- Email: [email protected]
266. Decade-Long Persistence of Adoptively Transferred Autologous Redirected CD4ζ Chimeric Receptor T Cells in HIV+ Study Subjects
HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I achieve long-term multi-lineage engraftment using transduced patient specific thalassemic G-CSF mobilized CD34+ cells expressing human beta-globin. These studies provide the basis for the upcoming phase I clinical trial of β-thalassemia major using LV transduced CD34+ cells encoding beta-globin.
264. Uncovering Haemopoietic System Dynamics and Tracking Fate and Long-Term Survival of Single Progenitor Clones In Vivo in Humans by Retroviral Tagging
Luca Biasco,1 Cristina Baricordi,2 Cynthia Bartholomae,3 Immacolata Brigida,1 Samantha Scaramuzza,1 Raffaele Fronza,4 Stefania Merella,1 Alessandro Ambrosi,5 Danilo Pellina,5 Clelia Di Serio,2,5 Eugenio Montini,1 Christof von Kalle,4 Manfred Schmidt,4 Alessandro Aiuti.1,6 1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; 2Vita-Salute University San Raffaele, Milan, Italy; 3 National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany; 4Alma Mater Studiorum University, Bologna, Italy; 5CUSSB Vita-Salute University, Milan, Italy; 6University of Rome “Tor Vergata”, Rome, Italy. Upon retroviral mediated gene correction of haemopoetic stem cells (HSC) each transduced progenitor is univocally marked by an integration site. Long-term studies in gene therapy (GT) patients allow for the first time to track haemopoietic clonal dynamics and survival of single progenitor clones in humans by retroviral tagging. We previously showed that ADA-SCID GT with CD34+ cells combined to reduced intensity conditioning resulted in full multilineage engraftment, in the absence of aberrant expansions. We now performed a comprehensive multilineage longitudinal insertion profile of bone marrow (BM) (CD34+, CD15+, CD19+, Glycophorin+) and peripheral blood (PB) (CD15+, CD19+, CD4+, CD8+ cells, naïve and memory T cells) cells in 4 patients 3-6 years after GT, retrieving to date 1055 and 1999 unique insertions from BM and PB cell lineages respectively. We could shape the “insertional landscape” of each lineage through a tri-factorial analysis based on insertional variables allowing to uncover the effects of selective advantages of gene-corrected cells in the periphery and the frequency of identical integrants in different haematopoietic compartments. BM cells and PB granulocytes displayed the highest proportion of shared integrants (up to 58.1%), reflecting the real-time repopulating activity of genecorrected progenitors. Strikingly, we detected “core integrants”, shared between CD34+ cells and both lymphoid and myeloid lineages at multiple time points, stably tagging active long-term multipotent progenitors overtime. Tracking two of these integrants (inside a fragile site of MLLT3 gene and downstream the LRRC30 gene) by specific PCRs we confirmed the multilineage contribution to haematopoiesis of these progenitor clones, showing fluctuating lineage outputs over a period of 5 years in the patient. We also retrieved 170 integrations from 4 T cell subtypes (Naive, TEMRA, Central and Effector memory) from one patient who received infusions of transduced PBL showing evidences that naive T cell clones may survive in humans for up to 10 years while maintaining their differentiation capacity into different T cell subpopulations. Additional analyses on transcriptional and chromatin status of integration sites collected from all lineages are ongoing. In conclusion, through retroviral tagging, we can uniquely track single transduced haemopoietic cells directly in vivo in humans. The application of mathematical models to our insertion datasets is allowing to uncover new information on the fate and activity of haematopoietic progenitors and their differentiated progeny years after transplantation in GT patients.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
265. Deep Sequencing Analyses of Small RNAs in HIV-1 Infected Cells Transduced with a Triple Inhibitory RNA Lentiviral Vector Haitang Li,1 John Rossi.1 Molecular and Cell Biology, City of Hope, Duarte, CA.
1
In an effort to develop a hematopoietic gene therapy approach to treat HIV infection, we created a lentiviral vector containing three different inhibitory agents including a short hairpin RNA (shRNA) targeting the HIV tat/rev region, a nucleolar localizing Tat binding decoy and a ribozyme against the endogenous CCR5 chemokine receptor mRNA. This lentiviral construct, has proven to be highly effective in inhibiting HIV replication in cell culture and is currently in use in gene therapy trial for AIDS/lymphoma patients. The potential deregulation of miRNA expression by ectopic expression of a short hairpin RNA or HIV-1 infection has been a matter of concern for gene therapy applications of RNAi. We therefore took advantage of next generation sequencing to monitor endogenous microRNA changes during the time course of HIV-1 infection in CEM T-cells transduced with the triple construct vector in the presence and absence of HIV-1 infection. Consistent with the idea that HIV replication was effetely inhibited, microRNAs generated from HIV-1 TAR and Nef were greatly reduced in triple construct treated CEM cells relative to vector backbone alone transduced cells, HIV-1 infected cells. Interestingly, the level of several endogenous microRNAs changed dramatically at different time points in the face of HIV-1 challenge, but the changes were less dramatic in cells expressing the anti-tat/rev shRNA. We also analyzed the ratios of the anti-HIV tat/rev guide strand versus the passenger strand over time in the triple construct transduced cells infected with HIV versus the uninfected triple construct transduced cells. Our results show that the guide strand/passenger strand ratio increases dramatically in HIV-1 infected cells whereas it remains stable in uninfected vector transduced cells. These results support a model in which engagement of an siRNA guide strand via targeting of the HIV transcripts leads to its stabilization. Finally, we analyzed the microRNA profile from a patient in the clinical trial at two time points-one pre-transduction and the other one year post transduction and post autologous stem cell transplant. The results of these analyses show marked changes in the miRNA profile as well, most likely a consequence of the stem cell transplant. We also see the HIV-1 TAR miRNA pre-transplant but it is absent post-transplant/transduction. In summary, our results provide the first in depth look at the microRNA profiles of HIV-1 infected cells in the presence of a protective gene therapy vector.
266. Decade-Long Persistence of Adoptively Transferred Autologous Redirected CD4ζ Chimeric Receptor T Cells in HIV+ Study Subjects
John Scholler,1 Naomi Aaronson,2 Gabriela Plesa,1 Richard Carroll,1 Gwendolyn Binder,1 Kristen Hege,3 Steven Deeks,4 Ronald Mitsuyasu,5 Ashley Vogel,1 Zhaohui Zheng,1 Julio Cotte,1 Wendy Bernstein,6 Troy Brady,1 Bruce Levine,1 Carl June.1 1 University of Pennsylvania, Philadelphia, PA; 2Walter Reed Army Medical Center, Washington, DC; 3Cell Genesys, South San Francisco; 4University of California, San Francisco; 5University of California, Los Angeles; 6National Cancer Institute, Bethesda. The ability of adoptively transferred T cells to persist long term and function in a homologous or enhanced fashion is a central goal of immunotherapy. To test the hypothesis that genetically modified T cells can have long term persistence, we measured the frequency of CD4ζ modified T cells in patients with HIV in three trials following adoptive transfer of CD4 and CD8 T cells transduced with the CD4ζ chimeric receptor over time. We developed a quantitative PCR (qPCR) assay to measure the persistence of adoptively transferred T lymphocytes transduced with a chimeric receptor consisting of S103
HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I extracellular CD4 linked to intracellular CD3-ζ (“CD4ζ” chimeric receptor). PBMC’s from a series of clinical trials in which HIV+ study subjects received autologous CD4ζ gene modified T cells from up to 11 years post-infusion were available for this long term persistence analysis. The data from three clinical trials demonstrated that 37 of 39 subjects continue to have CD4ζ modified cells detectable in PBMC, up to 11 years post infusion. While the majority of patients had an average CD4z copy level in their PBMC populations of 0.01 to 0.1%, a considerable proportion had levels exceeding 0.1% PBMC (i.e. ∼0.5% CD4 cells) and at relatively stable engraftment levels over time. The calculated half-life for the persistence of these cells in the three studies ranged from 13.8 to 28 years. RT-PCR analysis of RNA isolated from viably frozen cells representing 13 patients at 5 to 10 years post infusion, indicates that the CD4z gene is transcriptionally active throughout the period of CD4z gDNA detection. To determine the polyclonality in these long term persistent populations, 3 to 5 samples selected from five patients showing unique engraftment profiles between the 1st to the 10th anniversary after infusion were deep sequenced for integration site diversity. There are no clinical indications of delayed adverse events relating to the gene therapy in any of the patients. The data suggests that adoptively transferred retrovirally-modified T cells can have long term survival at levels equivalent to or exceeding levels of central memory T cells for at least 10 years.
267. Development of a New Strategy for X-CGD Gene Therapy Using a miRNA Regulated Lentiviral Vector
Maria Chiriaco,1 Giada Farinelli,1 Bernhard Gentner,2 Gigliola Di Matteo,1 Valentina Capo,1 Silvia Di Cesare,1 Samantha Scaramuzza,2 Lucia Sergi Sergi,2 Antonella Isgrò,3 Didier Trono,4 Manuel Grez,5 Andrea Finocchi,1 Paolo Rossi,1 Luigi Naldini,2,6 Alessandro Aiuti.1,2 1 Dep. of Pediatrics, Children’s Hospital Bambino Gesù and Univ. of Rome Tor Vergata, Rome, Italy; 2San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Scientific Institute HS Raffaele, Milan, Italy; 3Mediterranean Inst. of Hematology, Policlinico Tor Vergata, Rome, Italy; 4École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; 5Georg-Speyer Research Institute, Frankfurt, Germany; 6”Vita-Salute” S.Raffaele University, Milan, Italy. Chronic Granulomatous Disease (CGD) is caused by defective NADPH oxidase function in patients’ phagocytes leading to increased susceptibility to fungal and bacterial infections. Gene therapy with hematopoietic stem cells (HSC) may represent a valid alternative for gp91phox deficient patients (X-CGD). Previous clinical trials with gamma-retroviral vectors have achieved short-term benefit, which was hampered by low engraftment levels of gene modified cells, transgene silencing, and the occurrence of clonal dominance. Our goal was to develop a novel gene transfer approach into HSC for safe and efficacious therapy of X-CGD based on third generation SIN lentiviral vectors (LVVs) encoding gp91phox driven by an an internal cellular promoter (LVV-PGK.gp91). To reduce the potential risk of ectopic gp91phox expression in HSC, we introduced miR-126 target sequences (miR-126T) into the gp91phox expression cassette (LVV-PGKgp91_126T) to obtain post-transcriptional downregulation of transgene expression by miR-126 which is found to high levels in HSC and early progenitors but not in the myeloid progeny. Transduced myeloid (PLB985) and EBV-B cell lines defective for gp91phox showed effective gp91phox expression and restoration of a functional NADPH oxidase complex. After superinfection with a miR-126 expressing LVV, LVV-PGKgp91_126T-transduced gp91phox-deficient cells showed dramatically reduced gp91phox expression compared to LVV-PGKgp91-transduced cells, indicating specific regulation of gp91phox expression by miR-126. Transduction S104
of Lin-negative cells from X-CGD mice resulted in restoration of gp91 transgene expression and NAPDH activity after differentiation. We next investigated the effect of miR-126 mediated post-transcriptional regulation in healthy donor CD34+ cells. After short-term culture, gp91phox was detected ectopically in cells expressing CD34+ which were transduced with the LVV.PGK.gp91. Gp91phox ectopic expression in CD34+ expression was substantially reduced when cells were transduced with the LVV-PGKgp91_126T or a LVV vector encoding gp91phox driven by a synthetic myeloid specific promoter, but was similar in CD34-negative cells. Moreover, gene transfer into human CD34+ cells from XCGD patients with both LVV-PGKgp91 vectors resulted in expression of gp91phox in 30% of cells and restored NAPDH activity after myeloid differentiation. In summary these results indicate that that SIN-LVVs containing internal, cellular promoters represent an efficient approach for X-CGD gene therapy, and miRNA regulation can overcome potentially harmful, ectopic transgene expression in HSC.
268. Correction of Mouse Prkdc scid Deficiency by ZFN-Driven Gene Repair
Hayder H. Abdul-Razak,1 Céline Rocca,1 Francisco Javier Molina Estévez,2 Guillermo Güenechea,2 Steven J. Howe,3 Chih Hao V. Gan,3 Alison Roberts,4 Michael C. Holmes,5 Philip D. Gregory,5 Adrian J. Thrasher,3 Juan A. Bueren,2 Rafael J. Yáñez-Muñoz.1 1 School of Biological Sciences, Royal Holloway-University of London, Egham, Surrey, United Kingdom; 2Division of Hematopoiesis and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales, y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spa; 3UCL Institute of Child Health, University College London, London, United Kingdom; 4Medical and Molecular Genetics, King’s College London School of Medicine, London, United Kingdom; 5Sangamo BioSciences, Inc, Richmond, CA.
Recent reports have described successful correction of several diseases by ex vivo retroviral vector-mediated gene addition in hematopoietic stem cells (HSCs) followed by autologous transplantation. Unfortunately, in X-linked SCID (severe combined immunodeficiency), X-CGD (chronic granulomatous disease), WAS (Wiskott-Aldrich syndrome) and β-thalassemia the treatment has led to some insertional mutagenesis-related serious adverse events. For this reason and others, an improved way to treat both dominant and recessive genetic disorders would be to repair the mutant gene by homologous recombination-mediated gene targeting. To demonstrate therapeutic gene repair in a pre-clinical model of immunodeficiency we are developing an ex vivo system to correct the classical scid mouse, a model of human DNA-dependent protein kinase catalytic subunit (PRKDC) deficiency. We have obtained and optimised a zincfinger nuclease (ZFN) targeting mouse Prkdc in the vicinity of the scid mutation, produced targeting constructs to correct the mutation, and incorporated ZFN genes and targeting constructs into integrationdeficient lentiviral vectors (IDLVs). We have demonstrated specific ZFN activity in scid fibroblasts using the Surveyor assay, which detects modifications introduced at the target site upon repair by nonhomologous end-joining of the ZFN-induced DNA double strand break. Importantly, we have also demonstrated ZFN-mediated gene targeting of the scid mutation in fibroblasts through the incorporation of a diagnostic restriction site from the targeting construct into the targeted locus. We are currently conducting preliminary experiments of ZFN cutting and Prkdc gene correction in scid HSCs using optimised IDLV-mediated transduction.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
264. Uncovering Haemopoietic System Dynamics and Tracking Fate and Long-Term Survival of Single Progenitor Clones In Vivo in Humans by Retroviral Tagging
Luca Biasco,1 Cristina Baricordi,2 Cynthia Bartholomae,3 Immacolata Brigida,1 Samantha Scaramuzza,1 Raffaele Fronza,4 Stefania Merella,1 Alessandro Ambrosi,5 Danilo Pellina,5 Clelia Di Serio,2,5 Eugenio Montini,1 Christof von Kalle,4 Manfred Schmidt,4 Alessandro Aiuti.1,6 1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; 2Vita-Salute University San Raffaele, Milan, Italy; 3 National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany; 4Alma Mater Studiorum University, Bologna, Italy; 5CUSSB Vita-Salute University, Milan, Italy; 6University of Rome “Tor Vergata”, Rome, Italy. Upon retroviral mediated gene correction of haemopoetic stem cells (HSC) each transduced progenitor is univocally marked by an integration site. Long-term studies in gene therapy (GT) patients allow for the first time to track haemopoietic clonal dynamics and survival of single progenitor clones in humans by retroviral tagging. We previously showed that ADA-SCID GT with CD34+ cells combined to reduced intensity conditioning resulted in full multilineage engraftment, in the absence of aberrant expansions. We now performed a comprehensive multilineage longitudinal insertion profile of bone marrow (BM) (CD34+, CD15+, CD19+, Glycophorin+) and peripheral blood (PB) (CD15+, CD19+, CD4+, CD8+ cells, naïve and memory T cells) cells in 4 patients 3-6 years after GT, retrieving to date 1055 and 1999 unique insertions from BM and PB cell lineages respectively. We could shape the “insertional landscape” of each lineage through a tri-factorial analysis based on insertional variables allowing to uncover the effects of selective advantages of gene-corrected cells in the periphery and the frequency of identical integrants in different haematopoietic compartments. BM cells and PB granulocytes displayed the highest proportion of shared integrants (up to 58.1%), reflecting the real-time repopulating activity of genecorrected progenitors. Strikingly, we detected “core integrants”, shared between CD34+ cells and both lymphoid and myeloid lineages at multiple time points, stably tagging active long-term multipotent progenitors overtime. Tracking two of these integrants (inside a fragile site of MLLT3 gene and downstream the LRRC30 gene) by specific PCRs we confirmed the multilineage contribution to haematopoiesis of these progenitor clones, showing fluctuating lineage outputs over a period of 5 years in the patient. We also retrieved 170 integrations from 4 T cell subtypes (Naive, TEMRA, Central and Effector memory) from one patient who received infusions of transduced PBL showing evidences that naive T cell clones may survive in humans for up to 10 years while maintaining their differentiation capacity into different T cell subpopulations. Additional analyses on transcriptional and chromatin status of integration sites collected from all lineages are ongoing. In conclusion, through retroviral tagging, we can uniquely track single transduced haemopoietic cells directly in vivo in humans. The application of mathematical models to our insertion datasets is allowing to uncover new information on the fate and activity of haematopoietic progenitors and their differentiated progeny years after transplantation in GT patients.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
265. Deep Sequencing Analyses of Small RNAs in HIV-1 Infected Cells Transduced with a Triple Inhibitory RNA Lentiviral Vector Haitang Li,1 John Rossi.1 Molecular and Cell Biology, City of Hope, Duarte, CA.
1
In an effort to develop a hematopoietic gene therapy approach to treat HIV infection, we created a lentiviral vector containing three different inhibitory agents including a short hairpin RNA (shRNA) targeting the HIV tat/rev region, a nucleolar localizing Tat binding decoy and a ribozyme against the endogenous CCR5 chemokine receptor mRNA. This lentiviral construct, has proven to be highly effective in inhibiting HIV replication in cell culture and is currently in use in gene therapy trial for AIDS/lymphoma patients. The potential deregulation of miRNA expression by ectopic expression of a short hairpin RNA or HIV-1 infection has been a matter of concern for gene therapy applications of RNAi. We therefore took advantage of next generation sequencing to monitor endogenous microRNA changes during the time course of HIV-1 infection in CEM T-cells transduced with the triple construct vector in the presence and absence of HIV-1 infection. Consistent with the idea that HIV replication was effetely inhibited, microRNAs generated from HIV-1 TAR and Nef were greatly reduced in triple construct treated CEM cells relative to vector backbone alone transduced cells, HIV-1 infected cells. Interestingly, the level of several endogenous microRNAs changed dramatically at different time points in the face of HIV-1 challenge, but the changes were less dramatic in cells expressing the anti-tat/rev shRNA. We also analyzed the ratios of the anti-HIV tat/rev guide strand versus the passenger strand over time in the triple construct transduced cells infected with HIV versus the uninfected triple construct transduced cells. Our results show that the guide strand/passenger strand ratio increases dramatically in HIV-1 infected cells whereas it remains stable in uninfected vector transduced cells. These results support a model in which engagement of an siRNA guide strand via targeting of the HIV transcripts leads to its stabilization. Finally, we analyzed the microRNA profile from a patient in the clinical trial at two time points-one pre-transduction and the other one year post transduction and post autologous stem cell transplant. The results of these analyses show marked changes in the miRNA profile as well, most likely a consequence of the stem cell transplant. We also see the HIV-1 TAR miRNA pre-transplant but it is absent post-transplant/transduction. In summary, our results provide the first in depth look at the microRNA profiles of HIV-1 infected cells in the presence of a protective gene therapy vector.
266. Decade-Long Persistence of Adoptively Transferred Autologous Redirected CD4ζ Chimeric Receptor T Cells in HIV+ Study Subjects
John Scholler,1 Naomi Aaronson,2 Gabriela Plesa,1 Richard Carroll,1 Gwendolyn Binder,1 Kristen Hege,3 Steven Deeks,4 Ronald Mitsuyasu,5 Ashley Vogel,1 Zhaohui Zheng,1 Julio Cotte,1 Wendy Bernstein,6 Troy Brady,1 Bruce Levine,1 Carl June.1 1 University of Pennsylvania, Philadelphia, PA; 2Walter Reed Army Medical Center, Washington, DC; 3Cell Genesys, South San Francisco; 4University of California, San Francisco; 5University of California, Los Angeles; 6National Cancer Institute, Bethesda. The ability of adoptively transferred T cells to persist long term and function in a homologous or enhanced fashion is a central goal of immunotherapy. To test the hypothesis that genetically modified T cells can have long term persistence, we measured the frequency of CD4ζ modified T cells in patients with HIV in three trials following adoptive transfer of CD4 and CD8 T cells transduced with the CD4ζ chimeric receptor over time. We developed a quantitative PCR (qPCR) assay to measure the persistence of adoptively transferred T lymphocytes transduced with a chimeric receptor consisting of S103
HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I extracellular CD4 linked to intracellular CD3-ζ (“CD4ζ” chimeric receptor). PBMC’s from a series of clinical trials in which HIV+ study subjects received autologous CD4ζ gene modified T cells from up to 11 years post-infusion were available for this long term persistence analysis. The data from three clinical trials demonstrated that 37 of 39 subjects continue to have CD4ζ modified cells detectable in PBMC, up to 11 years post infusion. While the majority of patients had an average CD4z copy level in their PBMC populations of 0.01 to 0.1%, a considerable proportion had levels exceeding 0.1% PBMC (i.e. ∼0.5% CD4 cells) and at relatively stable engraftment levels over time. The calculated half-life for the persistence of these cells in the three studies ranged from 13.8 to 28 years. RT-PCR analysis of RNA isolated from viably frozen cells representing 13 patients at 5 to 10 years post infusion, indicates that the CD4z gene is transcriptionally active throughout the period of CD4z gDNA detection. To determine the polyclonality in these long term persistent populations, 3 to 5 samples selected from five patients showing unique engraftment profiles between the 1st to the 10th anniversary after infusion were deep sequenced for integration site diversity. There are no clinical indications of delayed adverse events relating to the gene therapy in any of the patients. The data suggests that adoptively transferred retrovirally-modified T cells can have long term survival at levels equivalent to or exceeding levels of central memory T cells for at least 10 years.
267. Development of a New Strategy for X-CGD Gene Therapy Using a miRNA Regulated Lentiviral Vector
Maria Chiriaco,1 Giada Farinelli,1 Bernhard Gentner,2 Gigliola Di Matteo,1 Valentina Capo,1 Silvia Di Cesare,1 Samantha Scaramuzza,2 Lucia Sergi Sergi,2 Antonella Isgrò,3 Didier Trono,4 Manuel Grez,5 Andrea Finocchi,1 Paolo Rossi,1 Luigi Naldini,2,6 Alessandro Aiuti.1,2 1 Dep. of Pediatrics, Children’s Hospital Bambino Gesù and Univ. of Rome Tor Vergata, Rome, Italy; 2San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Scientific Institute HS Raffaele, Milan, Italy; 3Mediterranean Inst. of Hematology, Policlinico Tor Vergata, Rome, Italy; 4École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; 5Georg-Speyer Research Institute, Frankfurt, Germany; 6”Vita-Salute” S.Raffaele University, Milan, Italy. Chronic Granulomatous Disease (CGD) is caused by defective NADPH oxidase function in patients’ phagocytes leading to increased susceptibility to fungal and bacterial infections. Gene therapy with hematopoietic stem cells (HSC) may represent a valid alternative for gp91phox deficient patients (X-CGD). Previous clinical trials with gamma-retroviral vectors have achieved short-term benefit, which was hampered by low engraftment levels of gene modified cells, transgene silencing, and the occurrence of clonal dominance. Our goal was to develop a novel gene transfer approach into HSC for safe and efficacious therapy of X-CGD based on third generation SIN lentiviral vectors (LVVs) encoding gp91phox driven by an an internal cellular promoter (LVV-PGK.gp91). To reduce the potential risk of ectopic gp91phox expression in HSC, we introduced miR-126 target sequences (miR-126T) into the gp91phox expression cassette (LVV-PGKgp91_126T) to obtain post-transcriptional downregulation of transgene expression by miR-126 which is found to high levels in HSC and early progenitors but not in the myeloid progeny. Transduced myeloid (PLB985) and EBV-B cell lines defective for gp91phox showed effective gp91phox expression and restoration of a functional NADPH oxidase complex. After superinfection with a miR-126 expressing LVV, LVV-PGKgp91_126T-transduced gp91phox-deficient cells showed dramatically reduced gp91phox expression compared to LVV-PGKgp91-transduced cells, indicating specific regulation of gp91phox expression by miR-126. Transduction S104
of Lin-negative cells from X-CGD mice resulted in restoration of gp91 transgene expression and NAPDH activity after differentiation. We next investigated the effect of miR-126 mediated post-transcriptional regulation in healthy donor CD34+ cells. After short-term culture, gp91phox was detected ectopically in cells expressing CD34+ which were transduced with the LVV.PGK.gp91. Gp91phox ectopic expression in CD34+ expression was substantially reduced when cells were transduced with the LVV-PGKgp91_126T or a LVV vector encoding gp91phox driven by a synthetic myeloid specific promoter, but was similar in CD34-negative cells. Moreover, gene transfer into human CD34+ cells from XCGD patients with both LVV-PGKgp91 vectors resulted in expression of gp91phox in 30% of cells and restored NAPDH activity after myeloid differentiation. In summary these results indicate that that SIN-LVVs containing internal, cellular promoters represent an efficient approach for X-CGD gene therapy, and miRNA regulation can overcome potentially harmful, ectopic transgene expression in HSC.
268. Correction of Mouse Prkdc scid Deficiency by ZFN-Driven Gene Repair
Hayder H. Abdul-Razak,1 Céline Rocca,1 Francisco Javier Molina Estévez,2 Guillermo Güenechea,2 Steven J. Howe,3 Chih Hao V. Gan,3 Alison Roberts,4 Michael C. Holmes,5 Philip D. Gregory,5 Adrian J. Thrasher,3 Juan A. Bueren,2 Rafael J. Yáñez-Muñoz.1 1 School of Biological Sciences, Royal Holloway-University of London, Egham, Surrey, United Kingdom; 2Division of Hematopoiesis and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales, y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spa; 3UCL Institute of Child Health, University College London, London, United Kingdom; 4Medical and Molecular Genetics, King’s College London School of Medicine, London, United Kingdom; 5Sangamo BioSciences, Inc, Richmond, CA.
Recent reports have described successful correction of several diseases by ex vivo retroviral vector-mediated gene addition in hematopoietic stem cells (HSCs) followed by autologous transplantation. Unfortunately, in X-linked SCID (severe combined immunodeficiency), X-CGD (chronic granulomatous disease), WAS (Wiskott-Aldrich syndrome) and β-thalassemia the treatment has led to some insertional mutagenesis-related serious adverse events. For this reason and others, an improved way to treat both dominant and recessive genetic disorders would be to repair the mutant gene by homologous recombination-mediated gene targeting. To demonstrate therapeutic gene repair in a pre-clinical model of immunodeficiency we are developing an ex vivo system to correct the classical scid mouse, a model of human DNA-dependent protein kinase catalytic subunit (PRKDC) deficiency. We have obtained and optimised a zincfinger nuclease (ZFN) targeting mouse Prkdc in the vicinity of the scid mutation, produced targeting constructs to correct the mutation, and incorporated ZFN genes and targeting constructs into integrationdeficient lentiviral vectors (IDLVs). We have demonstrated specific ZFN activity in scid fibroblasts using the Surveyor assay, which detects modifications introduced at the target site upon repair by nonhomologous end-joining of the ZFN-induced DNA double strand break. Importantly, we have also demonstrated ZFN-mediated gene targeting of the scid mutation in fibroblasts through the incorporation of a diagnostic restriction site from the targeting construct into the targeted locus. We are currently conducting preliminary experiments of ZFN cutting and Prkdc gene correction in scid HSCs using optimised IDLV-mediated transduction.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy