor negative. H1299 cells were stably transfected to express IL-24 receptors, IL20R1/IL-20R2 or IL-22R1/IL-20R2. The receptor positive cell lines isogenic to the parent H1299 cells were labeled H1299/IL-20R1 and H1299/IL-22R1 since the IL-20R2 subunit is common to both receptors. Expression of the receptors in these isogenic cell lines was confirmed by flow cytometry. Treatment with IL-24 protein resulted in dose- and time-dependent cell killing of H1299/IL-20R1 or H1299/ S382
IL-22R1 but not the parental H1299 cells. The specificity of IL-24 binding to its receptors to mediate cell killing was demonstrated by neutralization and blocking studies using anti-IL-24 antibody and anti-receptor antibodies. IL-24-mediated killing in receptorpositive cell lines was abrogated by more than eighty percent upon neutralization of IL-24. Additionally, Western blotting showed STAT3 phosphorylation only in IL-24-treated receptor positive cell lines but not in receptor-negative cells indicating receptor-ligand interaction involves the receptors and contributes to activation of the apoptotic signaling pathway. Furthermore, cell killing was independent of p53 as H1299 cells are null for p53. In summary, our studies demonstrate a novel IL-24-receptor mediated cell death pathway. Therefore IL-24 is the only IL-10 family member with tumor killing capability and is one of the few cytokines which induce apoptosis in tumor cells.
1019. A Versatile Viral Vector Platform Incorporating 2A Peptide Sequences for Multicistronic Gene Expression In Vitro and In Vivo
Charles G. Bailey,1 Cynthia Ng,1 Jessamy C. Tiffen,1 Jeff Holst,1 John E. J. Rasko.1,2 1 Gene and Stem Cell Therapy Program, Centenary Institute, Camperdown, NSW, Australia; 2Sydney Cancer Centre, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
Several viruses, including members of the Picornaviridiae family, use 2A peptides or 2A-like sequences to mediate protein cleavage. The incorporation of a 2A peptide linking sequences between genes encoded in the same open reading frame (ORF) results in near-complete separation and stoichometric production of encoded proteins via a ribosomal skipping mechanism. Cleavage occurs at a highly conserved consensus sequence at the C-terminal end of the 2A peptide encoded within the vector. The consequent retention of 20 amino acids at the carboxy terminus of proteins can be used as a tag for identification and cleavage efficiency determination using a specific anti-2A antibody. We have developed a vector platform for the assembly of multicistronic ORFs to facilitate shuttling into mammalian expression vectors, retroviral and lentiviral vectors. We have integrated autofluorescent and luminescent reporters, mammalian selectable markers and heterologous genes in various combinations into this vector platform for gene transfer experiments in vitro and in vivo. Importantly, selection of cassettes including tandem arrays of reporter genes or markers is tightly linked to the expression of our genes of interest. In an in vivo NOD/SCID tumour model, ACHN renal carcinoma cells and K562 erythroleukemia cells were transduced with lentiviral particles containing eGFP linked by a 2A peptide to firefly luciferase. GFP-positive cells were sorted by FACS, clonally isolated, expanded and then injected subcutaneously into mice. The development of tumours was then tracked in mice following injection of luciferin substrate intravenously and then imaging the anaesthetised mice using the Xenogen IVIS biophotonic imaging system. This ‘toolbox’ of 2A chimeric multicistronic vectors enables analysis and imaging of gene expression in vitro using multi-parametric flow cytometry, confocal microscopy or image streaming technology; and in vivo using bioluminescent or fluorescent imaging.
1020. Optimization of the Slc4a1 (Band 3) in Globin Gene Therapy Vectors
Faith Harrow, Stephanie Battle, Nancy E. Seidel, Amanda P. Cline, David M. Bodine. 1 Genetics and Molecular Biology Branch, Hematopoiesis Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD. Gene therapy holds the promise of a cure for the β globin disorders, sickle cell disease and β-thalassemia. Successful treatment would Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy