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Quintana-Bustamante O, Fañanas-Baquero S, Dessy-Rodriguez M, Ojeda-Pérez I, Segovia JC. Gene Editing for Inherited Red Blood Cell Diseases. Front Physiol 2022; 13:848261. [PMID: 35418876 PMCID: PMC8995967 DOI: 10.3389/fphys.2022.848261] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2022] [Accepted: 02/28/2022] [Indexed: 11/24/2022] Open
Abstract
Today gene therapy is a real therapeutic option to address inherited hematological diseases that could be beneficial for thousands of patients worldwide. Currently, gene therapy is used to treat different monogenic hematological pathologies, including several red blood cell diseases such as β-thalassemia, sickle cell disease and pyruvate kinase deficiency. This approach is based on addition gene therapy, which consists of the correction of hematopoietic stem cells (HSCs) using lentiviral vectors, which integrate a corrected version of the altered gene. Lentivirally-corrected HSCs generate healthy cells that compensate for the deficiency caused by genetic mutations. Despite its successful results, this approach lacks both control of the integration of the transgene into the genome and endogenous regulation of the therapeutic gene, both of which are important aspects that might be a cause for concern. To overcome these limitations, gene editing is able to correct the altered gene through more precise and safer approaches. Cheap and easy-to-design gene editing tools, such as the CRISPR/Cas9 system, allow the specific correction of the altered gene without affecting the rest of the genome. Inherited erythroid diseases, such as thalassemia, sickle cell disease and Pyruvate Kinase Deficiency, have been the test bed for these gene editing strategies, and promising results are currently being seen. CRISPR/Cas9 system has been successfully used to manipulate globin regulation to re-activate fetal globin chains in adult red blood cells and to compensate for hemoglobin defects. Knock-in at the mutated locus to express the therapeutic gene under the endogenous gene regulatory region has also been accomplished successfully. Thanks to the lessons learned from previous lentiviral gene therapy research and trials, gene editing for red blood cell diseases is rapidly moving from its proof-of-concept to its first exciting results in the clinic. Indeed, patients suffering from β-thalassemia and sickle cell disease have already been successfully treated with gene editing, which will hopefully inspire the use of gene editing to cure erythroid disorders and many other inherited diseases in the near future.
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Affiliation(s)
- Oscar Quintana-Bustamante
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Unidad Mixta de Terapias Avanzadas, Madrid, Spain
| | - Sara Fañanas-Baquero
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Unidad Mixta de Terapias Avanzadas, Madrid, Spain
| | - Mercedes Dessy-Rodriguez
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Unidad Mixta de Terapias Avanzadas, Madrid, Spain
| | - Isabel Ojeda-Pérez
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Unidad Mixta de Terapias Avanzadas, Madrid, Spain
| | - Jose-Carlos Segovia
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Unidad Mixta de Terapias Avanzadas, Madrid, Spain
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Amino acid modified [70] fullerene derivatives with high radical scavenging activity as promising bodyguards for chemotherapy protection. Sci Rep 2018; 8:16573. [PMID: 30410075 PMCID: PMC6224443 DOI: 10.1038/s41598-018-34967-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2017] [Accepted: 03/22/2018] [Indexed: 12/21/2022] Open
Abstract
Despite the great efforts for tumor therapy in the last decades, currently chemotherapy induced toxicity remains a formidable problem for cancer patients, and it usually prohibits the cancer therapy from successful completion due to severe side effects. In general, the main side effects of chemotherapeutic agents are from the as-produced reactive oxygen species (ROS) that not only harm the tumor cells but also damage the patients’ organs. Here we report the application of amino acid derivatives of fullerene (AADF) in the chemotherapy which strongly scavenge the excess ROS to protect the tested mice against the chemotherapy-induced hepatotoxicity and cardiotoxicity. Two amino acids, i.e., L-lysine and β-alanine were separately employed to chemically modify C70 fullerene, and L-lysine derivative of fullerene (C70-Lys) exhibits superior radical scavenging activity to β-alanine derivative of C70 (C70-Ala). As expected, C70-Lys show much better protective effect than C70-Ala against the chemotherapy injuries in vivo, which is verified by various histopathological, haematological examinations and antioxidative enzyme studies. Moreover, the L-glutathione level is increased and the cytochrome P-450 2E1 expression is inhibited. They are potentially developed as promising bodyguards for chemotherapy protection.
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Bhukhai K, de Dreuzy E, Giorgi M, Colomb C, Negre O, Denaro M, Gillet-Legrand B, Cheuzeville J, Paulard A, Trebeden-Negre H, Borwornpinyo S, Sii-Felice K, Maouche L, Down JD, Leboulch P, Payen E. Ex Vivo Selection of Transduced Hematopoietic Stem Cells for Gene Therapy of β-Hemoglobinopathies. Mol Ther 2018; 26:480-495. [PMID: 29221807 PMCID: PMC5835017 DOI: 10.1016/j.ymthe.2017.10.015] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2017] [Revised: 10/11/2017] [Accepted: 10/18/2017] [Indexed: 12/14/2022] Open
Abstract
Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the βAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure's suitability for human clinical applications while affording the additional safety of conditional suicide.
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Affiliation(s)
- Kanit Bhukhai
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France
| | - Edouard de Dreuzy
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France
| | - Marie Giorgi
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France
| | - Charlotte Colomb
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France
| | - Olivier Negre
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France; bluebird bio, Inc., Cambridge, MA 02141, USA; bluebird bio France, Fontenay aux Roses 92260, France
| | | | - Béatrix Gillet-Legrand
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France; bluebird bio France, Fontenay aux Roses 92260, France
| | - Joëlle Cheuzeville
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France; bluebird bio France, Fontenay aux Roses 92260, France
| | - Anaïs Paulard
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France; bluebird bio France, Fontenay aux Roses 92260, France
| | | | | | - Karine Sii-Felice
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France
| | - Leila Maouche
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France; INSERM, Paris 75013, France
| | - Julian D Down
- Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Phillippe Leboulch
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France; Ramathibodi Hospital, Bangkok 10400, Thailand; Harvard Medical School and Genetics Division, Department of Medicine, Brigham & Women's Hospital, Boston, MA 02115, USA.
| | - Emmanuel Payen
- CEA, Institute of Biology François Jacob, Fontenay aux Roses 92260, France; UMR_007, CEA and University of Paris Saclay, Fontenay aux Roses 92260, France; INSERM, Paris 75013, France.
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Zhou Y, Deng R, Zhen M, Li J, Guan M, Jia W, Li X, Zhang Y, Yu T, Zou T, Lu Z, Guo J, Sun L, Shu C, Wang C. Amino acid functionalized gadofullerene nanoparticles with superior antitumor activity via destruction of tumor vasculature in vivo. Biomaterials 2017; 133:107-118. [DOI: 10.1016/j.biomaterials.2017.04.025] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2017] [Revised: 04/05/2017] [Accepted: 04/13/2017] [Indexed: 11/16/2022]
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Dong AC, Rivella S. Gene Addition Strategies for β-Thalassemia and Sickle Cell Anemia. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 1013:155-176. [PMID: 29127680 DOI: 10.1007/978-1-4939-7299-9_6] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Beta-thalassemia and sickle cell anemia are two of the most common diseases related to the hemoglobin protein. In these diseases, the beta-globin gene is mutated, causing severe anemia and ineffective erythropoiesis. Patients can additionally present with a number of life-threatening co-morbidities, such as stroke or spontaneous fractures. Current treatment involves transfusion and iron chelation; allogeneic bone marrow transplant is the only curative option, but is limited by the availability of matching donors and graft-versus-host disease. As these two diseases are monogenic diseases, they make an attractive setting for gene therapy. Gene therapy aims to correct the mutated beta-globin gene or add back a functional copy of beta- or gamma-globin. Initial gene therapy work was done with oncoretroviral vectors, but has since shifted to lentiviral vectors. Currently, there are a few clinical trials underway to test the curative potential of some of these lentiviral vectors. This review will highlight the work done thus far, and present the challenges still facing gene therapy, such as genome toxicity concerns and achieving sufficient transgene expression to cure those with the most severe forms of thalassemia.
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Affiliation(s)
- Alisa C Dong
- Division of Hematology-Oncology, Department of Pediatrics, Weill Cornell Medical College, 515 E. 71st St., Room S-709, New York, NY, 10021, USA
| | - Stefano Rivella
- Division of Hematology-Oncology, Department of Pediatrics, Weill Cornell Medical College, 515 E. 71st St., S702, Box 284, New York, NY, 10021, USA.
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Palesch D, Boldt F, Müller JA, Eisele K, Stürzel CM, Wu Y, Münch J, Weil T. PEGylated Cationic Serum Albumin for Boosting Retroviral Gene Transfer. Chembiochem 2016; 17:1504-8. [PMID: 27239020 DOI: 10.1002/cbic.201600193] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2016] [Indexed: 01/28/2023]
Abstract
Retroviral vectors are common tools for introducing genes into the genome of a cell. However, low transduction rates are a major limitation in retroviral gene transfer, especially in clinical applications. We generated cationic human serum albumin (cHSA) protected by a shell of poly(ethylene glycol) (PEG); this significantly enhanced retroviral gene transduction with potentially attractive pharmacokinetics and low immunogenicity. By screening a panel of chemically optimized HSA compounds, we identified a very potent enhancer that boosted the transduction rates of viral vectors. Confocal microscopy revealed a drastically increased number of viral particles attached to the surfaces of target cells. In accordance with the positive net charge of cationic and PEGylated HSA, this suggests a mechanism of action in which the repulsion of the negatively charged cellular and viral vector membranes is neutralized, thereby promoting attachment and ultimately transduction. Importantly, the transduction-enhancing PEGylated HSA derivative evaded recognition by HSA-specific antibodies and macrophage activation. Our findings hold great promise for facilitating improved retroviral gene transfer.
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Affiliation(s)
- David Palesch
- Institute of Molecular Virology, Ulm University Medical Center, Meyerhofstrasse 1, 89081, Ulm, Germany
| | - Felix Boldt
- Institute of Organic Chemistry III/Macromolecular Chemistry, Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany
| | - Janis A Müller
- Institute of Molecular Virology, Ulm University Medical Center, Meyerhofstrasse 1, 89081, Ulm, Germany
| | - Klaus Eisele
- Institute of Organic Chemistry III/Macromolecular Chemistry, Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany
| | - Christina M Stürzel
- Institute of Molecular Virology, Ulm University Medical Center, Meyerhofstrasse 1, 89081, Ulm, Germany
| | - Yuzhou Wu
- Institute of Organic Chemistry III/Macromolecular Chemistry, Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany
| | - Jan Münch
- Institute of Molecular Virology, Ulm University Medical Center, Meyerhofstrasse 1, 89081, Ulm, Germany.
| | - Tanja Weil
- Institute of Organic Chemistry III/Macromolecular Chemistry, Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany.
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Nagree MS, López-Vásquez L, Medin JA. Towards in vivo amplification: Overcoming hurdles in the use of hematopoietic stem cells in transplantation and gene therapy. World J Stem Cells 2015; 7:1233-1250. [PMID: 26730268 PMCID: PMC4691692 DOI: 10.4252/wjsc.v7.i11.1233] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/08/2015] [Accepted: 11/25/2015] [Indexed: 02/06/2023] Open
Abstract
With the advent of safer and more efficient gene transfer methods, gene therapy has become a viable solution for many inherited and acquired disorders. Hematopoietic stem cells (HSCs) are a prime cell compartment for gene therapy aimed at correcting blood-based disorders, as well as those amenable to metabolic outcomes that can effect cross-correction. While some resounding clinical successes have recently been demonstrated, ample room remains to increase the therapeutic output from HSC-directed gene therapy. In vivo amplification of therapeutic cells is one avenue to achieve enhanced gene product delivery. To date, attempts have been made to provide HSCs with resistance to cytotoxic drugs, to include drug-inducible growth modules specific to HSCs, and to increase the engraftment potential of transduced HSCs. This review aims to summarize amplification strategies that have been developed and tested and to discuss their advantages along with barriers faced towards their clinical adaptation. In addition, next-generation strategies to circumvent current limitations of specific amplification schemas are discussed.
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Brennig S, Lachmann N, Buchegger T, Hetzel M, Schambach A, Moritz T. Chemoprotection of murine hematopoietic cells by combined gene transfer of cytidine deaminase (CDD) and multidrug resistance 1 gene (MDR1). JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2015; 34:148. [PMID: 26651614 PMCID: PMC4676838 DOI: 10.1186/s13046-015-0260-4] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/01/2015] [Accepted: 11/16/2015] [Indexed: 01/23/2023]
Abstract
Background Hematologic toxicity represents a major side effect of cytotoxic chemotherapy frequently preventing adequately dosed chemotherapy application and impeding therapeutic success. Transgenic (over)expression of chemotherapy resistance (CTX-R) genes in hematopoietic stem- and progenitor cells represents a potential strategy to overcome this problem. To apply this concept in the context of acute myeloid leukemia and myelodysplasia, we have investigated the overexpression of the multidrug resistance 1 (MDR1) and the cytidine deaminase (CDD) gene conferring resistance to anthracyclines and cytarabine (Ara-C), the two most important drugs in the treatment of these diseases. Methods State-of-the-art, third generation, self-inactivating (SIN) lentiviral vectors were utilized to overexpress a human CDD-cDNA and a codon-optimized human MDR1-cDNA corrected for cryptic splice sites from a spleen focus forming virus derived internal promoter. Studies were performed in myeloid 32D cells as well as primary lineage marker negative (lin−) murine bone marrow cells and flow cytometric analysis of suspension cultures and clonogenic analysis of vector transduced cells following cytotoxic drug challenge were utilized as read outs. Results Efficient chemoprotection of CDD and MDR1 transduced hematopoietic 32D as well as primary lin− cells was proven in the context of Ara-C and anthracycline application. Both, CTX-R transduced 32D as well as primary hematopoietic cells displayed marked resistance at concentrations 5–20 times the LD50 of non-transduced control cells. Moreover, simultaneous CDD/MDR1 gene transfer resulted in similar protection levels even when combined Ara-C anthracycline treatment was applied. Furthermore, significant enrichment of transduced cells was observed upon cytotoxic drug administration. Conclusions Our data demonstrate efficient chemoprotection as well as enrichment of transduced cells in hematopoietic cell lines as well as primary murine hematopoietic progenitor cells following Ara-C and/or anthracycline application, arguing for the efficacy as well as feasibility of our approach and warranting further evaluation of this concept. Electronic supplementary material The online version of this article (doi:10.1186/s13046-015-0260-4) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Sebastian Brennig
- Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, D-30625, Germany.,Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Nico Lachmann
- Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, D-30625, Germany.,Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,JRG Translational Hematology of Congenital Diseases, REBIRTH Cluster-of Excellence, Hannover Medical School, Hannover, Germany
| | - Theresa Buchegger
- Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, D-30625, Germany.,Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Miriam Hetzel
- Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, D-30625, Germany.,Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Axel Schambach
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, USA
| | - Thomas Moritz
- Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, D-30625, Germany. .,Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
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Schroeder JA, Chen Y, Fang J, Wilcox DA, Shi Q. In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection. J Thromb Haemost 2014; 12:1283-93. [PMID: 24931217 PMCID: PMC4127102 DOI: 10.1111/jth.12633] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2014] [Accepted: 06/02/2014] [Indexed: 12/25/2022]
Abstract
BACKGROUND Our previous studies have demonstrated that platelet-specific gene delivery to hematopoietic stem cells can induce sustained therapeutic levels of platelet factor VIII (FVIII) expression in mice with hemophilia A. OBJECTIVE In this study, we aimed to enhance platelet FVIII expression while minimizing potential toxicities. METHODS A novel lentiviral vector (LV), which harbors dual genes, the FVIII gene driven by the αIIb promoter (2bF8) and a drug-resistance gene, the MGMT(P140K) cassette, was constructed. Platelet FVIII expression in mice with hemophilia A was introduced by transduction of hematopoietic stem cells and transplantation. The recipients were treated with O(6)-benzylguanine followed by 1,3-bis-2 chloroethyl-1-nitrosourea monthly three or four times. Animals were analyzed by using polymerase chain reaction (PCR), quantitative PCR, FVIII:C assays, and inhibitor assays. Phenotypic correction was assessed by tail clipping tests and rotational thromboelastometry analysis. RESULTS Even using a low multiplicity of infection of 1 and a non-myeloablative conditioning regimen, after in vivo selection, the levels of platelet FVIII expression in recipients increased to 4.33 ± 5.48 mU per 10(8) platelets (n = 16), which were 19.7-fold higher than the levels obtained from the recipients before treatment. Quantitative PCR results confirmed that 2bF8/MGMT-LV-transduced cells were effectively enriched after drug-selective treatment. Fifteen of 16 treated animals survived tail clipping. Blood loss and whole blood clotting time were normalized in the treated recipients. Notably, no anti-FVIII antibodies were detected in the treated animals even after recombinant human B-domain deleted FVIII challenge. CONCLUSION we have established an effective in vivo selective system that allows us to enrich 2bF8LV-transduced cells, enhancing platelet FVIII expression while reducing the potential toxicities associated with platelet gene therapy.
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Affiliation(s)
- J A Schroeder
- Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA; Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA; Children's Research Institute, Children's Hospital of Wisconsin, Milwaukee, WI, USA
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Larochelle A, Dunbar CE. Hematopoietic stem cell gene therapy:assessing the relevance of preclinical models. Semin Hematol 2014; 50:101-30. [PMID: 24014892 DOI: 10.1053/j.seminhematol.2013.03.025] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
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Giannaccini M, Cuschieri A, Dente L, Raffa V. Non-mammalian vertebrate embryos as models in nanomedicine. NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE 2013; 10:703-19. [PMID: 24103306 DOI: 10.1016/j.nano.2013.09.010] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/22/2013] [Revised: 09/12/2013] [Accepted: 09/23/2013] [Indexed: 01/27/2023]
Abstract
UNLABELLED Various in vivo biological models have been proposed for studying the interactions of nano-materials in biological systems. Unfortunately, the widely used small mammalian animal models (rodents) are costly and labor intensive and generate ethical issues and antagonism from the anti-vivisectionist movement. Recently, there has been increasing interest in the scientific community in the interactions between nano-materials and non-mammalian developmental organisms, which are now being recognized as valid models for the study of human disease. This review examines and discusses the biomedical applications and the interaction of nano-materials with embryonic systems, focusing on non-mammalian vertebrate models, such as chicken, zebrafish and Xenopus. FROM THE CLINICAL EDITOR Animal models are critical components of preclinical biomedical research. This review discusses the feasibility and potential applications of non-mammalian vertebral animals, such as zebrafish, xenopus, and chicken as animal models in nanomedicine research.
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Affiliation(s)
- Martina Giannaccini
- Department of Biology, Cell and Developmental Biology Unit, Università di Pisa, Pisa, Italy; Institute of Life Science, Scuola Superiore Sant'Anna, Pisa, Italy.
| | - Alfred Cuschieri
- Institute of Life Science, Scuola Superiore Sant'Anna, Pisa, Italy
| | - Luciana Dente
- Department of Biology, Cell and Developmental Biology Unit, Università di Pisa, Pisa, Italy
| | - Vittoria Raffa
- Department of Biology, Cell and Developmental Biology Unit, Università di Pisa, Pisa, Italy; Institute of Life Science, Scuola Superiore Sant'Anna, Pisa, Italy
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Myeloprotection by cytidine deaminase gene transfer in antileukemic therapy. Neoplasia 2013; 15:239-48. [PMID: 23479503 DOI: 10.1593/neo.121954] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2012] [Revised: 01/03/2013] [Accepted: 01/07/2013] [Indexed: 12/22/2022] Open
Abstract
Gene transfer of drug resistance (CTX-R) genes can be used to protect the hematopoietic system from the toxicity of anticancer chemotherapy and this concept recently has been proven by overexpression of a mutant O(6)-methylguaninemethyltransferase in the hematopoietic system of glioblastoma patients treated with temozolomide. Given its protection capacity against such relevant drugs as cytosine arabinoside (ara-C), gemcitabine, decitabine, or azacytidine and the highly hematopoiesis-specific toxicity profile of several of these agents, cytidine deaminase (CDD) represents another interesting candidate CTX-R gene and our group recently has established the myeloprotective capacity of CDD gene transfer in a number of murine transplant studies. Clinically, CDD overexpression appears particularly suited to optimize treatment strategies for acute leukemias and myelodysplasias given the efficacy of ara-C (and to a lesser degree decitabine and azacytidine) in these disease entities. This article will review the current state of the art with regard to CDD gene transfer and point out potential scenarios for a clinical application of this strategy. In addition, risks and potential side effects associated with this approach as well as strategies to overcome these problems will be highlighted.
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Jonnalagadda M, Brown CE, Chang WC, Ostberg JR, Forman SJ, Jensen MC. Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy. PLoS One 2013; 8:e65519. [PMID: 23755242 PMCID: PMC3675038 DOI: 10.1371/journal.pone.0065519] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2012] [Accepted: 04/26/2013] [Indexed: 11/18/2022] Open
Abstract
Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.
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Affiliation(s)
- Mahesh Jonnalagadda
- Departments of Cancer Immunotherapeutics & Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope, Duarte, California, United States of America
| | - Christine E. Brown
- Departments of Cancer Immunotherapeutics & Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope, Duarte, California, United States of America
| | - Wen-Chung Chang
- Departments of Cancer Immunotherapeutics & Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope, Duarte, California, United States of America
| | - Julie R. Ostberg
- Departments of Cancer Immunotherapeutics & Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope, Duarte, California, United States of America
| | - Stephen J. Forman
- Departments of Cancer Immunotherapeutics & Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope, Duarte, California, United States of America
| | - Michael C. Jensen
- Departments of Cancer Immunotherapeutics & Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope, Duarte, California, United States of America
- Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Seattle, Washington, United States of America
- * E-mail:
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Choudhary R, Baturin D, Fosmire S, Freed B, Porter CC. Knockdown of HPRT for selection of genetically modified human hematopoietic progenitor cells. PLoS One 2013; 8:e59594. [PMID: 23555045 PMCID: PMC3598703 DOI: 10.1371/journal.pone.0059594] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2012] [Accepted: 02/15/2013] [Indexed: 12/14/2022] Open
Abstract
The inability to obtain sufficient numbers of transduced cells remains a limitation in gene therapy. One strategy to address this limitation is in vivo pharmacologic selection of transduced cells. We have previously shown that knockdown of HPRT using lentiviral delivered shRNA facilitates efficient selection of transduced murine hematopoietic progenitor cells (HPC) using 6-thioguanine (6TG). Herein, we now extend these studies to human HPC. We tested multiple shRNA constructs in human derived cell lines and identified the optimal shRNA sequence for knockdown of HPRT and 6TG resistance. We then tested this vector in human umbilical cord blood derived HPC in vitro and in NOD/SCID recipients. Knockdown of HPRT effectively provided resistance to 6TG in vitro. 6TG treatment of mice resulted in increased percentages of transduced human CD45(+) cells in the peripheral blood and in the spleen in particular, in both myeloid and lymphoid compartments. 6TG treatment of secondary recipients resulted in higher percentages of transduced human cells in the bone marrow, confirming selection from the progeny of long-term repopulating HPCs. However, the extent of selection of cells in the bone marrow at the doses of 6TG tested and the toxicity of higher doses, suggest that this strategy may be limited to selection of more committed progenitor cells. Together, these data suggest that human HPC can be programmed to be resistant to purine analogs, but that HPRT knockdown/6TG-based selection may not be robust enough for in vivo selection.
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Affiliation(s)
- Rashmi Choudhary
- Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, United States of America
| | - Dmitry Baturin
- Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, United States of America
| | - Susan Fosmire
- Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, United States of America
| | - Brian Freed
- Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado, United States of America
- Department of Immunology, University of Colorado School of Medicine, Aurora, Colorado, United States of America
| | - Christopher C. Porter
- Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, United States of America
- * E-mail:
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15
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Chemoselection of allogeneic HSC after murine neonatal transplantation without myeloablation or post-transplant immunosuppression. Mol Ther 2012; 20:2180-9. [PMID: 22871662 DOI: 10.1038/mt.2012.136] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
The feasibility of allogeneic transplantation, without myeloablation or post-transplant immunosuppression, was tested using in vivo chemoselection of allogeneic hematopoietic stem cells (HSCs) after transduction with a novel tricistronic lentiviral vector (MGMT(P140K)-2A-GFP-IRES-TK (MAGIT)). This vector contains P140K-O(6)-methylguanine-methyltransferase (MGMT(P140K)), HSV-thymidine kinase (TK(HSV)), and enhanced green fluorescent protein (eGFP) enabling (i) in vivo chemoselection of HSC by conferring resistance to benzylguanine (BG), an inhibitor of endogenous MGMT, and to chloroethylating agents such as 1,3-bis(2-chloroethyl)nitrosourea (BCNU) and, (ii) depletion of proliferating cells such as malignant clones or transduced donor T cells mediating graft versus host disease (GVHD), by expression of the suicide gene TK(HSV) and Ganciclovir (GCV) administration. Non-myeloablative transplantation of transduced, syngeneic, lineage-depleted (Lin(-)) BM in neonates resulted in 0.67% GFP(+) mononuclear cells in peripheral blood. BG/BCNU chemoselection, 4 and 8 weeks post-transplant, produced 50-fold donor cell enrichment. Transplantation and chemoselection of major histocompatibility complex (MHC)-mismatched MAGIT-transduced Lin(-) BM also produced similar expansion for >40 weeks. The efficacy of this allotransplant approach was validated in Hbb(th3) heterozygous mice by correction of β-thalassemia intermedia, without toxicity or GVHD. Negative selection, by administration of GCV resulted in donor cell depletion without graft ablation, as re-expansion of donor cells was achieved with BG/BCNU treatment. These studies show promise for developing non-ablative allotransplant approaches using in vivo positive/negative selection.
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16
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Mesenchymal stem cells: a potential targeted-delivery vehicle for anti-cancer drug, loaded nanoparticles. NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE 2012; 9:174-84. [PMID: 22772046 DOI: 10.1016/j.nano.2012.06.003] [Citation(s) in RCA: 117] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/05/2012] [Accepted: 06/04/2012] [Indexed: 12/22/2022]
Abstract
UNLABELLED The targeted delivery of anticancer agents is a promising field in anticancer therapy. Mesenchymal stem cells (MSCs) have inherent tumor-tropic and migratory properties, which allow them to serve as vehicles for targeted drug delivery systems for isolated tumors and metastatic diseases. MSCs have been successfully studied and discussed as a vehicle for cancer gene therapy. However, MSCs have not yet been discussed adequately as a potential vehicle for traditional anticancer drugs. In this review, we will examine the potential of MSCs as a targeted-delivery vehicle for anticancer drug-loaded nanoparticles (NPs), summarize various challenges, and discuss possible solutions for these challenges. FROM THE CLINICAL EDITOR In this review, the feasibility of mesenchymal stem cell-based targeted delivery of anticancer agents is discussed.
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Brennig S, Rattmann I, Lachmann N, Schambach A, Williams DA, Moritz T. In vivo enrichment of cytidine deaminase gene-modified hematopoietic cells by prolonged cytosine-arabinoside application. Cytotherapy 2012; 14:451-60. [DOI: 10.3109/14653249.2011.646043] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Abstract
Stem and progenitor cells are present in cord blood at a high frequency making these cells a major target population for experimental and clinical studies. Over the past decade there has been considerable developments in cord blood research and transplantation but despite the rapid progress many problems remain. The initial hope that cord blood would be an alternative source of haemopoietic cells for transplantation has been tempered by the fact that there are insufficient cells in most cord blood collections to engraft an adult of average weight. In attempts to increase the cell number, a plethora of techniques for ex-vivo expansion have been developed.These techniques have also proved useful for gene therapy. As cord blood cells possess unique properties this allows them to be utilised as suitable vehicles for gene therapy and long-term engraftment of transduced cells has been achieved. Current work examining the nature of the stem cells present in this haematological source indicates that cord blood contains not only haemopoietic stem cells but also primitive non-haemopoietic cells with high proliferative and developmental potential. As attention focuses on stem cell biology and the controversies surrounding the potential use of embryonic stem cells in treatment of disease, the properties of stem cells from other sources including cord blood are being re-appraised. The purpose of this article is to review some of the current areas of work and highlight biological problems associated with the use of cord blood cells.
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Affiliation(s)
- E A de Wynter
- Molecular Medicine Unit, University of Leeds, St. James's University Hospital, Leeds, U.K. (E-mail,
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19
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Uchida N, Hsieh MM, Hayakawa J, Madison C, Washington KN, Tisdale JF. Optimal conditions for lentiviral transduction of engrafting human CD34+ cells. Gene Ther 2011; 18:1078-86. [PMID: 21544097 DOI: 10.1038/gt.2011.63] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Cytokines are required for γ-retroviral transduction of human CD34+ cells. However, cytokines may reduce engraftment of CD34+ cells and may not be necessary for their lentiviral transduction. We sought to optimize transduction and engraftment of human CD34+ cells using lentiviral vectors. Single 24 h transduction of human CD34+ cells with human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors in media containing stem cell factor (SCF), FMS-like tyrosine kinase 3 (FLT3) ligand, thrombopoietin (each 100 ng ml⁻¹) and 10% fetal bovine serum was compared with various cytokine conditions during ex vivo culture and assayed using humanized xenograft mice for 6 months after transplantation. Serum-free media improved transduction efficiency of human CD34+ cells. Interleukin-3 (20 ng ml⁻¹) had little effect on transduction efficiency or engraftment. Threefold higher cytokine mixture (each 300 ng ml⁻¹) reduced engraftment of CD34+ cells. SCF alone (100 ng ml⁻¹) proved insufficient for maintaining engraftment ability and reduced transduction efficiency. Short-term prestimulation had little effect on transduction efficiency or engraftment, yet 24 h prestimulation showed higher transduction efficiency, higher gene expression levels and lower engraftment. In summary, 24 h prestimulation followed by single 24-h lentiviral transduction in serum-free media with SCF, FLT3 ligand and thrombopoietin yields high transduction efficiency to engrafting human CD34+ cells, and is applicable in human clinical gene therapy trials.
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Affiliation(s)
- N Uchida
- Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute-NHLBI/National Institute of Diabetes and Digestive and Kidney Diseases-NIDDK, National Institutes of Health-NIH, Bethesda, MD 20892, USA
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20
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21
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Mitsuhashi J, Hosoyama H, Tsukahara S, Katayama K, Noguchi K, Ito Y, Hatake K, Aiba K, Takahashi S, Sugimoto Y. In vivo expansion of MDR1-transduced cells accompanied by a post-transplantation chemotherapy regimen with mitomycin C and methotrexate. J Gene Med 2010; 12:596-603. [DOI: 10.1002/jgm.1474] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
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Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine. Anal Biochem 2010; 404:149-54. [PMID: 20450874 DOI: 10.1016/j.ab.2010.04.037] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2010] [Revised: 04/19/2010] [Accepted: 04/30/2010] [Indexed: 11/23/2022]
Abstract
Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O(6)-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders.
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23
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Gene therapy of benign gynecological diseases. Adv Drug Deliv Rev 2009; 61:822-35. [PMID: 19446586 DOI: 10.1016/j.addr.2009.04.023] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2009] [Accepted: 04/28/2009] [Indexed: 11/22/2022]
Abstract
Gene therapy is the introduction of genetic material into patient's cells to achieve therapeutic benefit. Advances in molecular biology techniques and better understanding of disease pathogenesis have validated the use of a variety of genes as potential molecular targets for gene therapy based approaches. Gene therapy strategies include: mutation compensation of dysregulated genes; replacement of defective tumor-suppressor genes; inactivation of oncogenes; introduction of suicide genes; immunogenic therapy and antiangiogenesis based approaches. Preclinical studies of gene therapy for various gynecological disorders have not only shown to be feasible, but also showed promising results in diseases such as uterine leiomyomas and endometriosis. In recent years, significant improvement in gene transfer technology has led to the development of targetable vectors, which have fewer side-effects without compromising their efficacy. This review provides an update on developing gene therapy approaches to treat common gynecological diseases such as uterine leiomyoma and endometriosis.
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24
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Wagner AM, Schoeberlein A, Surbek D. Fetal gene therapy: opportunities and risks. Adv Drug Deliv Rev 2009; 61:813-21. [PMID: 19426772 DOI: 10.1016/j.addr.2009.04.011] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2008] [Accepted: 04/28/2009] [Indexed: 10/20/2022]
Abstract
Advances in human prenatal medicine and molecular genetics have allowed the diagnosis of many genetic diseases early in gestation. In-utero transplantation of allogeneic hematopoietic stem cells (HSC) has been successfully used as a therapy in different animal models and recently also in human fetuses. Unfortunately, clinical success of this novel treatment is limited by the lack of donor cell engraftment in non-immunocompromised hosts and is thus restricted to diseases where the fetus is affected by severe immunodeficiency. Gene therapy using genetically modified autologous HSC circumvents allogeneic HLA barriers and constitutes one of the most promising new approaches to correct genetic deficits in the fetus. Recent developments of strategies to overcome failure of efficient transduction of quiescent hematopoietic cells include the use of new vector constructs and transduction protocols. These improvements open new perspectives for gene therapy in general and for prenatal gene transfer in particular. The fetus may be especially susceptible for successful gene therapy due to the immunologic naiveté of the immature hematopoietic system during gestation, precluding an immune reaction towards the transgene. Ethical issues, in particular those regarding treatment safety, must be taken into account before clinical trials with fetal gene therapy in human pregnancies can be initiated.
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25
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Lee ASJ, Kahatapitiya P, Kramer B, Joya JE, Hook J, Liu R, Schevzov G, Alexander IE, McCowage G, Montarras D, Gunning PW, Hardeman EC. Methylguanine DNA methyltransferase-mediated drug resistance-based selective enrichment and engraftment of transplanted stem cells in skeletal muscle. Stem Cells 2009; 27:1098-108. [PMID: 19415780 DOI: 10.1002/stem.28] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Cell replacement therapy using stem cell transplantation holds much promise in the field of regenerative medicine. In the area of hematopoietic stem cell transplantation, O(6)-methylguanine-DNA methyltransferase MGMT (P140K) gene-mediated drug resistance-based in vivo enrichment strategy of donor stem cells has been shown to achieve up to 75%-100% donor cell engraftment in the host's hematopoietic stem cell compartment following repeated rounds of selection. This strategy, however, has not been applied in any other organ system. We tested the feasibility of using this MGMT (P140K)-mediated enrichment strategy for cell transplantation in skeletal muscles of mice. We demonstrate that muscle cells expressing an MGMT (P140K) drug resistance gene can be protected and selectively enriched in response to alkylating chemotherapy both in vitro and in vivo. Upon transplantation of MGMT (P140K)-expressing male CD34(+ve) donor stem cells isolated from regenerating skeletal muscle into injured female muscle treated with alkylating chemotherapy, donor cells showed enhanced engraftment in the recipient muscle 7 days following transplantation as examined by quantitative-polymerase chain reaction using Y-chromosome specific primers. Fluorescent in situ hybridization analysis using a Y-chromosome paint probe revealed donor-derived de novo muscle fiber formation in the recipient muscle 14 days following transplantation, with approximately 12.5% of total nuclei within the regenerated recipient muscle being of donor origin. Following engraftment, the chemo-protected donor CD34(+ve) cells induced substantial endogenous regeneration of the chemo-ablated host muscle that is otherwise unable to self-regenerate. We conclude that the MGMT (P140K)-mediated enrichment strategy can be successfully implemented in muscle.
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Affiliation(s)
- Antonio S J Lee
- Oncology Research Unit, The Children's Hospital at Westmead, Westmead, New South Wales, Australia
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26
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Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene. Blood 2009; 113:5747-56. [PMID: 19365082 DOI: 10.1182/blood-2008-10-186684] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.
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27
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Bertino JR. Transfer of drug resistance genes into hematopoietic stem cells for marrow protection. Oncologist 2009; 13:1036-42. [PMID: 18952565 DOI: 10.1634/theoncologist.2008-0173] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Affiliation(s)
- Joseph R Bertino
- The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry, New Brunswick, New Jersey, USA.
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28
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Lee HJ, Lee YS, Kim HS, Kim YK, Kim JH, Jeon SH, Lee HW, Kim S, Miyoshi H, Chung HM, Kim DK. Retronectin enhances lentivirus-mediated gene delivery into hematopoietic progenitor cells. Biologicals 2009; 37:203-9. [PMID: 19264508 DOI: 10.1016/j.biologicals.2009.01.008] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2008] [Revised: 01/18/2009] [Accepted: 01/19/2009] [Indexed: 11/26/2022] Open
Abstract
Genetic modification of hematopoietic stem cells holds great promise in the treatment of hematopoietic disorders. However, clinical application of gene delivery has been limited, in part, by low gene transfer efficiency. To overcome this problem, we investigated the effect of retronectin (RN) on lentiviral-mediated gene delivery into hematopoietic progenitor cells (HPCs) derived from bone marrow both in vitro and in vivo. RN has been shown to enhance transduction by promoting colocalization of lentivirus and target cells. We found that RN enhanced lentiviral transfer of the VENUS transgene into cultured c-Kit(+) Lin(-) HPCs. As a complementary approach, in vivo gene delivery was performed by subjecting mice to intra-bone marrow injection of lentivirus or a mixture of RN and lentivirus. We found that co-injection with RN increased the number of VENUS-expressing c-Kit(+) Lin(-) HPCs in bone marrow by 2-fold. Further analysis of VENUS expression in colony-forming cells from the bone marrow of these animals revealed that RN increased gene delivery among these cells by 4-fold. In conclusion, RN is effective in enhancing lentivirus-mediated gene delivery into HPCs.
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Affiliation(s)
- Hyun-Joo Lee
- Graduate School of Life Science and Biotechnology, Pochon CHA University, CHA Stem Cell Institute, 605 Yeoksam 1-dong, Kangnam-gu, Seoul 135-081, Republic of Korea
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29
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Nordling D, Kaiser A, Reeves L. Release testing of retroviral vectors and gene-modified cells. Methods Mol Biol 2009; 506:265-279. [PMID: 19110632 DOI: 10.1007/978-1-59745-409-4_18] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
This chapter will review the design and execution of release testing requirements for retroviral vectors and gene-modified cells consistent with ensuring the success of the clinical trial on the basis of current US regulatory requirements. It is the ethical and legal responsibility of the clinical trial sponsor(s) to ensure safety of the patients through proper evaluation of the drug products prior to use. Any clinical trial drug product used in human subjects must be produced and evaluated for safety, quality, purity, and effectiveness according to Current Good Manufacturing Practices appropriate for the stage of clinical development.
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Affiliation(s)
- Diana Nordling
- Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
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30
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Abstract
Dose-limiting toxicity of chemotherapeutic agents, i.e., myelosuppression, can limit their effectiveness. The transfer and expression of drug-resistance genes might decrease the risks associated with acute hematopoietic toxicity. Protection of hematopoietic stem/progenitor cells by transfer of drug-resistance genes provides the possibility of intensification or escalation of antitumor drug doses and consequently an improved therapeutic index. This chapter reviews drug-resistance gene transfer strategies for either myeloprotection or therapeutic gene selection. Selecting candidate drug-resistance gene(s), gene transfer methodology, evaluating the safety and the efficiency of the treatment strategy, relevant in vivo models, and oncoretroviral transduction of human hematopoietic stem/progenitor cells under clinically applicable conditions are described.
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Affiliation(s)
- Tulin Budak-Alpdogan
- Department of Medicine, The Cancer Institute of New Jersey, Robert Wood Johson Medical School, University of Medicine & Dentistry of New Jersey, New Brunswick, NJ, USA
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31
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Budak-Alpdogan T, Rivière I. Genetic modification of human hematopoietic cells: preclinical optimization of oncoretroviral-mediated gene transfer for clinical trials. Methods Mol Biol 2009; 506:33-58. [PMID: 19110618 PMCID: PMC4360985 DOI: 10.1007/978-1-59745-409-4_4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
This chapter provides information about the oncoretroviral transduction of human hematopoietic stem/ progenitor cells under clinically applicable conditions. We describe in detail a short -60 h transduction protocol which consistently yields transduction efficiencies in the range of 30-50% with five different oncoretroviral vectors. We discuss a number of parameters that affect transduction efficiency, including the oncoretroviral vector characteristics, the vector stock collection, the source of CD34+ cells and transduction conditions.
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Affiliation(s)
- Tulin Budak-Alpdogan
- Department of Medicine, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ, USA
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32
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Persons DA. Hematopoietic stem cell gene transfer for the treatment of hemoglobin disorders. HEMATOLOGY. AMERICAN SOCIETY OF HEMATOLOGY. EDUCATION PROGRAM 2009; 2009:690-697. [PMID: 20008255 DOI: 10.1182/asheducation-2009.1.690] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
Hematopoietic stem cell (HSC)-targeted gene transfer is an attractive approach for the treatment of a number of hematopoietic disorders caused by single gene defects. Indeed, in a series of gene transfer trials for two different primary immunodeficiencies beginning early in this decade, outstanding success has been achieved. Despite generally low levels of engrafted, genetically modified HSCs, these trials were successful because of the marked selective advantage of gene-corrected lymphoid precursors that allowed reconstitution of the immune system. Unlike the immunodeficiencies, this robust level of in vivo selection is not available to hematopoietic repopulating cells or early progenitor cells following gene transfer of a therapeutic globin gene in the setting of beta-thalassemia and sickle cell disease. Both preclinical and clinical transplant studies involving bone marrow chimeras suggest that 20% or higher levels of engraftment of genetically modified HSCs will be needed for clinical success in the most severe of these disorders. Encouragingly, gene transfer levels in this range have recently been reported in a lentiviral vector gene transfer clinical trial for children with adrenoleukodystrophy. A clinical gene transfer trial for beta-thalassemia has begun in France, and one patient with transfusion-dependent HbE/beta-thalassemia has demonstrated a therapeutic effect after transplantation with autologous CD34(+) cells genetically modified with a beta-globin lentiviral vector. Here, the development and recent progress of gene therapy for the hemoglobin disorders is reviewed.
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Affiliation(s)
- Derek A Persons
- Division of Experimental Hematology, Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
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Surbek D, Schoeberlein A, Wagner A. Perinatal stem-cell and gene therapy for hemoglobinopathies. Semin Fetal Neonatal Med 2008; 13:282-90. [PMID: 18420474 DOI: 10.1016/j.siny.2008.03.002] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Most genetic diseases of the lymphohematopoietic system, including hemoglobinopathies, can now be diagnosed early in gestation. However, as yet, prenatal treatment is not available. Postnatal therapy by hematopoietic stem cell (HSC) transplantation from bone marrow, mobilized peripheral blood, or umbilical cord blood is possible for several of these diseases, in particular for the hemoglobinopathies, but is often limited by a lack of histocompatible donors, severe treatment-associated morbidity, and preexisting organ damage that developed before birth. In-utero transplantation of allogeneic HSC has been performed successfully in various animal models and recently in humans. However, the clinical success of this novel treatment is limited to diseases in which the fetus is affected by severe immunodeficiency. The lack of donor cell engraftment in nonimmunocompromised hosts is thought to be due to immunologic barriers, as well as to competitive fetal marrow population by host HSCs. Among the possible strategies to circumvent allogeneic HLA barriers, the use of gene therapy by genetically corrected autologous HSCs in the fetus is one of the most promising approaches. The recent development of strategies to overcome failure of efficient transduction of quiescent hematopoietic cells using new vector constructs and transduction protocols opens new perspectives for gene therapy in general, as well as for prenatal gene transfer in particular. The fetus might be especially susceptible for successful gene therapy approaches because of the developing, expanding hematopoietic system during gestation and the immunologic naiveté early in gestation, precluding immune reaction towards the transgene by inducing tolerance. Ethical issues, in particular regarding treatment safety, must be addressed more closely before clinical trials with fetal gene therapy in human pregnancies can be initiated.
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Affiliation(s)
- Daniel Surbek
- Department of Obstetrics and Gynecology, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland.
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Abstract
The concept of introducing genes into human cells for therapeutic purposes developed nearly 50 years ago as diseases due to defects in specific genes were recognized. Development of recombinant DNA techniques in the 1970s and their application to the study of mouse tumor viruses facilitated the assembly of the first gene transfer vectors. Vectors of several different types have now been developed for specific applications and over the past decade, efficacy has been demonstrated in many animal models. Clinical trials began in 1989 and by 2002 there was unequivocal evidence that children with severe combined immunodeficiency could be cured by gene transfer into primitive hematopoietic cells. Emerging from these successful trials was the realization that proto-oncogene activation by retroviral integration could contribute to leukemia. Much current effort is focused on development of safer vectors. Successful gene therapy applications have also been developed for control of graft-versus-host disease and treatment of various viral infections, leukemias, and lymphomas. The hemophilias seem amenable to gene therapy intervention and informative clinical trials have been conducted. The hemoglobin disorders, an early target for gene therapy, have proved particularly challenging although ongoing research is yielding new information that may ultimately lead to successful clinical trials.
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Corsten MF, Shah K. Therapeutic stem-cells for cancer treatment: hopes and hurdles in tactical warfare. Lancet Oncol 2008; 9:376-84. [PMID: 18374291 DOI: 10.1016/s1470-2045(08)70099-8] [Citation(s) in RCA: 111] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
As our understanding of stem-cell behaviour rapidly increases, more and more reports suggest that use of stem-cell therapy will extend well beyond regenerative medicine in the near future. Due to their inherent tumoritropic migratory properties, stem cells can serve as vehicles for the delivery of effective, targeted treatment to isolated tumours and to metastatic disease. In vitro, stem cells can readily be engineered by inserting specifically tailored transgenes with antitumour effects to create tumour-seeking therapeutic vehicles. Transgene effects include direct tumour-cell killing, promotion of local immune responses, oncolytic virus production, and prodrug activation schemes. Many of these strategies have been validated in a wide range of studies assessing treatment feasibility or efficacy and establishing methods for real-time monitoring of stem-cell migration and fate in vivo. New insights into avenues for stem-cell sourcing have shortened the probable time to realisation of such treatments for patients. In this Review, we provide an outline of the rationale and status of stem-cell-based treatments for tumours, and we discuss prospects for clinical implementation and the factors crucial for maintaining momentum towards this goal.
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Affiliation(s)
- Maarten F Corsten
- Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
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Schenk-Braat EAM, van Mierlo MMKB, Wagemaker G, Bangma CH, Kaptein LCM. An inventory of shedding data from clinical gene therapy trials. J Gene Med 2007; 9:910-21. [PMID: 17880045 DOI: 10.1002/jgm.1096] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Viruses are the most commonly used vectors for clinical gene therapy. The risk of dissemination of a viral vector into the environment via excreta from the treated patient, a phenomenon called shedding, is a major safety concern for the environment. Despite the significant number of clinical gene therapy trials that have been conducted worldwide, there is currently no overview of actual shedding data available. In this article, an inventory of shedding data obtained from a total of 100 publications on clinical gene therapy trials using retroviral, adenoviral, adeno-associated viral and pox viral vectors is presented. In addition, the experimental set-up for shedding analysis including the assays used and biological materials tested is summarized. The collected data based on the analysis of 1619 patients in total demonstrate that shedding of these vectors occurs in practice, mainly determined by the type of vector and the route of vector administration. Due to the use of non-quantitative assays, the lack of information on assay sensitivity in most publications, and the fact that assay sensitivity is expressed in various ways, general conclusions cannot be made as to the level of vector shedding. The evaluation of the potential impact and consequences of the observations is complicated by the high degree of variety in the experimental design of shedding analysis between trials. This inventory can be supportive to clinical gene therapy investigators for the establishment of an evidence-based risk assessment to be included in a clinical protocol application, as well as to national regulatory authorities for the ongoing development of regulatory guidelines regarding gene therapy.
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Louboutin JP, Liu B, Chekmasova AA, Reyes BAS, van Bockstaele EJ, Strayer DS. Delivering genes to the organ-localized immune system: long-term results of direct intramarrow transduction. J Gene Med 2007; 9:843-51. [PMID: 17694566 DOI: 10.1002/jgm.1084] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
We studied the distribution of transgene-expressing cells after direct gene transfer into the bone marrow (BM). Rats received direct injection into the femoral BM of SV(Nef-FLAG), a Tag-deleted recombinant SV40 carrying a marker gene (FLAG epitope). Controls received an unrelated rSV40 or saline. Blood cells (5%) and femoral marrow cells (25%) expressed FLAG throughout. FLAG expression was assessed in different organs at 1, 4 and 16 months. FLAG+ macrophages were seen throughout the body, and were prominent in the spleen. FLAG+ cells were common in pulmonary alveoli. The former included alveolar macrophages and type II pneumocytes. These cells were not detected at 1 month, occasional at 4 months and common at 16 months after intramarrow injection. Rare liver cells were positive for both FLAG and ferritin, indicating that some hepatocytes also expressed this BM-delivered transgene. Control animals were negative. Thus: (a) fixed tissue phagocytes may be accessible to gene delivery by intramarrow transduction of their progenitors; (b) transduced BM-resident cells or their derivatives may migrate to other organs (lungs) and may differentiate into epithelial cells; and (c) intramarrow injection of rSV40s does not detectably transduce parenchymal cells of other organs.
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38
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Huang KS, Lin YC, Su KC, Chen HY. An electroporation microchip system for the transfection of zebrafish embryos using quantum dots and GFP genes for evaluation. Biomed Microdevices 2007; 9:761-8. [PMID: 17541746 DOI: 10.1007/s10544-007-9087-x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
This study focuses on the design and experimental verification of an electroporation (EP) microchip system for the transfection of zebrafish (Danio rerio). For generating suitable pulses, a circuit is used to provide voltages between 0 and 700 V, with nearly 0-3,500 V/cm electric field. In addition, a proposed EP microchip, designed in a modular fashion, is fabricated using micro electromechanical system (MEMS) technology to allow for rapid and convenient replacement of each component. A numerical simulation is carried out to analyze the uniformity and strength of the EP electric fields generated in the microchip. Trypan blue dye, water-soluble quantum dots (MUA-QDs) and genes coding for green fluorescence protein (pEGFP-N1 plasmids) were employed to verify the successful delivery and transfection of zebrafish embryos. The experimental results show that the optimum delivery rate of trypan blue dyes and MUA-QDs were respectively up to 62 and 36% by using the proposed EP system. The successfully transfected embryos with the pEGFP-N1 plasmid used exhibit green fluorescence in the zebrafish embryos. The approach in the transfection of zebrafish embryos will provide many potential usages for cellular imaging areas, gene therapy research and medical applications.
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Affiliation(s)
- Keng-Shiang Huang
- Department of Engineering Science, National Cheng Kung University, 1 University Road, Tainan, 701, Taiwan, Republic of China.
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Mitsuhashi J, Tsukahara S, Suzuki R, Oh-hara Y, Nishi S, Hosoyama H, Katayama K, Noguchi K, Minowa S, Shibata H, Ito Y, Hatake K, Aiba K, Takahashi S, Sugimoto Y. Retroviral Integration Site Analysis and the Fate of Transduced Clones in anMDR1Gene Therapy Protocol Targeting Metastatic Breast Cancer. Hum Gene Ther 2007; 18:895-906. [PMID: 17907967 DOI: 10.1089/hum.2007.075] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
A clinical study of an MDR1 gene therapy protocol targeting metastatic breast cancer has been conducted in which the patients received high-dose chemotherapy, a transplant of MDR1-transduced autologous CD34(+) cells, and docetaxel. We herein report the molecular results of a 6-year follow-up of an individual in this study (patient 1). HaMDR-transduced cells, which had been initially detected in the peripheral blood of this individual, were found to have gradually decreased. After 10 cycles of docetaxel (days 71-316), MDR1 transgene levels were found to have increased, and then decreased to undetectable levels by day 1461. Thirty-eight MDR1-transduced clones were identified in patient 1, of which 11 showed a retroviral integration in close proximity to genes listed in the Retrovirus Tagged Cancer Gene Database (RTCGD). Four short-life clones in this group were found to harbor retroviral integrations close to the ZFHX1B, NOTCH1, BMI1, or HHEX gene; these genes have been frequently reported in the RTCGD. In addition, a long-lived RTCGD-hit clone, L-34, had a retroviral integration at a position 179 kb upstream of the EVI1 gene. L-34 was detectable on days 327-1154, but became undetectable 3 years after the docetaxel treatments had ceased. An additional three docetaxel-induced long-life clones showed comparable polymerase chain reaction profiles, which were also similar to that of the total MDR1-transduced cells. Our results thus show that docetaxel may have been effective in promoting the expansion of several MDR1-transduced clones in patient 1, but that they persist in the peripheral blood for only a few years.
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Affiliation(s)
- Junko Mitsuhashi
- Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan
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40
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Takahashi S, Aiba K, Ito Y, Hatake K, Nakane M, Kobayashi T, Minowa S, Shibata H, Mitsuhashi J, Tsukahara S, Ishikawa E, Suzuki R, Tsuruo T, Sugimoto Y. Pilot study of MDR1 gene transfer into hematopoietic stem cells and chemoprotection in metastatic breast cancer patients. Cancer Sci 2007; 98:1609-16. [PMID: 17683514 PMCID: PMC11158217 DOI: 10.1111/j.1349-7006.2007.00571.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022] Open
Abstract
A major problem in high-dose chemotherapy with autologous hematopoietic stem cell transplantation is insufficient function of reconstituted bone marrow that limits the efficacy of post-transplantation chemotherapy. Because transduction of hematopoietic stem cells with the multidrug resistance 1 (MDR1) gene might circumvent this problem, we conducted a pilot study of MDR1 gene therapy against metastatic breast cancer. Peripheral blood stem cells were harvested, and one-third of the cells were transduced with MDR1 retrovirus. After the reconstitution of bone marrow function, the patients received high-dose chemotherapy with transplantation of both MDR1-transduced and unprocessed peripheral blood stem cells. The patients then received docetaxel chemotherapy. Two patients received transplantation of the MDR1-transduced cells in 2001. Peripheral blood MDR1-transduced leukocytes were 3-5% of the total cells after transplantation, but decreased gradually. During docetaxel chemotherapy, an increase in the rate of MDR1-transduced leukocytes (up to 10%) was observed. Comparison of docetaxel-induced granulocytopenia in the two patients suggested a bone marrow-protective effect of the MDR1-transduced cells. No serious side-effect was observed, and the patients were in complete remission for more than 3 years. The MDR1-transduced cells gradually decreased and disappeared almost entirely by the end of 2004. Results of linear amplification-mediated polymerase chain reaction of the MDR1-transduced leukocytes suggested no sign of abnormal amplification of the transduced cells. A third patient received transplantation of the MDR1-transduced cells in 2004. These results suggest the feasibility of our MDR1 gene therapy against metastatic breast cancer, and follow-up is ongoing.
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Affiliation(s)
- Shunji Takahashi
- Division of Clinical Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 135-8500, Japan
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41
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Bozorgmehr F, Laufs S, Sellers SE, Roeder I, Zeller WJ, Zeller WJ, Dunbar CE, Fruehauf S. No Evidence of Clonal Dominance in Primates up to 4 Years Following Transplantation of Multidrug Resistance 1 Retrovirally Transduced Long-Term Repopulating Cells. Stem Cells 2007; 25:2610-8. [PMID: 17615269 DOI: 10.1634/stemcells.2007-0017] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Previous murine studies have suggested that retroviral multidrug resistance 1 (MDR1) gene transfer may be associated with a myeloproliferative disorder. Analyses at a clonal level and prolonged long-term follow-up in a model with more direct relevance to human biology were lacking. In this study, we analyzed the contribution of individual CD34-selected peripheral blood progenitor cells to long-term rhesus macaque hematopoiesis after transduction with a retroviral vector either expressing the multidrug resistance 1 gene (HaMDR1 vector) or expressing the neomycin resistance (NeoR) gene (G1Na vector). We found a total of 122 contributing clones from 8 weeks up to 4 years after transplantation. One hundred two clones contained the G1Na vector, whereas only 20 clones contained the HaMDR1 vector. Here, we show for the first time real-time polymerase chain reaction based quantification of individual transduced cell clones constituting 0.0008% +/- 0.0003% to 0.0041% +/- 0.00032% of primate peripheral blood cells. No clonal dominance was observed. Disclosure of potential conflicts of interest is found at the end of this article.
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Affiliation(s)
- Farastuk Bozorgmehr
- Research Group Pharmacology of Cancer Treatment, German Cancer Research Center, Heidelberg, Germany
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42
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Gori JL, Podetz-Pedersen K, Swanson D, Karlen AD, Gunther R, Somia NV, McIvor RS. Protection of mice from methotrexate toxicity by ex vivo transduction using lentivirus vectors expressing drug-resistant dihydrofolate reductase. J Pharmacol Exp Ther 2007; 322:989-97. [PMID: 17586722 DOI: 10.1124/jpet.107.123414] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTX-treated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs). In contrast, MTX-treated animals transplanted with control GFP-transduced marrow exhibited extremely reduced hematocrits with severe marrow hypoplasia and did not survive MTX dose escalation. To minimize cell manipulation, we treated unfractionated marrow in an overnight exposure. Transduction at a multiplicity of infection of 10 resulted in up to 11% vector-modified PBMCs in primary recipients and successful repopulation of secondary recipients with vector-marked cells. Experimental cohorts exhibited sustained proviral expression with stable GFP fluorescence intensity. These results demonstrate the effectiveness of lentivirus vectors for chemoprotection in a well developed animal model, with the potential for further preclinical development toward human application.
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Affiliation(s)
- Jennifer L Gori
- Institute of Human Genetics, Department of Genetics, Cell Biology, and Development, 6-160 Jackson Hall, 321 Church St. SE, University of Minnesota, Minneapolis, MN 55455, USA
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Milsom MD, Williams DA. Live and let die: in vivo selection of gene-modified hematopoietic stem cells via MGMT-mediated chemoprotection. DNA Repair (Amst) 2007; 6:1210-21. [PMID: 17482893 PMCID: PMC2064866 DOI: 10.1016/j.dnarep.2007.03.020] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Gene transfer into hematopoietic stem cells (HSC) provides a potential means of correcting monogenic defects and altering drug sensitivity of normal bone marrow to cytotoxic agents. These applications have significant therapeutic potential but the translation of successful murine studies into human therapies has been hindered by low gene transfer in large animals (including humans), and recent serious side effects in a human immunodeficiency trial related to insertional mutagenesis. The latter trial, along with other subsequent trials, while bringing into focus the potential risks of integrating vector systems, also clearly demonstrate the potential usefulness of in vivo selection as it relates to inefficient stem cell transduction. Developing from initial studies by our group and other investigators in which drug resistance was utilized to demonstrate the feasibility of using gene transfer to effect protection from myelotoxicity of chemotherapeutic agents, expression of mutant forms of O(6)-methyguanine-DNA-methytransferase (MGMT) coupled with the simultaneous use of pharmacologic inhibitors and chemotherapeutic agents has been shown to provide a powerful method to select HSC in vivo. While stem and progenitor cell protection and resulting selection in vivo has potential applications for the treatment of selected cancers (allowing dose escalation) and for correction of monogenic disease (allowing an iatrogenic survival advantage of transduced cells in vivo), such an in vivo selection may have untoward effects on stem cell behavior. These deleterious effects may include stem cell exhaustion; lineage skewing; accumulation of genotoxic lesions; and clonal dominance driven towards a pro-leukemic phenotype. Knowledge of the likelihood of such deleterious events occurring as well as their potential implications will be critical to future clinical applications and may also enhance our understanding of both normal stem cell behavior and the evolution of hematopoietic malignancies.
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Affiliation(s)
- Michael D Milsom
- Cincinnati Children's Research Foundation, Cincinnati Children's Hospital Medical Center, Division of Experimental Hematology, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
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44
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Raaijmakers MHGP. ATP-binding-cassette transporters in hematopoietic stem cells and their utility as therapeutical targets in acute and chronic myeloid leukemia. Leukemia 2007; 21:2094-102. [PMID: 17657220 DOI: 10.1038/sj.leu.2404859] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
ATP-binding-cassette (ABC) transporters are evolutionary extremely well-conserved transmembrane proteins that are highly expressed in hematopoietic stem cells (HSCs). The physiological function in human stem cells is believed to be protection against genetic damage caused by both environmental and naturally occurring xenobiotics. Additionally, ABC transporters have been implicated in the maintenance of quiescence and cell fate decisions of stem cells. These physiological roles suggest a potential role in the pathogenesis and biology of stem cell-derived hematological malignancies such as acute and chronic myeloid leukemia. This paper reviews the (patho)physiological role of ABC transporters in human normal and malignant HSCs and discusses its implications for their utility as therapeutical targets to eradicate leukemic stem cells in these diseases.
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Affiliation(s)
- M H G P Raaijmakers
- Department of Hematology, University Medical Center Nijmegen St. Radboud, Nijmegen Center for Molecular Life Sciences, Nijmegen, The Netherlands.
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45
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Abstract
A major side effect of cancer chemotherapy is myelosuppression. Expression of drug-resistance genes in hematopoietic stem cells (HSC) using gene transfer methodologies holds the promise of overcoming marrow toxicity in cancer chemotherapy. Adequate protection of marrow cells in cancer patients from myelotoxicity in this way would permit the use of escalating doses of chemotherapy for eradicating residual disease. A second use of drug-resistance genes is for coexpression with a therapeutic gene in HSCs to provide a selection advantage to gene-modified cells. In this review, we discuss several drug resistance genes, which are well suited for in vivo selection as well as other newer candidate genes with potential for use in this manner.
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Affiliation(s)
- M Zaboikin
- Division of Hematology/Oncology, Department of Medicine, Vanderbilt University, Nashville, TN 37232, USA
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46
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Ball CR, Pilz IH, Schmidt M, Fessler S, Williams DA, von Kalle C, Glimm H. Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression. Blood 2007; 110:1779-87. [PMID: 17496202 PMCID: PMC1976372 DOI: 10.1182/blood-2006-11-053710] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Efficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis, even if initial gene transfer efficiency is low. Moreover, selection of a limited number of transduced HSCs lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However, a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore, understanding the impact of in vivo selection on proliferation and lineage differentiation of stem-cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K)-expressing HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K-expressing stem-cell clones in mice that underwent serial transplantation and did not lead to clonal exhaustion. Interestingly, not all gene-modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced-intensity long-term in vivo selection, thus underscoring the potential value of MGMT P140K selection for clinical gene therapy.
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Affiliation(s)
- Claudia R Ball
- National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany
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47
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Schambach A, Baum C. Vector design for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells. DNA Repair (Amst) 2007; 6:1187-96. [PMID: 17482894 PMCID: PMC2128767 DOI: 10.1016/j.dnarep.2007.03.017] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Enhancing DNA repair activity of hematopoietic cells by stably integrating gene vectors that express O(6)-methylguanine-DNA-methyltransferase (MGMT) is of major interest for innovative approaches in tumor chemotherapy and for the control of hematopoietic chimerism in the treatment of multiple other acquired or inherited disorders. Crucial determinants of this selection principle are the stringency of treatment with O(6)-alkylating agents and the level of transgenic MGMT expression. Attempts to generate clinically useful MGMT vectors focus on the design of potent expression cassettes, an important component of which is formed by enhancer sequences that are active in primitive as well as more differentiated hematopoietic cells. However, recent studies have revealed that vectors harboring strong enhancer sequences are more likely to induce adverse events related to insertional mutagenesis. Safety-improved vectors that maintain high levels of MGMT expression may be constructed based on the following principles: choice of enhancer-promoter sequences with relatively mild long-distance effects despite a high transcription rate, improved RNA processing (export, stability and translation), and protein design. The need for optimizing MGMT protein design is supported by recent observations suggesting that the P140K mutant of MGMT, developed to be resistant to inhibitors such as O(6)-benzylguanine, may confer a selective disadvantage when expressed at high levels. Here, we provide a review of the literature exploring MGMT expression vectors for bone marrow chemoprotection, and describe experimental evidence suggesting that high expression of MGMT P140K induces a selective disadvantage in the absence of alkylating agents. We conclude that the appropriate design of expression vectors and MGMT protein features will be crucial for the long-term prospects of this promising selection principle.
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Affiliation(s)
- Axel Schambach
- Department of Experimental Hematology, Hannover Medical School, Hannover, Germany
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48
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Shoda H, Fujio K, Yamaguchi Y, Okamoto A, Sawada T, Kochi Y, Yamamoto K. Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases. Arthritis Res Ther 2007; 8:R166. [PMID: 17078892 PMCID: PMC1794509 DOI: 10.1186/ar2074] [Citation(s) in RCA: 106] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2006] [Revised: 10/05/2006] [Accepted: 11/01/2006] [Indexed: 12/17/2022] Open
Abstract
IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFα). We examined the in vivo relationship between IL-32 and TNFα, and the pathologic role of IL-32 in the TNFα-related diseases – arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFα reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFα, we prepared an overexpression model mouse of human IL-32β (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFα, IL-1β, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFα concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFα was increased in resting F4/80+ macrophages, and the expression of TNFα, IL-1β and IL-6 was increased in lipopolysaccharide-stimulated F4/80+ macrophages and CD11c+ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32β-producing CD4+ T cells significantly exacerbated collagen-induced arthritis, and a TNFα blockade cancelled the exacerbating effects of hIL-32β. We therefore conclude that IL-32 is closely associated with TNFα, and contributes to the exacerbation of TNFα-related inflammatory arthritis and colitis.
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Affiliation(s)
- Hirofumi Shoda
- Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Keishi Fujio
- Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Yumi Yamaguchi
- Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Akiko Okamoto
- Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Tetsuji Sawada
- Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Yuta Kochi
- Laboratory for Rheumatic Diseases, SNP Research Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
| | - Kazuhiko Yamamoto
- Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Matsuura S, Koto H, Ide K, Fujino Y, Setoguchi-Mukai A, Ohno K, Tsujimoto H. Induction of chemoresistance in a cultured canine cell line by retroviral transduction of the canine multidrug resistance 1 gene. Am J Vet Res 2007; 68:95-100. [PMID: 17199425 DOI: 10.2460/ajvr.68.1.95] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
OBJECTIVE To induce chemoresistance in a normal canine cell line through the transduction of the canine multidrug resistance 1 gene (mdr1). SAMPLE POPULATION Madin-Darby canine kidney (MDCK) epithelial cell line. PROCEDURES The full-length canine mdr1 cDNA clone isolated in our laboratory was inserted into a Moloney murine leukemia virus-based vector to construct the retroviral vector, pLNC-cMDR1. After retroviral transduction of pLNC-cMDR1 into MDCK cells, the expression and function of the P-glycoprotein, a product of mdr1, were assessed by immunoblotting, measurement of rhodamine123 (Rh123) retention, and drug sensitivity assays. RESULTS P-glycoprotein was strongly expressed in cells transduced with pLNC-cMDR1. This P-glycoprotein was fully functional, as demonstrated by the decreased Rh123 retention and the increased resistance to chemotherapeutic drugs. Measured as 50% inhibitory concentrations, resistance increased 59 times to vincristine and 25 times to doxorubicin in MDCK cells after transduction of pLNC-cMDR1. CONCLUSIONS AND CLINICAL RELEVANCE Transduction of canine mdr1 is an effective method for inducing chemoresistance in normal canine cells. This system may be applicable to the induction of drug resistance in hematopoietic cells.
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MESH Headings
- ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis
- ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics
- ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism
- Animals
- Antineoplastic Agents/pharmacology
- Base Sequence
- COS Cells
- Cell Line
- Chlorocebus aethiops
- DNA, Complementary/genetics
- Dogs/genetics
- Dogs/metabolism
- Doxorubicin/pharmacology
- Drug Resistance, Neoplasm
- Flow Cytometry/veterinary
- Fluorescent Dyes/metabolism
- Genetic Vectors/genetics
- Inhibitory Concentration 50
- Molecular Sequence Data
- Retroviridae/genetics
- Rhodamine 123/metabolism
- Sequence Analysis, DNA
- Transduction, Genetic/veterinary
- Vincristine/pharmacology
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Affiliation(s)
- Shinobu Matsuura
- Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
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50
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Kustikova OS, Geiger H, Li Z, Brugman MH, Chambers SM, Shaw CA, Pike-Overzet K, de Ridder D, Staal FJT, von Keudell G, Cornils K, Nattamai KJ, Modlich U, Wagemaker G, Goodell MA, Fehse B, Baum C. Retroviral vector insertion sites associated with dominant hematopoietic clones mark "stemness" pathways. Blood 2006; 109:1897-907. [PMID: 17119121 PMCID: PMC1801061 DOI: 10.1182/blood-2006-08-044156] [Citation(s) in RCA: 80] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Evidence from model organisms and clinical trials reveals that the random insertion of retrovirus-based vectors in the genome of long-term repopulating hematopoietic cells may increase self-renewal or initiate malignant transformation. Clonal dominance of nonmalignant cells is a particularly interesting phenotype as it may be caused by the dysregulation of genes that affect self-renewal and competitive fitness. We have accumulated 280 retrovirus vector insertion sites (RVISs) from murine long-term studies resulting in benign or malignant clonal dominance. RVISs (22.5%) are located in or near (up to 100 kb [kilobase]) to known proto-oncogenes, 49.6% in signaling genes, and 27.9% in other or unknown genes. The resulting insertional dominance database (IDDb) shows substantial overlaps with the transcriptome of hematopoietic stem/progenitor cells and the retrovirus-tagged cancer gene database (RTCGD). RVISs preferentially marked genes with high expression in hematopoietic stem/progenitor cells, and Gene Ontology revealed an overrepresentation of genes associated with cell-cycle control, apoptosis signaling, and transcriptional regulation, including major "stemness" pathways. The IDDb forms a powerful resource for the identification of genes that stimulate or transform hematopoietic stem/progenitor cells and is an important reference for vector biosafety studies in human gene therapy.
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Affiliation(s)
- Olga S Kustikova
- Department of Experimental Hematology, Hannover Medical School, Germany
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