77 CCR5 positive cell specific targeted transduction of a CCR5 shRNA lentiviral vector for HIV gene therapy

77 CCR5 Positive Cell Specific Targeted Transduction of a CCR5 shRNA Lentiviral Vector for HIV Gene Therapy Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene[.]

RNA VIRUS VECTORS I copy/cell in the various organs ranged from 1/1 to 1/100,000 There was no difference in EGFP biodistribution and expression pattern between the E14.5 and E16.5 lentiviral deliveries, although high mortality was associated with E12.5 delivery Together, our results demonstrate efficient gene transfer to and expression in the respiratory epithelium in the predicted therapeutic range RNA Virus Vectors I 73 TTRAP Enhance the Lentiviral Gene Expression Xu Wang, Jinzhong Chen, Jinglun Xue Institute of Genetics, Fudan University, Shanghai, China The lentivirus integrase HIV-IN plays a crucial role in the viral life cycle, especially the step of integration However, the cellular response to the incoming viral protein and potential interaction between IN and cellular proteins is largely unknown This study reveals that HIV integrase interacts with a recently identified PML-NBs associated protein TTRAP by yeast two hybrid, coimmunoprecipitation and intracellular co-location Knocking down the expression of endogenous TTRAP with specific siRNA duplex decreased the lentiviral integration rate, while TTRAP over expression increased the integration apparently Interestingly, TTRAP facilitates the lentiviral vector integration in a PML–independent manner Knocking down the expression of PML presents no difference in integration comparing with control group This is the first time that HIV integrase is reported to interact with a PML-NBs protein which facilitates the lentivirus integration The result provided a clue to improve the lentiviral vector integration or to develop the therapy methods against HIV infection 74 A Daxx-Binding Tetrapeptide 870KELK873 of HIV-1 Integrase Is Critical for Lentiviral Gene Expression Limin Wan, Jinzhong Chen Insititute of Genetics, Fudan University, Shanghai, China Our recent studies have found cellular protein Daxx to interact with HIV-1 integrase, but the region critical for this interaction has not been identified Here we found that a tetrapeptide 870KELK873 in HIV-1 integrase C-terminus is crucial for Daxx-binding Further investigation on the functions of this region suggested that deleting the tetrapeptide affects neither virus packaging nor viral gene integration into the host genome However, the reporter gene EGFP expression was significantly lower in cells infected by the HIV-1derived lentivirus without the tetrapeptide compared to those infected by wild-type lentivirus Our results suggest that the Daxx-binding tetrapeptide 870KELK873 of HIV-1 integrase is critical for the HIV1-derived lentiviral reporter gene expression, which may shed some light on the gene therapy based on lentiviral vectors 75 Sp100 Inhibited the Lentiviral Gene Expression Liping Qu, Jinzhong Chen Institute of Genetics, Fudan University, Shanghai, China The lentivirus integrase HIV-IN plays a crucial role in the viral life cycle, especially the step of integration But the cellular response to the incoming viral protein and potential interaction between IN and cellular proteins is unknown This study reveals that HIV integrase interacts with a PML-NBs associated protein Sp100 by yeast two hybrid, co-immunoprecipitation and intracellular co-location Knocking down the expression of endogenous Sp100 with specific siRNA duplex increased the lentiviral gene expression, while Sp100 over expression decreased the lentiviral gene expression apparently Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy The result provided a clue to improve the lentiviral vector integration or to develop the therapy methods against HIV infection 76 Enhancing Retroviral Transduction of Cord Blood CD34+ Cells by MIP-1α Leili Moezzi,1 Kamran Alimoghaddam,2 Seyed Hamidolah Ghaffari,2 Alireza Ardjmand,2 Pantea Ghodsi,2 Bahram Chahardouli,2 Somayeh Shahrokhi,3 Ardeshir Ghavamzadeh.2 Tehran University of Medical Sciences, Faculty of Paramedical Sciences, Tehran, Islamic Republic of Iran; 2HematologyOncology and Stem Cell Research Center, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran; 3Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Islamic Republic of Iran Introduction: Hematopoietic stem cells are in quiescence state and because of requirement of retroviral transduction to infect dividing cells; they are resistant to retrovirus transduction and needs to pre-stimulation by a cytokine cocktail For proliferation without maturation, we suggest MIP-1α as a novel factor Material and methods: retroviral vector produced by PG13/LN C8 cells tittered on hela cells Then CD34+ cells of cord blood prestimulated in serum free media supplemented with 50 ng/ml SCF,Flt3,TPO,IL6 in the presence and absence of 50 ng/ml MIP-1plus 8g/ml protamin sulfate and viral supernatant media) with and reefed them after 24 and 72 hours Then cells were harvested from the plate and seeded into new dishes in the absence of viral supernatant for one week Transduction efficiency by semi sensitive PCR for Neomycin resistance gene was assessed Results: Multiplicity of infection was 1.7105 PCR analysis of neomycin resistance gene in cord blood CD34+ cells in the presence and absence of MIP-1revealed improved Transduction of cord blood cells (CD34+ = 40.7%, CD34+MIP=65%) Conclusion: Addition of MIP-1α to cytokine cocktail may improve transduction efficiency of cord blood hematopoietic progenitor cells Further studies required for clarify its effect on functional properties of CD34+ cells 77 CCR5 Positive Cell Specific Targeted Transduction of a CCR5 shRNA Lentiviral Vector for HIV Gene Therapy Joseph S Anderson,1 Jan Nolta,1 Gerhard Bauer.1 Internal Medicine/Stem Cell Program, University of CaliforniaDavis, Sacramento, CA HIV gene therapy offers a potential alternative to current small molecule antiretroviral treatments which, after prolonged use, can become toxic and allow the generation of escape mutants Current HIV gene therapy protocols rely on ex vivo transductions of hematopoietic stem cells or peripheral blood mononuclear cells (PBMCs) These procedures require either apheresis of PBMCs, mobilization of peripheral blood stem cells, or bone marrow aspirations, methods to isolate the target cells, clinical grade tissue culture methods to introduce the vector, and finally re-administration of the gene modified cells into the patient The development of cell specific targeting vectors capable of selectively transducing cells of interest, in vivo, would greatly simplify and enhance gene therapy applications, by bringing them to areas where sophisticated laboratories and clinics are not available As a first step towards achieving in vivo transduction protocols for HIV gene therapy, a promising strategy is currently being evaluated in our laboratory Based on previous work using a Protein-A ZZ-domain/monoclonal antibody (mAb) conjugated Sindbis virus glycoprotein pseudotyped lentiviral vector, here we demonstrated the utility of this strategy for HIV gene therapy by specifically targeting HIV susceptible cells and making them resistant to HIV infection Using a CCR5 mAb conjugated envelope, CCR5 positive cells were specifically targeted for transduction by a lentiviral vector containing a highly potent CCR5 S31 RNA VIRUS VECTORS I shRNA capable of >93% knockdown of CCR5 expression Mixed cell assays of HEK-293T cells (CCR5-) and Ghost-X4/R5/R3 cells (CCR5+) as well as freshly isolated PBMCs were used to determine the specificity of the CCR5-targeting vector These populations of different cells were transduced and analyzed by FACS for EGFP expression to determine transduction efficiency and specificity After transduction with the CCR5 targeting vector, only CCR5 expressing cells were positive for EGFP In the mixed culture of HEK-293T and Ghost-X4/R5/R3 cells, we could demonstrate that only the CCR5 positive Ghost-X4/R5/R3 cells were selectively transduced As for the PBMC cultures, only T cells (CCR5+) and monocytes (CCR5+) were transduced as compared to B cells (CCR5-) which were not transduced Transduced cell populations were further analyzed for CCR5 knockdown As observed by FACS, a substantial knockdown of CCR5 expression (>93%) was observed in both cultured cells and PBMCs Transgenic cells were subsequently challenged with BaL-1, an R5-tropic strain of HIV-1 CCR5 shRNA transduced cells were resistant to HIV-1 infection as compared to nontransduced and EGFP-alone control transduced cells A selective survival advantage of CCR5 shRNA transgenic cells was also observed in a mixed population of cells containing both nontransduced and transduced cells These data represent an initial proof-of-concept evaluation of this targeting vector strategy for HIV gene therapy Further in vivo experiments will allow us to evaluate the preclinical efficacy for possible therapeutic applications in humans 78 Potent Pre-Integration Inhibition of HIV1 Infection by a Triple Combination Anti-HIV Lentiviral Vector Containing a CCR5 shRNA, a Chimeric TRIM5α Restriction Factor, and a TAR Decoy for HIV Gene Therapy Joseph S Anderson,1 John Javien,1 Jan Nolta,1 Gerhard Bauer.1 Internal Medicine/Stem Cell Program, University of CaliforniaDavis, Sacramento, CA HIV continues to be a major public health problem with continued infections occurring worldwide Current antiretroviral therapies (ART) can suppress HIV infection in patients who adhere to their daily regimen However, with continued use, toxic side effects and the generation of escape mutants can render ART ineffective Hematopoietic stem cell gene therapy for HIV offers a potential alternative to these drugs and can offer the possibility of a onetime treatment with constitutive or controlled expression of the respective anti-HIV genes in HIV target cells over the patient’s lifetime Various stages of HIV’s life cycle including pre-entry, preintegration, post-integration, and post-transcription can be targeted by gene therapy to inhibit viral infection As demonstrated in combination antretroviral therapy, the generation of escape mutants can be avoided by combining multiple anti-HIV molecules into a single treatment We therefore combined various highly potent anti-HIV genes functioning at separate stages of the viral life cycle into a single lentiviral vector These genes include a CCR5 siRNA/ miRNA, a human/rhesus macaque chimeric TRIM5α, and a TAR decoy Our anti-HIV strategy particularly focuses on combating the virus at the pre-entry and pre-integration stages in order to minimize any proviral formation using the CCR5 and TRIM5α anti HIV genes As an additional safety mechanism we added the TAR decoy with a post integration anti-HIV action, in the rare case of HIV overcoming the first two lines of defense Here we demonstrate the in vitro efficacy of this combination anti-HIV lentiviral vector in both cultured and primary cells including transduced CD34 hematopoietic progenitor cell (HPC) derived macrophages After transduction and maturation, cells were analyzed for CCR5 cell surface expression by FACS Combination lentiviral vector transgenic cells displayed a substantial knockdown (>95%) of CCR5 expression as compared to nontransduced and control GFP-alone transduced cells Subsequent S32 viral challenge assays were performed with various strains of HIV-1, R5-tropic BaL-1, X4-tropic NL4-3, and dual-tropic 89.6, to determine the levels of viral inhibition Combination lentiviral vector transgenic cells, both cultured and primary cells, displayed potent protection from HIV challenge, as compared to nontransduced and GFP-alone transduced cells Vector titers of the combination construct were consistent with titers of control GFP-alone vector averaging 5x109 TU/ml, indicating the absence of any detrimental effects caused by three inserted transgenes Our data demonstrate the strong inhibition of HIV conferred by this novel anti-HIV lentiviral vector offering the potential for its therapeutic use in clinical applications Future in vivo studies will allow for evaluation of both the safety and efficacy of this combination vector in a preclinical setting 79 Lentiviral Vectors with Leukocyte Integrin CD11b Promoter Leads to Efficient Transduction of Canine Leukocyte Adhesion Deficiency CD34+ Cells Cedar J Fowler,1,2 Michael J Hunter,1 Everette J R Nelson,1 Laura M Tuschong,1 Thomas R Bauer, Jr.,1 Dennis D Hickstein.1 Experimental Transplantation and Immunology, National Cancer Institute, Bethesda, MD; 2HHMI/NIH Research Scholars Program, Howard Hughes Medical Institute, Chevy Chase, MD When compared to retroviral and lentiviral vectors utilizing viral promoter/enhancers, vectors with internal cellular promoters provide the potential to achieve efficient transduction of hematopoietic cells while reducing the risk of genotoxicity The optimal approach would use promoters that are not only tightly regulated, but also specific for the target cell type We tested the SIN lentiviral vector pRRLSIN cPPT.cCD18 with the human leukocyte integrin CD11a or CD11b promoters to transfer canine CD18 cDNA into CD34+ bone marrow cells from dogs with Canine Leukocyte Adhesion Deficiency (CLAD) In CLAD, mutations in CD18 results in the inability to express CD11/ CD18 heterodimers on the cell surface of leukocytes; rescue with CD18 results in CD11/CD18 surface expression The CD11a and CD11b subunits are expressed exclusively on leukocytes; CD11a is expressed primarily on lymphocytes, whereas CD11b is expressed primarily on neutrophils We used a third generation lentivirus vector system with 10 different upstream fragments from the human CD11a and CD11b promoters to express canine CD18 in CLAD CD34+ cells in vitro: the human CD11a promoters ranged from 356 bp to 1.7 kb, and the human CD11b promoters ranged from 388 bp to 1.6 kb All promoters included the transcription start sites Vectors were titered on an LAD EBV B-cell line that lacks endogenous CD18 CLAD CD34+ cells were transduced at two different MOIs (10 and 100) for each vector and analyzed by flow cytometry for CD18 expression days following transduction None of the five human CD11a promoters resulted in greater than 10% CD18+ cells measured days after transduction In contrast, greater than 20 % CD18+ cells were obtained with the 639 bp human CD11b promoter days after transduction This was the highest percentage of any of the CD11b promoter inserts tested These results support the use of SIN lentiviral vectors with the 639 bp human CD11b promoter to express canine CD18 in animals with CLAD Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy ... transduced cells A selective survival advantage of CCR5 shRNA transgenic cells was also observed in a mixed population of cells containing both nontransduced and transduced cells These data represent... Factor, and a TAR Decoy for HIV Gene Therapy Joseph S Anderson,1 John Javien,1 Jan Nolta,1 Gerhard Bauer.1 Internal Medicine/Stem Cell Program, University of CaliforniaDavis, Sacramento, CA HIV continues... effects and the generation of escape mutants can render ART ineffective Hematopoietic stem cell gene therapy for HIV offers a potential alternative to these drugs and can offer the possibility of a