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389. Directed Integration of Insulated Lentiviral Vectors to the Heterochromatin towards Safer Gene Transfer to Stem Cells
RNA VIRUS VECTORS II 387. Engineered Lentiviral Vectors Pseudotyped with a CD4 Receptor and a Fusogenic Protein Can Target Cells Expressing HIV-1 Envelope Proteins Chi-Lin Lee,1 Jason Dang,1 Kye-Il Joo,1 Pin Wang.1 Department of Chemical Engineering, University of Southern California, Los Angeles, CA.
1
Lentiviral vectors (LVs) derived from human immunodeficiency virus type 1 (HIV-1) are promising vehicles for gene delivery because they not only efficiently transduce both dividing and non-dividing cells, but also maintain long-term transgene expression. Development of an LV system capable of transducing cells in a cell type-specific manner can be beneficial for certain applications that rely on targeted gene delivery. Previously it was shown that an inverse fusion strategy that incorporated an HIV-1 receptor (CD4) and its co-receptor (CXCR4 or CCR5) onto vector surfaces could confer to LVs the ability to selectively deliver genes to HIV-1 envelope-expressing cells. To build upon this work, we aim to improve its relatively low transduction efficiency and circumvent its inability to target multiple tropisms of HIV-1 by a single vector. We investigated a method to create LVs co-enveloped with the HIV-1 cellular receptor CD4 and a fusogenic protein derived from the Sindbis virus glycoprotein and tested its efficiency to selectively deliver genes into cells expressing HIV-1 envelope proteins. The engineered LV system yields a higher level of transduction efficiency and a broader tropism towards cells displaying the HIV-1 envelope protein (Env) than the previously developed system. Furthermore, we demonstrated in vitro that this engineered LV can preferentially deliver suicide gene therapy to HIV-1 envelopeexpressing cells. We conclude that it is potentially feasible to target LVs towards HIV-1-infected cells by functional co-incorporation of the CD4 and fusogenic proteins, and provide preliminary evidence for further investigation on a potential alternative treatment for eradicating HIV-1-infected cells that produce drug-resistant viruses after highly active antiretroviral therapy (HAART).
388. A Multi Platform HIV-2 Derived Lentiviral Vector System and Its Potential Use as a Research Tool and for Gene Therapy Development
Ajit G. Chande,1 Hemant R. Dhamne,1 Rohan H. Kamat,1 Santhosh V. Chakkaramakkil,1 Rabindranath Mukhopadhyaya.1 1 Virology, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai, Maharashtra, India. We developed an Indian human immunodeficiency virus type 2 (HIV-2) isolate derived lentiviral vector (LV) with a versatile multiple cloning site (MCS) that also allows direct sub-cloning of a PCR amplified transgene cassette. Efficiency of the vector systems has been validated by development of transgenic cell lines as well as long term expression of transgene in vivo (mouse models) and effective down regulation of target gene by delivery of specific shRNA, both in vitro and in vivo. Multiple user friendly formats have been developed now to expand utility of the LV. These include different antibiotics selection (G-418, puromycin and hygromycin), blue-white colony selection, streptavidin and HA tag for pull down and detection in interactome study, inclusion of additional MCS in LTR (dual MCS format) and use of a novel promoter for sustained transgene expression in vivo. In LV mediated gene transfer only those cells where the LV integrates receive the benefit of transgene expression and fewer number of target cell transduction in vivo is a major limitation. To bypass this limitation we developed a novel format where the product coded by the transgene can be secreted out with a cell penetrating peptide allowing entry to nearby untransduced cells thereby resulting in enhanced biodistribution of LV mediated transgene product. Efficacy of the system was evaluated using two different LV mediated transgene coded proteins, a fluorescent protein and a cytokine. We are also developing novel pseudotyping Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
as an approach to target specific cell types. Towards this we are using viral envelop protein to pseudotype LV for appropriate gene delivery to cells which are natural target for a particular virus and also pseudotype LV with particular carbohydrate binding proteins to target tumour cells over expressing specific interacting sugar moieties. Simultaneous development of appropriate vector formats with novel of target cell specific gene delivery will help in better gene therapy strategy evaluation.
389. Directed Integration of Insulated Lentiviral Vectors to the Heterochromatin towards Safer Gene Transfer to Stem Cells
Alexandre Artus,1 Caroline Duros,1 Yaïr Botbol,2 Simone Scholz,3 Manfred Schmidt,3 Christof von Kalle,3 Marc Lavigne,2 Odile Y. D. Cohen-Haguenauer,1 Odile Y. D. Cohen-Haguenauer.4 1 LBPA and CLINIGENE, Ecole Normale Superieure, Cachan, France; 2Laboratoire Joliot Curie, ENS de Lyon, Lyon, France; 3Laboratory of Translational Oncology, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany; 4Medical Oncology and Oncogenetics, Hopital Saint-Louis and University Paris-Diderot, Paris, France. We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing integration to heterochromatin. We have previously identify a specific GIE combination in collaboration with Nic Mermod, which translates into high titers and boundary effect in both gammaretro and lentivectors (DCaro4). In target cells, the expression profile becomes homogenous; its level is strictly conditioned by the promoter. These data remain stable in both HeLa cells and cord blood HSCs for over three months, irrespective of the multiplicity of infection (MOI). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, 4xIns2-DCaro4 has been further tested and compared to its non-insulated counterpart, with chimeric HIV-1 derived integrases targeting heterochromatin through either histone H3 or methylated CpG islands (ML6 & ML10 chimeras, respectively). With DCaro4 and ML6 chimeras, a homogeneous expression is sustained over time. With the control, GFP expression is just over background double-mutant in catalytic and ledgf binding-sites while expression can be induced with HDAC. In CD34+ cells from cord-blood, these data are partially recapitulated with the ML6 chimera and entirely with the ML14, which includes an additional mutation translating in an enhanced specificity by one order of magnitude. High throughput integration sites analysis reveals a distinct profile with histone H3 targeting chimeras. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent risk for insertional mutagenesis through undesired enhancer-activation, in combination with a potent and stable genetic insulation, regardless of the integration site.
390.
Retroviral Delivery of Episomal Vectors
Julie Lemay,1 Claudia Hagedorn,2 Elena Kuzmenko,1 HansJoachim Lipps,2 Reinhard Seger,1 Janine Reichenbach,1 Ulrich Siler.1 1 Division of Immunology/Hematology/BMT, PRC, University Children’s Hospital Zürich, Zürich, Switzerland; 2ZBAF, Institute of Cell Biology, University Witten/Herdecke, Witten, Germany. Recent clinical successes in human retroviral gene therapy were accompanied by unexpected side effects due to transactivation events. One approach to mediate transgene expression without the risk of transactivation is the expression from episomally maintained plasmids. As chemical transfection of hematopoietic stem cells is inefficient, we developed a new strategy for episomal vector delivery, using non-integrating retroviral vectors. We incorporated various S151
1
Lentiviral vectors (LVs) derived from human immunodeficiency virus type 1 (HIV-1) are promising vehicles for gene delivery because they not only efficiently transduce both dividing and non-dividing cells, but also maintain long-term transgene expression. Development of an LV system capable of transducing cells in a cell type-specific manner can be beneficial for certain applications that rely on targeted gene delivery. Previously it was shown that an inverse fusion strategy that incorporated an HIV-1 receptor (CD4) and its co-receptor (CXCR4 or CCR5) onto vector surfaces could confer to LVs the ability to selectively deliver genes to HIV-1 envelope-expressing cells. To build upon this work, we aim to improve its relatively low transduction efficiency and circumvent its inability to target multiple tropisms of HIV-1 by a single vector. We investigated a method to create LVs co-enveloped with the HIV-1 cellular receptor CD4 and a fusogenic protein derived from the Sindbis virus glycoprotein and tested its efficiency to selectively deliver genes into cells expressing HIV-1 envelope proteins. The engineered LV system yields a higher level of transduction efficiency and a broader tropism towards cells displaying the HIV-1 envelope protein (Env) than the previously developed system. Furthermore, we demonstrated in vitro that this engineered LV can preferentially deliver suicide gene therapy to HIV-1 envelopeexpressing cells. We conclude that it is potentially feasible to target LVs towards HIV-1-infected cells by functional co-incorporation of the CD4 and fusogenic proteins, and provide preliminary evidence for further investigation on a potential alternative treatment for eradicating HIV-1-infected cells that produce drug-resistant viruses after highly active antiretroviral therapy (HAART).
388. A Multi Platform HIV-2 Derived Lentiviral Vector System and Its Potential Use as a Research Tool and for Gene Therapy Development
Ajit G. Chande,1 Hemant R. Dhamne,1 Rohan H. Kamat,1 Santhosh V. Chakkaramakkil,1 Rabindranath Mukhopadhyaya.1 1 Virology, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai, Maharashtra, India. We developed an Indian human immunodeficiency virus type 2 (HIV-2) isolate derived lentiviral vector (LV) with a versatile multiple cloning site (MCS) that also allows direct sub-cloning of a PCR amplified transgene cassette. Efficiency of the vector systems has been validated by development of transgenic cell lines as well as long term expression of transgene in vivo (mouse models) and effective down regulation of target gene by delivery of specific shRNA, both in vitro and in vivo. Multiple user friendly formats have been developed now to expand utility of the LV. These include different antibiotics selection (G-418, puromycin and hygromycin), blue-white colony selection, streptavidin and HA tag for pull down and detection in interactome study, inclusion of additional MCS in LTR (dual MCS format) and use of a novel promoter for sustained transgene expression in vivo. In LV mediated gene transfer only those cells where the LV integrates receive the benefit of transgene expression and fewer number of target cell transduction in vivo is a major limitation. To bypass this limitation we developed a novel format where the product coded by the transgene can be secreted out with a cell penetrating peptide allowing entry to nearby untransduced cells thereby resulting in enhanced biodistribution of LV mediated transgene product. Efficacy of the system was evaluated using two different LV mediated transgene coded proteins, a fluorescent protein and a cytokine. We are also developing novel pseudotyping Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
as an approach to target specific cell types. Towards this we are using viral envelop protein to pseudotype LV for appropriate gene delivery to cells which are natural target for a particular virus and also pseudotype LV with particular carbohydrate binding proteins to target tumour cells over expressing specific interacting sugar moieties. Simultaneous development of appropriate vector formats with novel of target cell specific gene delivery will help in better gene therapy strategy evaluation.
389. Directed Integration of Insulated Lentiviral Vectors to the Heterochromatin towards Safer Gene Transfer to Stem Cells
Alexandre Artus,1 Caroline Duros,1 Yaïr Botbol,2 Simone Scholz,3 Manfred Schmidt,3 Christof von Kalle,3 Marc Lavigne,2 Odile Y. D. Cohen-Haguenauer,1 Odile Y. D. Cohen-Haguenauer.4 1 LBPA and CLINIGENE, Ecole Normale Superieure, Cachan, France; 2Laboratoire Joliot Curie, ENS de Lyon, Lyon, France; 3Laboratory of Translational Oncology, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany; 4Medical Oncology and Oncogenetics, Hopital Saint-Louis and University Paris-Diderot, Paris, France. We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing integration to heterochromatin. We have previously identify a specific GIE combination in collaboration with Nic Mermod, which translates into high titers and boundary effect in both gammaretro and lentivectors (DCaro4). In target cells, the expression profile becomes homogenous; its level is strictly conditioned by the promoter. These data remain stable in both HeLa cells and cord blood HSCs for over three months, irrespective of the multiplicity of infection (MOI). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, 4xIns2-DCaro4 has been further tested and compared to its non-insulated counterpart, with chimeric HIV-1 derived integrases targeting heterochromatin through either histone H3 or methylated CpG islands (ML6 & ML10 chimeras, respectively). With DCaro4 and ML6 chimeras, a homogeneous expression is sustained over time. With the control, GFP expression is just over background double-mutant in catalytic and ledgf binding-sites while expression can be induced with HDAC. In CD34+ cells from cord-blood, these data are partially recapitulated with the ML6 chimera and entirely with the ML14, which includes an additional mutation translating in an enhanced specificity by one order of magnitude. High throughput integration sites analysis reveals a distinct profile with histone H3 targeting chimeras. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent risk for insertional mutagenesis through undesired enhancer-activation, in combination with a potent and stable genetic insulation, regardless of the integration site.
390.
Retroviral Delivery of Episomal Vectors
Julie Lemay,1 Claudia Hagedorn,2 Elena Kuzmenko,1 HansJoachim Lipps,2 Reinhard Seger,1 Janine Reichenbach,1 Ulrich Siler.1 1 Division of Immunology/Hematology/BMT, PRC, University Children’s Hospital Zürich, Zürich, Switzerland; 2ZBAF, Institute of Cell Biology, University Witten/Herdecke, Witten, Germany. Recent clinical successes in human retroviral gene therapy were accompanied by unexpected side effects due to transactivation events. One approach to mediate transgene expression without the risk of transactivation is the expression from episomally maintained plasmids. As chemical transfection of hematopoietic stem cells is inefficient, we developed a new strategy for episomal vector delivery, using non-integrating retroviral vectors. We incorporated various S151