63 results on '"Hollis RP"'
Search Results
2. A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors
- Author
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De Oliveira, SN, Wang, J, Ryan, C, Morrison, SL, Kohn, DB, and Hollis, RP
- Abstract
Background: Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. CARs displayed on the surface of transduced cells perform non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of mature T-lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B-lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models.Methods: A specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T-cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The CD19sIg proteins were produced from 293 T cells by stable lentiviral vector transduction and purification from culture medium.Results: ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining.Conclusions: This fusion molecule is a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells. © 2013 De Oliveira et al.; licensee BioMed Central Ltd.
- Published
- 2013
3. Lentiviral vectors with amplified β cell-specific gene expression
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Shaw, KL, Pais, E, Ge, S, Hardee, C, Skelton, D, Hollis, RP, Crooks, GM, and Kohn, DB
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Medical Biotechnology ,Biochemistry and Cell Biology ,Biomedical and Clinical Sciences ,Biological Sciences ,Rare Diseases ,Genetics ,Biotechnology ,Diabetes ,Gene Therapy ,5.2 Cellular and gene therapies ,Development of treatments and therapeutic interventions ,Animals ,Cell Line ,Gene Expression ,Gene Transfer Techniques ,Genetic Vectors ,Green Fluorescent Proteins ,Humans ,Insulin ,Insulin-Secreting Cells ,Islets of Langerhans ,Lentivirus ,Mice ,Organ Specificity ,Phosphoglycerate Kinase ,Promoter Regions ,Genetic ,Transcriptional Activation ,lentiviral vector ,insulin promoter ,transcriptional activation ,lineage-specificity ,gene amplification ,Medical and Health Sciences ,Biological sciences ,Biomedical and clinical sciences ,Health sciences - Abstract
An important goal of gene therapy is to be able to deliver genes, so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Although tissue-specific promoters confer selectivity, in a vector-based system, their activity may be too weak to mediate detectable levels in gene-expression studies. We have used a two-step transcriptional amplification system to amplify gene expression from lentiviral vectors using the human insulin promoter. In this system, the human insulin promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA-binding domain fused to the activation domain of the Herpes simplex virus-1 VP16 activator), which in turn activates a GAL4-responsive promoter, driving the enhanced green fluorescent protein reporter gene. Vectors carrying the human insulin promoter did not express in non-beta-cell lines, but expressed in murine insulinoma cell lines, indicating that the human insulin promoter was capable of conferring cell specificity of expression. The insulin-amplifiable vector was able to amplify gene expression five to nine times over a standard insulin-promoter vector. In primary human islets, gene expression from the insulin-promoted vectors was coincident with insulin staining. These vectors will be useful in gene-expression studies that require a detectable signal and tissue specificity.
- Published
- 2009
4. Lentiviral vectors with amplified beta cell-specific gene expression.
- Author
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Shaw, KL, Pais, E, Ge, S, Hardee, C, Skelton, D, Hollis, RP, Crooks, GM, and Kohn, DB
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Islets of Langerhans ,Cell Line ,Animals ,Humans ,Mice ,Lentivirus ,Insulin ,Phosphoglycerate Kinase ,Green Fluorescent Proteins ,Gene Transfer Techniques ,Organ Specificity ,Gene Expression ,Genetic Vectors ,Insulin-Secreting Cells ,Promoter Regions ,Genetic ,Transcriptional Activation ,lentiviral vector ,insulin promoter ,transcriptional activation ,lineage-specificity ,gene amplification ,Promoter Regions ,Genetic ,Biotechnology ,Biological Sciences ,Medical and Health Sciences - Abstract
An important goal of gene therapy is to be able to deliver genes, so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Although tissue-specific promoters confer selectivity, in a vector-based system, their activity may be too weak to mediate detectable levels in gene-expression studies. We have used a two-step transcriptional amplification system to amplify gene expression from lentiviral vectors using the human insulin promoter. In this system, the human insulin promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA-binding domain fused to the activation domain of the Herpes simplex virus-1 VP16 activator), which in turn activates a GAL4-responsive promoter, driving the enhanced green fluorescent protein reporter gene. Vectors carrying the human insulin promoter did not express in non-beta-cell lines, but expressed in murine insulinoma cell lines, indicating that the human insulin promoter was capable of conferring cell specificity of expression. The insulin-amplifiable vector was able to amplify gene expression five to nine times over a standard insulin-promoter vector. In primary human islets, gene expression from the insulin-promoted vectors was coincident with insulin staining. These vectors will be useful in gene-expression studies that require a detectable signal and tissue specificity.
- Published
- 2009
5. Effects of Nonionic Surfactants on Properties of Water Dispersible Granule Formulations
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Utz, CG, primary, Sekmistrz, JM, additional, and Hollis, RP, additional
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6. Using Nonionic Surfactants in Aqueous Formulations
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Utz, CG, primary, Drewno, GW, additional, and Hollis, RP, additional
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7. Lentiviral vectors for precise expression to treat X-linked lymphoproliferative disease.
- Author
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Ayoub PG, Gensheimer J, Lathrop L, Juett C, Quintos J, Tam K, Reid J, Ma F, Tam C, McAuley GE, Brown D, Wu X, Zhang R, Bradford K, Hollis RP, Crooks GM, and Kohn DB
- Abstract
X-linked lymphoproliferative disease (XLP1) results from SH2D1A gene mutations affecting the SLAM-associated protein (SAP). A regulated lentiviral vector (LV), XLP-SMART LV, designed to express SAP at therapeutic levels in T, NK, and NKT cells, is crucial for effective gene therapy. We experimentally identified 34 genomic regulatory elements of the SH2D1A gene and designed XLP-SMART LVs to emulate the lineage and stage-specific control of SAP. We screened them for their on-target enhancer activity in T, NK, and NKT cells and their off-target enhancer activity in B cell and myeloid populations. In combination, three enhancer elements increased SAP promoter expression up to 4-fold in on-target populations in vitro . NSG-Tg(Hu-IL15) xenograft studies with XLP-SMART LVs demonstrated up to 7-fold greater expression in on-target cells over a control EFS-LV, with no off-target expression. The XLP-SMART LVs exhibited stage-specific T and NK cell expression in peripheral blood, bone marrow, spleen, and thymic tissues (mimicking expression patterns of SAP). Transduction of XLP1 patient CD8+ T cells or BM CD34+ cells with XLP-SMART LVs restored restimulation-induced cell death and NK cytotoxicity to wild-type levels, respectively. These data demonstrate that it is feasible to create a lineage and stage-specific LV to restore the XLP1 phenotype by gene therapy., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)
- Published
- 2024
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8. A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease.
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Hart KL, Liu B, Brown D, Campo-Fernandez B, Tam K, Orr K, Hollis RP, Brendel C, Williams DA, and Kohn DB
- Abstract
A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34
+ hematopoietic stem and progenitor cells (HSPCs). This LVV contains locus control region cores expressing an anti-sickling βAS3 -globin gene and two microRNA-adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently decreasing sickle hemoglobin (HbS). The decrease in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo . With high titer and enhanced transduction of HSPC at a low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to decrease costs and help improve accessibility to gene therapy for SCD., Competing Interests: The authors declare a patent related to this work (Application Number: USSN 63/319,152). D.A.W. has received research funding from bluebird bio research in hemoglobinopathies. Boston Children’s Hospital has licensed certain intellectual property (IP) relevant to hemoglobinopathies to bluebird bio. The bifunctional anti-sickling LVVs described here are the subject of patent applied for by University of California, Los Angeles and Boston Children’s Hospital., (© 2024 The Author(s).)- Published
- 2024
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9. Lentiviral gene therapy for X-linked chronic granulomatous disease recapitulates endogenous CYBB regulation and expression.
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Wong RL, Sackey S, Brown D, Senadheera S, Masiuk K, Quintos JP, Colindres N, Riggan L, Morgan RA, Malech HL, Hollis RP, and Kohn DB
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- Animals, Mice, NADPH Oxidases genetics, NADPH Oxidases metabolism, NADPH Oxidase 2 genetics, Genetic Therapy methods, Mutation, Granulomatous Disease, Chronic genetics, Granulomatous Disease, Chronic therapy
- Abstract
X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.
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- 2023
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10. Improved lentiviral vector titers from a multi-gene knockout packaging line.
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Han J, Tam K, Tam C, Hollis RP, and Kohn DB
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Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2'-5'-oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to ∼2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1 , LDLR , and PKR , a previously identified factor impeding LV titers. Packaging in CHEDAR yielded ∼7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by ∼2-fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to ∼11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)
- Published
- 2021
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11. Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency.
- Author
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Reinhardt B, Habib O, Shaw KL, Garabedian E, Carbonaro-Sarracino DA, Terrazas D, Fernandez BC, De Oliveira S, Moore TB, Ikeda AK, Engel BC, Podsakoff GM, Hollis RP, Fernandes A, Jackson C, Shupien S, Mishra S, Davila A, Mottahedeh J, Vitomirov A, Meng W, Rosenfeld AM, Roche AM, Hokama P, Reddy S, Everett J, Wang X, Luning Prak ET, Cornetta K, Hershfield MS, Sokolic R, De Ravin SS, Malech HL, Bushman FD, Candotti F, and Kohn DB
- Subjects
- Adenosine Deaminase genetics, Adolescent, Agammaglobulinemia genetics, Child, Child, Preschool, Follow-Up Studies, Hematopoietic Stem Cell Transplantation methods, Humans, Infant, Severe Combined Immunodeficiency genetics, Transplantation, Autologous methods, Treatment Outcome, Agammaglobulinemia therapy, Genetic Therapy methods, Severe Combined Immunodeficiency therapy
- Abstract
Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)-ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508., (© 2021 by The American Society of Hematology.)
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- 2021
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12. Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency.
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Kohn DB, Booth C, Shaw KL, Xu-Bayford J, Garabedian E, Trevisan V, Carbonaro-Sarracino DA, Soni K, Terrazas D, Snell K, Ikeda A, Leon-Rico D, Moore TB, Buckland KF, Shah AJ, Gilmour KC, De Oliveira S, Rivat C, Crooks GM, Izotova N, Tse J, Adams S, Shupien S, Ricketts H, Davila A, Uzowuru C, Icreverzi A, Barman P, Campo Fernandez B, Hollis RP, Coronel M, Yu A, Chun KM, Casas CE, Zhang R, Arduini S, Lynn F, Kudari M, Spezzi A, Zahn M, Heimke R, Labik I, Parrott R, Buckley RH, Reeves L, Cornetta K, Sokolic R, Hershfield M, Schmidt M, Candotti F, Malech HL, Thrasher AJ, and Gaspar HB
- Subjects
- Adenosine Deaminase deficiency, Adolescent, Child, Child, Preschool, Genetic Therapy adverse effects, Humans, Infant, Lymphocyte Count, Progression-Free Survival, Prospective Studies, Transplantation, Autologous, Agammaglobulinemia therapy, Genetic Therapy methods, Genetic Vectors, Hematopoietic Stem Cell Transplantation, Lentivirus genetics, Severe Combined Immunodeficiency therapy
- Abstract
Background: Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency., Methods: We treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA . Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up., Results: Overall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade., Conclusions: Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.)., (Copyright © 2021 Massachusetts Medical Society.)
- Published
- 2021
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13. β-Globin Lentiviral Vectors Have Reduced Titers due to Incomplete Vector RNA Genomes and Lowered Virion Production.
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Han J, Tam K, Ma F, Tam C, Aleshe B, Wang X, Quintos JP, Morselli M, Pellegrini M, Hollis RP, and Kohn DB
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- Gene Transfer Techniques, Genetic Vectors genetics, HEK293 Cells, Humans, beta-Globins metabolism, tat Gene Products, Human Immunodeficiency Virus genetics, tat Gene Products, Human Immunodeficiency Virus metabolism, Genetic Vectors metabolism, Lentivirus genetics, RNA metabolism, Virion physiology, beta-Globins genetics
- Abstract
Lentiviral vectors (LVs) commonly used for the treatment of hemoglobinopathies often have low titers and sub-optimal gene transfer efficiency for human hematopoietic stem and progenitor cells (HSPCs), hindering clinical translation and commercialization for ex vivo gene therapy. We observed that a high percentage of β-globin LV viral genomic RNAs were incomplete toward the 3' end in packaging cells and in released vector particles. The incomplete vector genomes impeded reverse transcription in target cells, limiting stable gene transfer to HSPCs. By combining three modifications to vector design and production (shortening the vector length to 5.3 kb; expressing HIV-1 Tat protein during packaging; and packaging in PKR-/- cells) there was a 30-fold increase in vector titer and a 3-fold increase in vector infectivity in HSPCs. These approaches may improve the manufacturing of β-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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14. Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells.
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Benitez EK, Lomova Kaufman A, Cervantes L, Clark DN, Ayoub PG, Senadheera S, Osborne K, Sanchez JM, Crisostomo RV, Wang X, Reuven N, Shaul Y, Hollis RP, Romero Z, and Kohn DB
- Abstract
Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Benitez, Lomova Kaufman, Cervantes, Clark, Ayoub, Senadheera, Osborne, Sanchez, Crisostomo, Wang, Reuven, Shaul, Hollis, Romero and Kohn.)
- Published
- 2020
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15. Human CLEC9A antibodies deliver NY-ESO-1 antigen to CD141 + dendritic cells to activate naïve and memory NY-ESO-1-specific CD8 + T cells.
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Masterman KA, Haigh OL, Tullett KM, Leal-Rojas IM, Walpole C, Pearson FE, Cebon J, Schmidt C, O'Brien L, Rosendahl N, Daraj G, Caminschi I, Gschweng EH, Hollis RP, Kohn DB, Lahoud MH, and Radford KJ
- Subjects
- Animals, Female, Healthy Volunteers, Humans, Mice, Antigens, Neoplasm metabolism, CD8-Positive T-Lymphocytes metabolism, Dendritic Cells immunology, Lectins, C-Type metabolism, Membrane Proteins metabolism, Receptors, Mitogen metabolism, Thrombomodulin metabolism
- Abstract
Background: Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8
+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs., Methods: Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1β, tumor necrosis factor and CD107a and by lysis of target tumor cells., Results: CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro., Conclusions: These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)- Published
- 2020
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16. Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences.
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Morgan RA, Ma F, Unti MJ, Brown D, Ayoub PG, Tam C, Lathrop L, Aleshe B, Kurita R, Nakamura Y, Senadheera S, Wong RL, Hollis RP, Pellegrini M, and Kohn DB
- Abstract
Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis -regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic β
AS3 -globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types., (© 2020 The Author(s).)- Published
- 2020
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17. Improved Titer and Gene Transfer by Lentiviral Vectors Using Novel, Small β-Globin Locus Control Region Elements.
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Morgan RA, Unti MJ, Aleshe B, Brown D, Osborne KS, Koziol C, Ayoub PG, Smith OB, O'Brien R, Tam C, Miyahira E, Ruiz M, Quintos JP, Senadheera S, Hollis RP, and Kohn DB
- Subjects
- Animals, Bone Marrow Cells metabolism, Disease Models, Animal, HEK293 Cells, Healthy Volunteers, Hematopoietic Stem Cells metabolism, Humans, Mice, Phenotype, Transfection, Anemia, Sickle Cell therapy, Genetic Therapy methods, Genetic Vectors, Lentivirus genetics, Locus Control Region genetics, Transduction, Genetic methods, beta-Globins genetics
- Abstract
β-globin lentiviral vectors (β-LV) have faced challenges in clinical translation for gene therapy of sickle cell disease (SCD) due to low titer and sub-optimal gene transfer to hematopoietic stem and progenitor cells (HSPCs). To overcome the challenge of preserving efficacious expression while increasing vector performance, we used published genomic and epigenomic data available through ENCODE to redefine enhancer element boundaries of the β-globin locus control region (LCR) to construct novel ENCODE core sequences. These novel LCR elements were used to design a β-LV of reduced proviral length, termed CoreGA-AS3-FB, produced at higher titers and possessing superior gene transfer to HSPCs when compared to the full-length parental β-LV at equal MOI. At low vector copy number, vectors containing the ENCODE core sequences were capable of reversing the sickle phenotype in a mouse model of SCD. These studies provide a β-LV that will be beneficial for gene therapy of SCD by significantly reducing the cost of vector production and extending the vector supply., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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18. Development of Hematopoietic Stem Cell-Engineered Invariant Natural Killer T Cell Therapy for Cancer.
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Zhu Y, Smith DJ, Zhou Y, Li YR, Yu J, Lee D, Wang YC, Di Biase S, Wang X, Hardoy C, Ku J, Tsao T, Lin LJ, Pham AT, Moon H, McLaughlin J, Cheng D, Hollis RP, Campo-Fernandez B, Urbinati F, Wei L, Pang L, Rezek V, Berent-Maoz B, Macabali MH, Gjertson D, Wang X, Galic Z, Kitchen SG, An DS, Hu-Lieskovan S, Kaplan-Lefko PJ, De Oliveira SN, Seet CS, Larson SM, Forman SJ, Heath JR, Zack JA, Crooks GM, Radu CG, Ribas A, Kohn DB, Witte ON, and Yang L
- Subjects
- Animals, Cells, Cultured, Genetic Engineering, Humans, Mice, Mice, SCID, Natural Killer T-Cells transplantation, Neoplasms immunology, Receptors, Antigen, T-Cell genetics, Xenograft Model Antitumor Assays, Hematopoietic Stem Cells physiology, Immunotherapy, Adoptive methods, Natural Killer T-Cells physiology, Neoplasms therapy
- Abstract
Invariant natural killer T (iNKT) cells are potent immune cells for targeting cancer; however, their clinical application has been hindered by their low numbers in cancer patients. Here, we developed a proof-of-concept for hematopoietic stem cell-engineered iNKT (HSC-iNKT) cell therapy with the potential to provide therapeutic levels of iNKT cells for a patient's lifetime. Using a human HSC engrafted mouse model and a human iNKT TCR gene engineering approach, we demonstrated the efficient and long-term generation of HSC-iNKT cells in vivo. These HSC-iNKT cells closely resembled endogenous human iNKT cells, could deploy multiple mechanisms to attack tumor cells, and effectively suppressed tumor growth in vivo in multiple human tumor xenograft mouse models. Preclinical safety studies showed no toxicity or tumorigenicity of the HSC-iNKT cell therapy. Collectively, these results demonstrated the feasibility, safety, and cancer therapy potential of the proposed HSC-iNKT cell therapy and laid a foundation for future clinical development., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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19. Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates.
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Romero Z, Lomova A, Said S, Miggelbrink A, Kuo CY, Campo-Fernandez B, Hoban MD, Masiuk KE, Clark DN, Long J, Sanchez JM, Velez M, Miyahira E, Zhang R, Brown D, Wang X, Kurmangaliyev YZ, Hollis RP, and Kohn DB
- Subjects
- Anemia, Sickle Cell metabolism, Anemia, Sickle Cell therapy, CRISPR-Cas Systems, Dependovirus, Endonucleases genetics, Gene Expression, Gene Targeting, Genetic Therapy, Genetic Vectors genetics, Humans, Parvovirinae genetics, Tissue Donors, Transduction, Genetic, Zinc Finger Nucleases genetics, Anemia, Sickle Cell genetics, Gene Editing, Hematopoietic Stem Cells metabolism, Mutation, beta-Globins genetics
- Abstract
Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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20. PGE2 and Poloxamer Synperonic F108 Enhance Transduction of Human HSPCs with a β-Globin Lentiviral Vector.
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Masiuk KE, Zhang R, Osborne K, Hollis RP, Campo-Fernandez B, and Kohn DB
- Abstract
Lentiviral vector (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. Clinical trials are currently underway to treat sickle cell disease using LVs expressing designed anti-sickling globin genes. However, because of the large size and complexity of the human β-globin gene, LV products often have low titers and transduction efficiency, requiring large amounts to treat a single patient. Furthermore, transduction of patient HSPCs often fails to achieve a sufficiently high vector copy number (VCN) and transgene expression for clinical benefit. We therefore investigated the combination of two compounds (PGE2 and poloxamer synperonic F108) to enhance transduction of HSPCs with a clinical-scale preparation of Lenti/G-AS3-FB. Here, we found that transduction enhancers increased the in vitro VCN of bulk myeloid cultures ∼10-fold while using a 10-fold lower LV dose. This was accompanied by an increased percentage of transduced colony-forming units. Importantly, analysis of immune-deficient NSG xenografts revealed that the combination of PGE2/synperonic F108 increased LV gene transfer in a primitive HSC population , with no effects on lineage distribution or engraftment. The use of transduction enhancers may greatly improve efficacy for LV-based HSPC gene therapy.
- Published
- 2019
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21. Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.
- Author
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Masiuk KE, Laborada J, Roncarolo MG, Hollis RP, and Kohn DB
- Subjects
- Animals, Diabetes Mellitus, Type 1 genetics, Diabetes Mellitus, Type 1 immunology, Diabetes Mellitus, Type 1 therapy, Diarrhea genetics, Disease Models, Animal, Forkhead Transcription Factors genetics, Genes, Reporter, Genetic Diseases, X-Linked genetics, Humans, Immune System Diseases genetics, Immune System Diseases immunology, Immune System Diseases therapy, Mice, T-Lymphocytes, Regulatory immunology, Autoimmunity, Cell Lineage, Diabetes Mellitus, Type 1 congenital, Diarrhea immunology, Diarrhea therapy, Forkhead Transcription Factors therapeutic use, Genetic Diseases, X-Linked immunology, Genetic Diseases, X-Linked therapy, Genetic Therapy, Hematopoietic Stem Cells metabolism, Immune System Diseases congenital, Lentivirus genetics
- Abstract
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a devastating autoimmune disease caused by mutations in FoxP3, a transcription factor required for the development and function of regulatory T cells (Treg cells). Allogeneic hematopoietic stem cell transplant (HSCT) can be curative, but suitable donors are often unavailable. Here, we demonstrate a strategy for autologous HSCT and gene therapy utilizing a lentiviral vector (LV) to restore FoxP3 expression under the control of endogenous human FOXP3 regulatory elements. Both murine transplant models and humanized mice engrafted with LV-modified HSCs show high levels of LV expression selective for CD4+CD25+FoxP3+ Treg cells. LV transduction of scurfy (FoxP3
mut ) HSCs restores development of functional FoxP3+ Treg cells that suppress T cell proliferation in vitro and rescue the scurfy autoimmune phenotype in vivo. These findings demonstrate preclinical efficacy for the treatment of IPEX patients by autologous HSC transplant and may provide valuable insights into new cell therapies for autoimmunity., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2019
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22. Improving Gene Editing Outcomes in Human Hematopoietic Stem and Progenitor Cells by Temporal Control of DNA Repair.
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Lomova A, Clark DN, Campo-Fernandez B, Flores-Bjurström C, Kaufman ML, Fitz-Gibbon S, Wang X, Miyahira EY, Brown D, DeWitt MA, Corn JE, Hollis RP, Romero Z, and Kohn DB
- Subjects
- Animals, Humans, Mice, DNA Repair genetics, Gene Editing methods, Hematopoietic Stem Cell Transplantation methods, Stem Cells metabolism, Transplantation Conditioning methods
- Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas9)-mediated gene editing of human hematopoietic stem cells (hHSCs) is a promising strategy for the treatment of genetic blood diseases through site-specific correction of identified causal mutations. However, clinical translation is hindered by low ratio of precise gene modification using the corrective donor template (homology-directed repair, HDR) to gene disruption (nonhomologous end joining, NHEJ) in hHSCs. By using a modified version of Cas9 with reduced nuclease activity in G1 phase of cell cycle when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieved a four-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement after xenotransplantation of edited hHSCs into immunodeficient mice. This strategy for improving gene editing outcomes in hHSCs has important implications for the field of gene therapy, and can be applied to diseases where increased HDR/NHEJ ratio is critical for therapeutic success. Stem Cells 2019;37:284-294., (© AlphaMed Press 2018.)
- Published
- 2019
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23. IND-Enabling Studies for a Clinical Trial to Genetically Program a Persistent Cancer-Targeted Immune System.
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Puig-Saus C, Parisi G, Garcia-Diaz A, Krystofinski PE, Sandoval S, Zhang R, Champhekar AS, McCabe J, Cheung-Lau GC, Truong NA, Vega-Crespo A, Komenan MDS, Pang J, Macabali MH, Saco JD, Goodwin JL, Bolon B, Seet CS, Montel-Hagen A, Crooks GM, Hollis RP, Campo-Fernandez B, Bischof D, Cornetta K, Gschweng EH, Adelson C, Nguyen A, Yang L, Witte ON, Baltimore D, Comin-Anduix B, Kohn DB, Wang X, Cabrera P, Kaplan-Lefko PJ, Berent-Maoz B, and Ribas A
- Subjects
- Animals, Antigens, Neoplasm genetics, Cells, Cultured, Clinical Trials as Topic, Drugs, Investigational therapeutic use, HLA-A2 Antigen genetics, Hematopoietic Stem Cells metabolism, Humans, Membrane Proteins genetics, Mice, Inbred C57BL, Mice, Transgenic, Neoplasms genetics, Neoplasms immunology, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell metabolism, T-Lymphocytes metabolism, Genetic Therapy methods, Hematopoietic Stem Cells immunology, Immunotherapy, Adoptive methods, Neoplasms therapy, Receptors, Antigen, T-Cell immunology, T-Lymphocytes immunology
- Abstract
Purpose: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application., Experimental Design: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/K
b mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use., Results: TCR genetically modified and ex vivo -cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality., Conclusions: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471., (©2018 American Association for Cancer Research.)- Published
- 2019
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24. Pre-clinical Development of a Lentiviral Vector Expressing the Anti-sickling βAS3 Globin for Gene Therapy for Sickle Cell Disease.
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Poletti V, Urbinati F, Charrier S, Corre G, Hollis RP, Campo Fernandez B, Martin S, Rothe M, Schambach A, Kohn DB, and Mavilio F
- Abstract
Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) β-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the β-globin promoter and a reduced version of the β-globin locus-control region. The vector (GLOBE-AS3) transduced 60%-80% of mobilized CD34
+ hematopoietic stem-progenitor cells (HSPCs) and drove βAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12-14 weeks after transplantation. Vector integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral integration pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3.- Published
- 2018
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25. Gene Therapy for Sickle Cell Disease: A Lentiviral Vector Comparison Study.
- Author
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Urbinati F, Campo Fernandez B, Masiuk KE, Poletti V, Hollis RP, Koziol C, Kaufman ML, Brown D, Mavilio F, and Kohn DB
- Subjects
- Animals, Cell Differentiation, Colony-Forming Units Assay, Disease Models, Animal, Gene Expression, Gene Order, Genetic Vectors chemistry, Genetic Vectors genetics, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Humans, Lentivirus genetics, Mice, Phenotype, RNA, Messenger genetics, Transduction, Genetic, Treatment Outcome, Anemia, Sickle Cell genetics, Anemia, Sickle Cell therapy, Genetic Therapy methods, beta-Globins genetics
- Abstract
Sickle cell disease (SCD) is an inherited blood disorder caused by a single amino acid substitution in the β-globin chain of hemoglobin. Gene therapy is a promising therapeutic alternative, particularly in patients lacking an allogeneic bone marrow (BM) donor. One of the major challenges for an effective gene therapy approach is the design of an efficient vector that combines high-level and long-term β-globin expression with high infectivity in primary CD34+ cells. Two lentiviral vectors carrying an anti-sickling β-globin transgene (AS3) were directly compared: the Lenti/βAS3-FB, and Globe-AS3 with and without the FB insulator. The comparison was performed initially in human BM CD34+ cells derived from SCD patients in an in vitro model of erythroid differentiation. Additionally, the comparison was carried out in two in vivo models: First, an NOD SCID gamma mouse model was used to compare transduction efficiency and β-globin expression in human BM CD34+ cells after transplant. Second, a sickle mouse model was used to analyze β-globin expression produced from the vectors tested, as well as hematologic correction of the sickle phenotype. While minor differences were found in the vectors in the in vitro study (2.4-fold higher vector copy number in CD34+ cells when using Globe-AS3), no differences were noted in the overall correction of the SCD phenotype in the in vivo mouse model. This study provides a comprehensive in vitro and in vivo analysis of two globin lentiviral vectors, which is useful for determining the optimal candidate for SCD gene therapy.
- Published
- 2018
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26. Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome.
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Kuo CY, Long JD, Campo-Fernandez B, de Oliveira S, Cooper AR, Romero Z, Hoban MD, Joglekar AV, Lill GR, Kaufman ML, Fitz-Gibbon S, Wang X, Hollis RP, and Kohn DB
- Subjects
- Animals, Antigens, CD34 metabolism, Base Sequence, CD40 Ligand metabolism, CRISPR-Associated Protein 9 metabolism, CRISPR-Cas Systems genetics, Cell Differentiation, Cell Line, Colony-Forming Units Assay, DNA Repair, DNA, Complementary genetics, Humans, Mice, T-Lymphocytes metabolism, Transcription Activator-Like Effector Nucleases metabolism, Gene Editing, Genetic Diseases, X-Linked genetics, Hematopoietic Stem Cells metabolism, Hyper-IgM Immunodeficiency Syndrome genetics
- Abstract
X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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27. Characterization of Gene Alterations following Editing of the β-Globin Gene Locus in Hematopoietic Stem/Progenitor Cells.
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Long J, Hoban MD, Cooper AR, Kaufman ML, Kuo CY, Campo-Fernandez B, Lumaquin D, Hollis RP, Wang X, Kohn DB, and Romero Z
- Subjects
- Gene Conversion, Gene Rearrangement, Gene Targeting, High-Throughput Nucleotide Sequencing, Humans, Nucleic Acid Amplification Techniques, Translocation, Genetic, Gene Editing, Genetic Variation, Hematopoietic Stem Cells metabolism, beta-Globins genetics
- Abstract
The use of engineered nucleases combined with a homologous DNA donor template can result in targeted gene correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. However, because of the high homology between the adjacent human β- and δ-globin genes, off-target cleavage is observed at δ-globin when using some endonucleases targeted to the sickle mutation in β-globin. Introduction of multiple double-stranded breaks by endonucleases has the potential to induce intergenic alterations. Using a novel droplet digital PCR assay and high-throughput sequencing, we characterized the frequency of rearrangements between the β- and δ-globin paralogs when delivering these nucleases. Pooled CD34
+ cells and colony-forming units from sickle bone marrow were treated with nuclease only or including a donor template and then analyzed for potential gene rearrangements. It was observed that, in pooled CD34+ cells and colony-forming units, the intergenic β-δ-globin deletion was the most frequent rearrangement, followed by inversion of the intergenic fragment, with the inter-chromosomal translocation as the least frequent. No rearrangements were observed when endonuclease activity was restricted to on-target β-globin cleavage. These findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted gene alterations., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)- Published
- 2018
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28. Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells.
- Author
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Masiuk KE, Brown D, Laborada J, Hollis RP, Urbinati F, and Kohn DB
- Subjects
- ADP-ribosyl Cyclase 1 metabolism, Animals, Antigens, CD34 metabolism, Gene Expression, Gene Transfer Techniques, Genes, Reporter, Genetic Therapy, Genetic Vectors genetics, Graft Survival, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells cytology, Humans, Immunomagnetic Separation, Immunophenotyping, Lentivirus genetics, Mice, Transgenes, Hematopoietic Stem Cells metabolism, Transduction, Genetic
- Abstract
Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 10
8 to 1 × 109 CD34+ cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34+ cells uses LV inefficiently, because the majority of CD34+ cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34+ CD38- cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34+ CD38- cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34+ purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34+ CD38- cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38+ cells. Importantly, LV modification and transplantation of IB-purified CD34+ CD38- cells/non-modified CD38+ cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34+ cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)- Published
- 2017
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29. Preclinical studies for a phase 1 clinical trial of autologous hematopoietic stem cell gene therapy for sickle cell disease.
- Author
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Urbinati F, Wherley J, Geiger S, Fernandez BC, Kaufman ML, Cooper A, Romero Z, Marchioni F, Reeves L, Read E, Nowicki B, Grassman E, Viswanathan S, Wang X, Hollis RP, and Kohn DB
- Subjects
- Adult, Animals, Antigens, CD34 metabolism, Bone Marrow Cells, Bone Marrow Transplantation, Case-Control Studies, Clinical Trials, Phase I as Topic, Genetic Vectors, Humans, Lentivirus genetics, Mice, Inbred NOD, Transduction, Genetic, Transplantation, Autologous methods, Anemia, Sickle Cell therapy, Genetic Therapy methods, Hematopoietic Stem Cell Transplantation methods, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells physiology
- Abstract
Background Aims: Gene therapy by autologous hematopoietic stem cell transplantation (HSCT) represents a new approach to treat sickle cell disease (SCD). Optimization of the manufacture, characterization and testing of the transduced hematopoietic stem cell final cell product (FCP), as well as an in depth in vivo toxicology study, are critical for advancing this approach to clinical trials., Methods: Data are shown to evaluate and establish the feasibility of isolating, transducing with the Lenti/β
AS3 -FB vector and cryopreserving CD34+ cells from human bone marrow (BM) at clinical scale. In vitro and in vivo characterization of the FCP was performed, showing that all the release criteria were successfully met. In vivo toxicology studies were conducted to evaluate potential toxicity of the Lenti/βAS3 -FB LV in the context of a murine BM transplant., Results: Primary and secondary transplantation did not reveal any toxicity from the lentiviral vector. Additionally, vector integration site analysis of murine and human BM cells did not show any clonal skewing caused by insertion of the Lenti/βAS3 -FB vector in cells from primary and secondary transplanted mice., Conclusions: We present here a complete protocol, thoroughly optimized to manufacture, characterize and establish safety of a FCP for gene therapy of SCD., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)- Published
- 2017
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30. CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells.
- Author
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Hoban MD, Lumaquin D, Kuo CY, Romero Z, Long J, Ho M, Young CS, Mojadidi M, Fitz-Gibbon S, Cooper AR, Lill GR, Urbinati F, Campo-Fernandez B, Bjurstrom CF, Pellegrini M, Hollis RP, and Kohn DB
- Subjects
- Anemia, Sickle Cell therapy, Base Sequence, Cell Line, DNA Cleavage, Gene Targeting, Genetic Loci, Humans, Protein Binding, RNA, Guide, CRISPR-Cas Systems, Transcription Activator-Like Effector Nucleases metabolism, Anemia, Sickle Cell genetics, CRISPR-Cas Systems, Gene Editing, Hematopoietic Stem Cells metabolism, Mutation, Targeted Gene Repair, beta-Globins genetics
- Abstract
Targeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.
- Published
- 2016
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31. Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases.
- Author
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Bjurström CF, Mojadidi M, Phillips J, Kuo C, Lai S, Lill GR, Cooper A, Kaufman M, Urbinati F, Wang X, Hollis RP, and Kohn DB
- Abstract
We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34
+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID -IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs., (Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy. Published by Elsevier Inc. All rights reserved.)- Published
- 2016
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32. Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy.
- Author
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Baldwin K, Urbinati F, Romero Z, Campo-Fernandez B, Kaufman ML, Cooper AR, Masiuk K, Hollis RP, and Kohn DB
- Subjects
- ADP-ribosyl Cyclase 1 metabolism, Animals, Antigens, CD34 metabolism, Cell Differentiation, Cell Line, Cell Proliferation, Cell Separation, Erythroid Cells cytology, Genetic Vectors metabolism, Hematopoietic Stem Cells metabolism, Humans, Lentivirus genetics, Mice, Inbred NOD, Receptors, LDL metabolism, Genetic Therapy, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells cytology, Transduction, Genetic
- Abstract
Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising ∼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-β(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy., Competing Interests: of Potential Conflicts of Interest The authors indicate no potential conflicts of interest., (© 2015 AlphaMed Press.)
- Published
- 2015
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33. Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34+ cells.
- Author
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Urbinati F, Hargrove PW, Geiger S, Romero Z, Wherley J, Kaufman ML, Hollis RP, Chambers CB, Persons DA, Kohn DB, and Wilber A
- Subjects
- Anemia, Sickle Cell genetics, Anemia, Sickle Cell therapy, Antigens, CD34 metabolism, Fetal Hemoglobin genetics, Flow Cytometry, Gene Expression, Gene Transfer Techniques, Genetic Therapy methods, Genetic Vectors genetics, Hemoglobins genetics, Humans, Reverse Transcriptase Polymerase Chain Reaction, beta-Globins metabolism, gamma-Globins metabolism, Bone Marrow Cells metabolism, Lentivirus genetics, beta-Globins genetics, gamma-Globins genetics
- Abstract
Sickle cell disease (SCD) can be cured by allogeneic hematopoietic stem cell transplant. However, this is only possible when a matched donor is available, making the development of gene therapy using autologous hematopoietic stem cells a highly desirable alternative. We used a culture model of human erythropoiesis to directly compare two insulated, self-inactivating, and erythroid-specific lentiviral vectors, encoding for γ-globin (V5m3-400) or a modified β-globin (βAS3-FB) for production of antisickling hemoglobin (Hb) and correction of red cell deformability after deoxygenation. Bone marrow CD34+ cells from three SCD patients were transduced using V5m3-400 or βAS3-FB and compared with mock-transduced SCD or healthy donor CD34+ cells. Lentiviral transduction did not impair cell growth or differentiation, as gauged by proliferation and acquisition of erythroid markers. Vector copy number averaged approximately one copy per cell, and corrective globin mRNA levels were increased more than sevenfold over mock-transduced controls. Erythroblasts derived from healthy donor and mock-transduced SCD cells produced a low level of fetal Hb that was increased to 23.6 ± 4.1% per vector copy for cells transduced with V5m3-400. Equivalent levels of modified normal adult Hb of 17.6 ± 3.8% per vector copy were detected for SCD cells transduced with βAS3-FB. These levels of antisickling Hb production were sufficient to reduce sickling of terminal-stage red blood cells upon deoxygenation. We concluded that the achieved levels of fetal Hb and modified normal adult Hb would likely prove therapeutic to SCD patients who lack matched donors., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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34. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.
- Author
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Hoban MD, Cost GJ, Mendel MC, Romero Z, Kaufman ML, Joglekar AV, Ho M, Lumaquin D, Gray D, Lill GR, Cooper AR, Urbinati F, Senadheera S, Zhu A, Liu PQ, Paschon DE, Zhang L, Rebar EJ, Wilber A, Wang X, Gregory PD, Holmes MC, Reik A, Hollis RP, and Kohn DB
- Subjects
- Anemia, Sickle Cell pathology, Animals, Antigens, CD34 analysis, Base Sequence, Bone Marrow Cells metabolism, Bone Marrow Cells pathology, Cells, Cultured, Endodeoxyribonucleases metabolism, Fetal Blood transplantation, Genetic Loci, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells pathology, Humans, Mice, Mice, Inbred NOD, Mice, SCID, Molecular Sequence Data, Zinc Fingers, Anemia, Sickle Cell genetics, Anemia, Sickle Cell therapy, Genetic Therapy, Hematopoietic Stem Cells metabolism, Mutation, beta-Globins genetics
- Abstract
Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers., (© 2015 by The American Society of Hematology.)
- Published
- 2015
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35. The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells.
- Author
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Romero Z, Campo-Fernandez B, Wherley J, Kaufman ML, Urbinati F, Cooper AR, Hoban MD, Baldwin KM, Lumaquin D, Wang X, Senadheera S, Hollis RP, and Kohn DB
- Abstract
Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies.
- Published
- 2015
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- View/download PDF
36. HSV-sr39TK positron emission tomography and suicide gene elimination of human hematopoietic stem cells and their progeny in humanized mice.
- Author
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Gschweng EH, McCracken MN, Kaufman ML, Ho M, Hollis RP, Wang X, Saini N, Koya RC, Chodon T, Ribas A, Witte ON, and Kohn DB
- Subjects
- Animals, Antigens, CD34 blood, Antigens, CD34 immunology, Antigens, Neoplasm genetics, Antigens, Neoplasm immunology, Disease Models, Animal, Genetic Therapy, Genetic Vectors genetics, Hematopoietic Stem Cells diagnostic imaging, Hematopoietic Stem Cells immunology, Humans, Membrane Proteins genetics, Membrane Proteins immunology, Mice, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell immunology, Transduction, Genetic, Genes, Transgenic, Suicide, Hematopoietic Stem Cells physiology, Herpesvirus 1, Human genetics, Immunotherapy methods, Positron-Emission Tomography methods, T-Lymphocytes immunology
- Abstract
Engineering immunity against cancer by the adoptive transfer of hematopoietic stem cells (HSC) modified to express antigen-specific T-cell receptors (TCR) or chimeric antigen receptors generates a continual supply of effector T cells, potentially providing superior anticancer efficacy compared with the infusion of terminally differentiated T cells. Here, we demonstrate the in vivo generation of functional effector T cells from CD34-enriched human peripheral blood stem cells modified with a lentiviral vector designed for clinical use encoding a TCR recognizing the cancer/testes antigen NY-ESO-1, coexpressing the PET/suicide gene sr39TK. Ex vivo analysis of T cells showed antigen- and HLA-restricted effector function against melanoma. Robust engraftment of gene-modified human cells was demonstrated with PET reporter imaging in hematopoietic niches such as femurs, humeri, vertebrae, and the thymus. Safety was demonstrated by the in vivo ablation of PET signal, NY-ESO-1-TCR-bearing cells, and integrated lentiviral vector genomes upon treatment with ganciclovir, but not with vehicle control. Our study provides support for the efficacy and safety of gene-modified HSCs as a therapeutic modality for engineered cancer immunotherapy. Cancer Res; 74(18); 5173-83. ©2014 AACR., (©2014 American Association for Cancer Research.)
- Published
- 2014
- Full Text
- View/download PDF
37. Envelope, please. And the award goes to….
- Author
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Kohn DB and Hollis RP
- Subjects
- Animals, Humans, Betaretrovirus genetics, Genetic Therapy methods, Genetic Vectors genetics, Hematopoietic Stem Cells, Lentivirus genetics, Transduction, Genetic, Viral Envelope Proteins genetics
- Published
- 2014
- Full Text
- View/download PDF
38. Dissecting the mechanism of histone deacetylase inhibitors to enhance the activity of zinc finger nucleases delivered by integrase-defective lentiviral vectors.
- Author
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Joglekar AV, Stein L, Ho M, Hoban MD, Hollis RP, and Kohn DB
- Subjects
- Humans, K562 Cells, Zinc Fingers, Deoxyribonucleases biosynthesis, Deoxyribonucleases genetics, Genetic Vectors, Histone Deacetylase Inhibitors pharmacology, Integrases, Lentivirus, Transduction, Genetic, Viral Proteins
- Abstract
Integrase-defective lentiviral vectors (IDLVs) have been of limited success in the delivery of zinc finger nucleases (ZFNs) to human cells, due to low expression. A reason for reduced gene expression has been proposed to involve the epigenetic silencing of vector genomes, carried out primarily by histone deacetylases (HDACs). In this study, we tested valproic acid (VPA), a known HDAC inhibitor (HDACi), for its ability to increase transgene expression from IDLVs, especially in the context of ZFN delivery. Using ZFNs targeting the human adenosine deaminase (ADA) gene in K562 cells, we demonstrated that treatment with VPA enhanced ZFN expression by up to 3-fold, resulting in improved allelic disruption at the ADA locus. Furthermore, three other U.S. Food and Drug Administration-approved HDACis (vorinostat, givinostat, and trichostatin-A) exhibited a similar effect on the activity of ZFN-IDLVs in K562 cells. In primary human CD34(+) cells, VPA- and vorinostat-treated cells showed higher levels of expression of both green fluorescent protein (GFP) as well as ZFNs from IDLVs. A major mechanism for the effects of HDAC inhibitors on improving expression was from their modulation of the cell cycle, and the influence of heterochromatinization was determined to be a lesser contributing factor.
- Published
- 2014
- Full Text
- View/download PDF
39. Erythropoiesis from human embryonic stem cells through erythropoietin-independent AKT signaling.
- Author
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Kim WS, Zhu Y, Deng Q, Chin CJ, He CB, Grieco AJ, Dravid GG, Parekh C, Hollis RP, Lane TF, Bouhassira EE, Kohn DB, and Crooks GM
- Subjects
- Cell Cycle, Cell Differentiation, Cell Line, Cell Proliferation, Cell Survival, Erythroid Precursor Cells cytology, Erythroid Precursor Cells metabolism, Humans, Megakaryocytes cytology, Megakaryocytes metabolism, Protein Multimerization, Protein Structure, Tertiary, Receptors, Thrombopoietin chemistry, Receptors, Thrombopoietin metabolism, Embryonic Stem Cells cytology, Embryonic Stem Cells enzymology, Erythropoiesis, Erythropoietin metabolism, Proto-Oncogene Proteins c-akt metabolism, Signal Transduction
- Abstract
Unlimited self renewal capacity and differentiation potential make human pluripotent stem cells (PSC) a promising source for the ex vivo manufacture of red blood cells (RBCs) for safe transfusion. Current methods to induce erythropoiesis from PSC suffer from low yields of RBCs, most of which are immature and contain embryonic and fetal rather than adult hemoglobins. We have previously shown that homodimerization of the intracellular component of MPL (ic-MPL) induces erythropoiesis from human cord blood progenitors. The goal of this study was to investigate the potential of ic-MPL dimerization to induce erythropoiesis from human embryonic stem cells (hESCs) and to identify the signaling pathways activated by this strategy. We present here the evidence that ic-MPL dimerization induces erythropoietin (EPO)-independent erythroid differentiation from hESC by inducing the generation of erythroid progenitors and by promoting more efficient erythroid maturation with increased RBC enucleation as well as increased gamma:epsilon globin ratio and production of beta-globin protein. ic-MPL dimerization is significantly more potent than EPO in inducing erythropoiesis, and its effect is additive to EPO. Signaling studies show that dimerization of ic-MPL, unlike stimulation of the wild type MPL receptor, activates AKT in the absence of JAK2/STAT5 signaling. AKT activation upregulates GATA-1 and FOXO3 transcriptional pathways with resulting inhibition of apoptosis, modulation of cell cycle, and enhanced maturation of erythroid cells. These findings open up potential new targets for the generation of therapeutically relevant RBC products from hPSC., (© 2014 AlphaMed Press.)
- Published
- 2014
- Full Text
- View/download PDF
40. Preclinical demonstration of lentiviral vector-mediated correction of immunological and metabolic abnormalities in models of adenosine deaminase deficiency.
- Author
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Carbonaro DA, Zhang L, Jin X, Montiel-Equihua C, Geiger S, Carmo M, Cooper A, Fairbanks L, Kaufman ML, Sebire NJ, Hollis RP, Blundell MP, Senadheera S, Fu PY, Sahaghian A, Chan RY, Wang X, Cornetta K, Thrasher AJ, Kohn DB, and Gaspar HB
- Subjects
- Adenosine Deaminase immunology, Adenosine Deaminase metabolism, Agammaglobulinemia genetics, Agammaglobulinemia pathology, Animals, B-Lymphocytes immunology, Cells, Cultured, Disease Models, Animal, Female, HEK293 Cells, HT29 Cells, Humans, Male, Mice, Mice, Inbred C57BL, Promoter Regions, Genetic, Severe Combined Immunodeficiency genetics, Severe Combined Immunodeficiency pathology, T-Lymphocytes immunology, Transduction, Genetic, Virus Integration, Adenosine Deaminase deficiency, Adenosine Deaminase genetics, Agammaglobulinemia immunology, Agammaglobulinemia therapy, Genetic Vectors adverse effects, Lentivirus genetics, Peptide Elongation Factor 1 genetics, Severe Combined Immunodeficiency immunology, Severe Combined Immunodeficiency therapy
- Abstract
Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA(-/-) mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA(-/-) mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34(+) cells transduced with 1-5 × 10(7) TU/ml had 1-3 vector copies/cell and expressed 1-2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis.
- Published
- 2014
- Full Text
- View/download PDF
41. Modification of hematopoietic stem/progenitor cells with CD19-specific chimeric antigen receptors as a novel approach for cancer immunotherapy.
- Author
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De Oliveira SN, Ryan C, Giannoni F, Hardee CL, Tremcinska I, Katebian B, Wherley J, Sahaghian A, Tu A, Grogan T, Elashoff D, Cooper LJ, Hollis RP, and Kohn DB
- Subjects
- Animals, Cell Differentiation, Cell Line, Cytotoxicity, Immunologic, Disease Models, Animal, Flow Cytometry, Gene Order, Genetic Vectors genetics, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells cytology, Humans, Immunotherapy, Killer Cells, Natural cytology, Killer Cells, Natural immunology, Killer Cells, Natural metabolism, Lentivirus genetics, Lymphocyte Activation genetics, Lymphocyte Activation immunology, Mice, Transgenic, Myeloid Cells cytology, Myeloid Cells immunology, Myeloid Cells metabolism, Neoplasms therapy, Receptors, Antigen immunology, T-Lymphocytes cytology, T-Lymphocytes immunology, T-Lymphocytes metabolism, Transduction, Genetic, Antigens, CD19 immunology, Hematopoietic Stem Cells immunology, Hematopoietic Stem Cells metabolism, Neoplasms genetics, Neoplasms immunology, Receptors, Antigen genetics
- Abstract
Chimeric antigen receptors (CARs) against CD19 have been shown to direct T-cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. However, in some cases, there has been insufficient persistence of effector cells, limiting clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to deliver the CD19-specific CAR, with potential for ensuring persistent production of effector cells of multiple lineages targeting B-lineage malignant cells. Assessments were performed using in vitro myeloid or natural killer (NK) cell differentiation of human HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. Gene transfer did not impair hematopoietic differentiation and cell proliferation when transduced at 1-2 copies/cell. CAR-bearing myeloid and NK cells specifically lysed CD19-positive cells, with second-generation CAR including CD28 domains being more efficient in NK cells. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a strategy for generating multiple lineages of effector cells for immunotherapy against B-lineage malignancies to augment graft-versus-leukemia activity.
- Published
- 2013
- Full Text
- View/download PDF
42. Integrase-defective lentiviral vectors as a delivery platform for targeted modification of adenosine deaminase locus.
- Author
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Joglekar AV, Hollis RP, Kuftinec G, Senadheera S, Chan R, and Kohn DB
- Subjects
- Endonucleases genetics, Genetic Loci, Humans, K562 Cells, Lentivirus metabolism, Zinc Fingers, Adenosine Deaminase genetics, Endonucleases metabolism, Genetic Vectors, Integrases genetics, Lentivirus genetics, Transduction, Genetic, Virus Integration
- Abstract
We investigated the use of integrase-defective lentiviral vectors (IDLVs) for transient delivery of zinc finger nucleases (ZFNs) and donor templates for site-specific modification of the human adenosine deaminase (hADA) gene. Initially, we constructed IDLVs carrying ZFN monomers (Single-IDLVs) and found them to be able to deliver their gene-editing payload to K562 cells successfully upon cotransduction, with minimal cytotoxicity. To simplify delivery, we designed an IDLV construct to deliver both ZFN monomers from the same vector (Double-IDLV). However, this construct in its original state was prone to rearrangements of the vector genome, resulting in greatly reduced functionality; this was due to recombination between highly similar ZFN monomers arranged in tandem. We modified the Double-IDLV constructs to reduce recombination and restored simultaneous delivery of both ZFNs. We also tested an IDLV construct for delivery of donor templates and demonstrated its efficacy for gene modification. In summary, we highlighted the importance of modifying vector design for co-delivery of highly similar sequences inherent to genome-editing nucleases, and demonstrated significant improvement in the use of IDLVs for delivery of ZFNs and donor templates for genome modification.
- Published
- 2013
- Full Text
- View/download PDF
43. β-globin gene transfer to human bone marrow for sickle cell disease.
- Author
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Romero Z, Urbinati F, Geiger S, Cooper AR, Wherley J, Kaufman ML, Hollis RP, de Assin RR, Senadheera S, Sahagian A, Jin X, Gellis A, Wang X, Gjertson D, Deoliveira S, Kempert P, Shupien S, Abdel-Azim H, Walters MC, Meiselman HJ, Wenby RB, Gruber T, Marder V, Coates TD, and Kohn DB
- Abstract
Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-βAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-βAS3-FB LV transduced BM CD34+ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total β-globin-like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34+ cells were transplanted into immunodeficient mice, and the human cells recovered after 2-3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34+ cells that were directly differentiated in vitro. These results demonstrate that the CCL-βAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling β-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells.
- Published
- 2013
- Full Text
- View/download PDF
44. Highly efficient large-scale lentiviral vector concentration by tandem tangential flow filtration.
- Author
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Cooper AR, Patel S, Senadheera S, Plath K, Kohn DB, and Hollis RP
- Subjects
- Animals, Antigens, CD34 metabolism, Cell Culture Techniques, Cell Line, Tumor, Fibroblasts metabolism, HT29 Cells, Hematopoietic Stem Cells metabolism, Humans, Induced Pluripotent Stem Cells metabolism, Lentivirus genetics, Mice, Transduction, Genetic, Centrifugation, Filtration methods, Genetic Vectors isolation & purification, Lentivirus isolation & purification
- Abstract
Large-scale lentiviral vector (LV) concentration can be inefficient and time consuming, often involving multiple rounds of filtration and centrifugation. This report describes a simpler method using two tangential flow filtration (TFF) steps to concentrate liter-scale volumes of LV supernatant, achieving in excess of 2000-fold concentration in less than 3h with very high recovery (>97%). Large volumes of LV supernatant can be produced easily through the use of multi-layer flasks, each having 1720 cm(2) surface area and producing ∼560 mL of supernatant per flask. Combining the use of such flasks and TFF greatly simplifies large-scale production of LV. As a demonstration, the method is used to produce a very high titer LV (>10(10)TU/mL) and transduce primary human CD34+ hematopoietic stem/progenitor cells at high final vector concentrations with no overt toxicity. A complex LV (STEMCCA) for induced pluripotent stem cell (iPSC) generation is also concentrated from low initial titer and used to transduce and reprogram primary human fibroblasts with no overt toxicity. Additionally, a generalized and simple multiplexed real-time PCR assay is described for lentiviral vector titer and copy number determination., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
45. Stable transgene expression in primitive human CD34+ hematopoietic stem/progenitor cells, using the Sleeping Beauty transposon system.
- Author
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Sumiyoshi T, Holt NG, Hollis RP, Ge S, Cannon PM, Crooks GM, and Kohn DB
- Subjects
- Animals, Antigens, CD34 immunology, Cell Separation, Fetal Blood cytology, Gene Expression, Gene Transfer Techniques, Green Fluorescent Proteins genetics, Humans, Jurkat Cells, Mice, Mice, SCID, DNA Transposable Elements, Genetic Therapy methods, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells metabolism, Transgenes, Transposases genetics
- Abstract
Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. In this study, we improved the SB transposon-mediated gene transfer system for transduction of human CD34(+) stem/progenitor cells by two approaches: (1) to increase the transposition efficacy, a hyperactive mutant of SB, HSB, was used; (2) to improve the expression of the SB transposase and the transgene cassette carried by the transposon, different viral and cellular promoters were evaluated. SB components were delivered in trans into the target cells by Nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression both in vitro and in vivo. In purified human cord blood CD34(+) cells, HSB achieved long-term transgene expression in nearly 7-fold more cells than the original SB transposase. Significantly brighter levels of eGFP expression (5-fold) were achieved with the human elongation factor 1alpha (EF1-alpha) promoter in Jurkat human T cells, compared with that achieved with the modified myeloproliferative sarcoma virus long terminal repeat enhancer-promoter (MNDU3); in contrast, the MNDU3 promoter expressed eGFP at the highest level in K-562 myeloid cells. In human CD34(+) cord blood cells studied under conditions directing myeloid differentiation, the highest transgene integration and expression were achieved using the EF1-alpha promoter to express the SB transposase combined with the MNDU3 promoter to express the eGFP reporter. Stable transgene expression was achieved at levels up to 27% for more than 4 weeks of culture after improved gene transfer to CD34(+) cells (average, 17%; n = 4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34(+) cells demonstrated that SB-modified human CD34(+) cells engrafted in NOD/SCID/gamma chain(null) (NSG) mice and differentiated into multilineage cell types with eGFP expression. More importantly, secondary transplantation studies demonstrated that the integrated transgene was stably expressed in more primitive CD34(+) hematopoietic stem cells (HSCs) with long-term repopulating capability. This study demonstrates that an improved HSB gene transfer system can stably integrate genes into primitive human HSCs while maintaining the pluripotency of the stem cells, which shows promise for further advancement of non-virus-based gene therapy using hematopoietic stem cells.
- Published
- 2009
- Full Text
- View/download PDF
46. Stable gene transfer to human CD34(+) hematopoietic cells using the Sleeping Beauty transposon.
- Author
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Hollis RP, Nightingale SJ, Wang X, Pepper KA, Yu XJ, Barsky L, Crooks GM, and Kohn DB
- Subjects
- Humans, K562 Cells, Time Factors, Transposases genetics, Antigens, CD34, DNA Transposable Elements genetics, Electroporation methods, Gene Expression, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Mutagenesis, Insertional methods, Transposases biosynthesis
- Abstract
Objective: Methods of gene transfer to hematopoietic stem cells that result in stable integration may provide treatments for many inherited and acquired blood diseases. It has been demonstrated previously that a gene delivery system based on the Sleeping Beauty (SB) transposon can be derived where a plasmid transiently expressing the SB transposase can mediate the stable chromosomal integration of a codelivered second plasmid containing a gene expression unit flanked by the inverted repeats derived from the transposon., Methods: Plasmid DNA containing the elements required for SB transposition was delivered to hematopoietic cells via electroporation. Integrated transgene (enhanced green fluorescent protein [eGFP]) expression was assessed in vitro and in vivo., Results: In the K562 human hematopoietic cell line, we observed stable expression of eGFP in >60% of cells for over 2 months after electroporation of the two plasmids; in contrast, in control cells either not treated with transposase or exposed to a defective mutant transposase, the level of gene expression had fallen to near background (<0.1%) by 2 weeks. In purified human cord blood CD34(+) progenitor cells, the transposase led to stable gene transfer at levels up to 6% for over 4 weeks, but gene transfer to more primitive nonobese diabetic/severe combined immunodeficient repopulating cells or CD34(+)/CD38(-) in long-term culture was low and electroporation of the cells with plasmid DNA caused significant cell death., Conclusion: The long-term stable expression highlights the potential of this transposase-based gene delivery method for ameliorating diseases affecting the hematopoietic system, although further improvements in gene transfer efficacy are needed.
- Published
- 2006
- Full Text
- View/download PDF
47. Transient gene expression by nonintegrating lentiviral vectors.
- Author
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Nightingale SJ, Hollis RP, Pepper KA, Petersen D, Yu XJ, Yang C, Bahner I, and Kohn DB
- Subjects
- Antigens, CD34 metabolism, Base Sequence, Cells, Cultured, Drug Resistance, Genetic Therapy methods, Green Fluorescent Proteins genetics, HIV-1 genetics, HT29 Cells, Hematopoietic Stem Cells metabolism, Humans, Jurkat Cells, Kanamycin Kinase genetics, Molecular Sequence Data, Neomycin pharmacology, Gene Expression Regulation, Genetic Vectors genetics, Lentivirus genetics, Virus Integration
- Abstract
Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34(+) hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eGFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (approximately 90% of cells), but expression was transient, diminishing toward background (<0.5%) within less than 1 month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 10(4)-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.
- Published
- 2006
- Full Text
- View/download PDF
48. Phage integrases for the construction and manipulation of transgenic mammals.
- Author
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Hollis RP, Stoll SM, Sclimenti CR, Lin J, Chen-Tsai Y, and Calos MP
- Subjects
- Animals, Attachment Sites, Microbiological genetics, Base Sequence, Cell Line, Chromosome Deletion, Genome, Humans, Mammals embryology, Mice, Mice, Transgenic, Mutagenesis, Site-Directed genetics, Bacillus Phages enzymology, Gene Targeting methods, Integrases metabolism, Mammals metabolism, Recombination, Genetic genetics, Transgenes genetics
- Abstract
Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites. Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule. Here we demonstrate the ability of the phiC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos. Transgenic early embryos and a mid-gestation mouse are reported. We also demonstrate the ability of the phiC31, R4, and TP901-1 phage integrases to recombine two introduced att sites on the same chromosome in human cells, resulting in deletion of the intervening material. We compare the frequencies of mammalian chromosomal deletion catalyzed by these three integrases in different chromosomal locations. The results reviewed here introduce these bacteriophage integrases as tools for site-specific modification of the genome for the creation and manipulation of transgenic mammals.
- Published
- 2003
- Full Text
- View/download PDF
49. Site-specific genomic integration produces therapeutic Factor IX levels in mice.
- Author
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Olivares EC, Hollis RP, Chalberg TW, Meuse L, Kay MA, and Calos MP
- Subjects
- Animals, Gene Expression Regulation, Genetic Therapy methods, Genome, Injections, Intravenous, Integrases genetics, Integrases metabolism, Liver drug effects, Liver physiology, Mice, Mice, Inbred C57BL, Plasmids administration & dosage, Plasmids genetics, Reproducibility of Results, Sensitivity and Specificity, Sequence Analysis, DNA, Factor IX biosynthesis, Factor IX genetics, Liver metabolism, Mutagenesis, Site-Directed
- Abstract
We used the integrase from phage phiC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to approximately 4 microg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration.
- Published
- 2002
- Full Text
- View/download PDF
50. Phage R4 integrase mediates site-specific integration in human cells.
- Author
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Olivares EC, Hollis RP, and Calos MP
- Subjects
- Base Sequence, Cell Division drug effects, Cell Division genetics, Cell Line, DNA, Viral genetics, Drug Resistance, Microbial genetics, Genome, Human, Humans, Integrases genetics, Luciferases genetics, Luciferases metabolism, Molecular Sequence Data, Neomycin pharmacology, Plasmids genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Recombination, Genetic genetics, Transfection, Virus Integration, Attachment Sites, Microbiological genetics, Bacteriophages enzymology, Integrases metabolism
- Abstract
The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.
- Published
- 2001
- Full Text
- View/download PDF
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