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HIV: Targeted Disruption of CCR5 Using Engineered Zinc Finger Protein Nucleases (ZFNs)
GENE THERAPY FOR HIV & AIDS 756. Targeting Viral Entry in Gene Therapy of HIV-Infection Jan van Lunzen,1 Tobias Glaunsinger,1 Felix Hermann,2 Klaus Kuehlcke,3 Alexander Alexandrov,3 Dorothee von Laer.2 1 Infectious Diseases, University Hospital Eppendorf, Hamburg, Germany; 2Applied Virology and Gene Therapy, Georg-SpeyerHaus, Frankfurt am Main, Germany; 3EUFETS, Fresenius Biotech GmbH, Bad Homburg, Germany. Despite significant improvements of the survival of HIV-infected patients during HAART, treatment limitations such as the development of drug-resistant HIV strains and long-term toxicities call for innovative treatment strategies. In this study, a novel antiviral gene that effectively inhibits virus entry was tested in a phase I/II clinical trial. The antiviral gene is expressed from a retroviral vector (M87o) and encodes for the membrane-anchored antiviral peptide C46. C46 comprises 46 amino acids, is derived from the second heptad repeat of the HIV-1 envelope glycoprotein gp41 and effectively inhibits fusion of the viral and cellular membranes during virus entry. The membrane-anchored form (maC46) was shown to effectively block entry of a broad range of HIV isolates, including viruses resistant to the fusion inhibitory peptide C36 (T20). C36 (T20) corresponds to the 36 C-terminal amino acids of C46. The gene was introduced into autolous CD4 T lymphocytes from 10 patients with severe immunodeficiency and HAART failure. Cells were expanded by co-stimulation with anti-CD3 and anti-CD28 immobilized on beads. Between 2 and 15 x 10E9 cells were infused. No major toxicities were observed and neither cellular nor humoral immune responses to the transgene product were detected. While a significant rise in T helper cell counts was seen, viral load was not affected. Gene marking could be detected throughout the one-year follow-up, but the levels were too low to account for the marked rise in CD4 counts.
757. Engineered Expression of Novel TRIM5 Isoforms Inhibits HIV-1 Infection in CD34 Hematopoietic Stem Cell Derived Macrophages Joseph S. Anderson,1 Ramesh Akkina.1 1 Microbiology, Immunology, Pathology, Colorado State University, Fort Collins, CO. Species specific innate resistance against viral infections offers novel avenues for antiviral therapeutic and prophylactic approaches. The retroviral and lentiviral restriction factors Ref1 and Lv1 are variants of the tripartite motif protein, TRIM5a, a component of cytoplasmic bodies. TRIM5a severely restricts productive retroviral infections at the level of post-entry and pre-integration by destabilizing the incoming viral capsid via ubiquitination. Using this approach, resistance to HIV-1 infection could be conferred by TRIM5arh expression in otherwise susceptible cells. Here we show that stable expression of simian TRIM5arh via a lentiviral vector in a permissive cell culture line, Magi-CXC4, conferred resistance to HIV-1. To translate these findings into a stem cell gene therapy setting, the TRIM5arh transgene was stable introduced into CD34+ hematopoietic progenitor cells to derive transgenic macrophages. Upon viral challenge, TRIM5arh expressing macrophages were highly resistant to HIV-1 infection compared to control cells. Human macrophages expressing TRIM5arh were also found to be phenotypically and functionally normal expressing the characteristic surface markers CD14, CD4, CCR5, CXCR4, MHCII, and B7.1. Based on these promising results, we sought to further improve the TRIM5a inhibiting action by designing more novel constructs by modifying the native human isoform of TRIM5a. These modified isoforms have also been shown to be effective in conferring viral resistance. These results demonstrate that the species specific restriction factor TRIM5arh is effective in conferring HIV-1 resistance in a stem cell setting thus paving the way for its application in AIDS gene therapy. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
758. Towards Gene Knock out Therapy for AIDS/HIV: Targeted Disruption of CCR5 Using Engineered Zinc Finger Protein Nucleases (ZFNs) Elena Perez,2 Yann Jouvenot,1 Jeffrey C. Miller,1 Olga Liu,2 Jianbin Wang,1 Dmitry Guschin,1 Victor Bartsevich,1 Kenneth Kim,1 Ya-Li Lee,1 Edward J. Rebar,1 Dale Ando,1 Philip D. Gregory,1 James Riley,2 Michael C. Holmes,1 Carl June.2 1 Research and Development, Sangamo BioSciences, Richmond, CA; 2Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA. HIV entry into target cells requires binding of the envelope protein GP120 to the CD4 co-receptors CCR5 or CXCR4. Loss of these HIV co-receptors is therefore attractive as a potential therapy for HIV patients, yet no current methods permit the efficient therapeutic disruption of a chosen gene in the human genome. To this end, we have developed designed zinc-finger protein nucleases (ZFNs) to target predetermined sequences within the CCR5 gene. Once these ZFNs create a double strand break (DSB) in the targeted DNA, natural DNA repair pathways, including Non-Homologous End Joining (NHEJ), subsequently repair the DSB. Importantly, however, NHEJ is error prone and thus can result in permanent disruption of the targeted gene. Designed ZFNs that recognize coding sequences within the CCR5 gene were generated and their DNA-binding and cleavage properties were determined in vitro. Vectors encoding the validated ZFNs were introduced into human cells for in vivo assessment of function. ZFNs targeted to the CCR5 co-receptor efficiently cleaved their expected DNA target sites, leading to high efficiency target gene disruption (>5%) in transiently transfected cells even in the absence of selective pressure. These results have been confirmed in relevant primary cell types (monocytes, CD4+ T cells, CD34+ hematopoietic precursors). We also demonstrated that PM-1 and GHOST cells modified by the CCR5-ZFNs became resistant to HIV infection. Challenge assays using T cells pre-treated with these ZFNs are in progress, although it is well established that a patients cells carrying a deletion within the CCR5 gene are resistant to infection despite repeated exposure to R5-tropic virus. Moreover, the frequency of gene disruption observed supports its examination as a possible method for the therapeutic modification of isolated patient cells to generate HIV resistant T cells or hematopoietic precursors. Full-time employee of Sangamo BioSciences.
759. In Search of an Effective Target: In Vivo Approach Using HIV-1 Specific siRNA and Ribozyme Libraries Hoshang J. Unwalla,1 Atilla A. Seyhan,2 Brian H. Johnston,2 John J. Rossi.1 1 Molecular Biology, Beckman Research Institute of the City of Hope National Medical Center, Duarte, CA; 2SomaGenics, Inc., Santa Cruz, CA. Short interfering RNA (siRNA) and ribozymes have been shown to silence HIV-1 gene expression by site-specific cleavage of viral mRNA. A major factor determining whether ribozymes or siRNAs will be effective for post-transcriptional gene silencing is the choice of an appropriate target site on the mRNA. Conventional approaches for target site selection often do not work due to structural motifs in the mRNA that preclude accessibility of these RNA-based antivirals. An effective screening strategy for potential targets on the viral genome is use of HIV specific siRNA or ribozyme libraries in cell culture coupled with HIV challenge assays and selective survival. For siRNA site selection, an HIV specific siRNA library spanning all possible HIV-specific siRNA sequences of 21-mer length were created and inserted between convergent human U6 and H1 S293
757. Engineered Expression of Novel TRIM5 Isoforms Inhibits HIV-1 Infection in CD34 Hematopoietic Stem Cell Derived Macrophages Joseph S. Anderson,1 Ramesh Akkina.1 1 Microbiology, Immunology, Pathology, Colorado State University, Fort Collins, CO. Species specific innate resistance against viral infections offers novel avenues for antiviral therapeutic and prophylactic approaches. The retroviral and lentiviral restriction factors Ref1 and Lv1 are variants of the tripartite motif protein, TRIM5a, a component of cytoplasmic bodies. TRIM5a severely restricts productive retroviral infections at the level of post-entry and pre-integration by destabilizing the incoming viral capsid via ubiquitination. Using this approach, resistance to HIV-1 infection could be conferred by TRIM5arh expression in otherwise susceptible cells. Here we show that stable expression of simian TRIM5arh via a lentiviral vector in a permissive cell culture line, Magi-CXC4, conferred resistance to HIV-1. To translate these findings into a stem cell gene therapy setting, the TRIM5arh transgene was stable introduced into CD34+ hematopoietic progenitor cells to derive transgenic macrophages. Upon viral challenge, TRIM5arh expressing macrophages were highly resistant to HIV-1 infection compared to control cells. Human macrophages expressing TRIM5arh were also found to be phenotypically and functionally normal expressing the characteristic surface markers CD14, CD4, CCR5, CXCR4, MHCII, and B7.1. Based on these promising results, we sought to further improve the TRIM5a inhibiting action by designing more novel constructs by modifying the native human isoform of TRIM5a. These modified isoforms have also been shown to be effective in conferring viral resistance. These results demonstrate that the species specific restriction factor TRIM5arh is effective in conferring HIV-1 resistance in a stem cell setting thus paving the way for its application in AIDS gene therapy. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
758. Towards Gene Knock out Therapy for AIDS/HIV: Targeted Disruption of CCR5 Using Engineered Zinc Finger Protein Nucleases (ZFNs) Elena Perez,2 Yann Jouvenot,1 Jeffrey C. Miller,1 Olga Liu,2 Jianbin Wang,1 Dmitry Guschin,1 Victor Bartsevich,1 Kenneth Kim,1 Ya-Li Lee,1 Edward J. Rebar,1 Dale Ando,1 Philip D. Gregory,1 James Riley,2 Michael C. Holmes,1 Carl June.2 1 Research and Development, Sangamo BioSciences, Richmond, CA; 2Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA. HIV entry into target cells requires binding of the envelope protein GP120 to the CD4 co-receptors CCR5 or CXCR4. Loss of these HIV co-receptors is therefore attractive as a potential therapy for HIV patients, yet no current methods permit the efficient therapeutic disruption of a chosen gene in the human genome. To this end, we have developed designed zinc-finger protein nucleases (ZFNs) to target predetermined sequences within the CCR5 gene. Once these ZFNs create a double strand break (DSB) in the targeted DNA, natural DNA repair pathways, including Non-Homologous End Joining (NHEJ), subsequently repair the DSB. Importantly, however, NHEJ is error prone and thus can result in permanent disruption of the targeted gene. Designed ZFNs that recognize coding sequences within the CCR5 gene were generated and their DNA-binding and cleavage properties were determined in vitro. Vectors encoding the validated ZFNs were introduced into human cells for in vivo assessment of function. ZFNs targeted to the CCR5 co-receptor efficiently cleaved their expected DNA target sites, leading to high efficiency target gene disruption (>5%) in transiently transfected cells even in the absence of selective pressure. These results have been confirmed in relevant primary cell types (monocytes, CD4+ T cells, CD34+ hematopoietic precursors). We also demonstrated that PM-1 and GHOST cells modified by the CCR5-ZFNs became resistant to HIV infection. Challenge assays using T cells pre-treated with these ZFNs are in progress, although it is well established that a patients cells carrying a deletion within the CCR5 gene are resistant to infection despite repeated exposure to R5-tropic virus. Moreover, the frequency of gene disruption observed supports its examination as a possible method for the therapeutic modification of isolated patient cells to generate HIV resistant T cells or hematopoietic precursors. Full-time employee of Sangamo BioSciences.
759. In Search of an Effective Target: In Vivo Approach Using HIV-1 Specific siRNA and Ribozyme Libraries Hoshang J. Unwalla,1 Atilla A. Seyhan,2 Brian H. Johnston,2 John J. Rossi.1 1 Molecular Biology, Beckman Research Institute of the City of Hope National Medical Center, Duarte, CA; 2SomaGenics, Inc., Santa Cruz, CA. Short interfering RNA (siRNA) and ribozymes have been shown to silence HIV-1 gene expression by site-specific cleavage of viral mRNA. A major factor determining whether ribozymes or siRNAs will be effective for post-transcriptional gene silencing is the choice of an appropriate target site on the mRNA. Conventional approaches for target site selection often do not work due to structural motifs in the mRNA that preclude accessibility of these RNA-based antivirals. An effective screening strategy for potential targets on the viral genome is use of HIV specific siRNA or ribozyme libraries in cell culture coupled with HIV challenge assays and selective survival. For siRNA site selection, an HIV specific siRNA library spanning all possible HIV-specific siRNA sequences of 21-mer length were created and inserted between convergent human U6 and H1 S293