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1.
Mol Ther ; 27(6): 1126-1138, 2019 06 05.
Article in English | MEDLINE | ID: mdl-31005597

ABSTRACT

Clinical success of autologous CD19-directed chimeric antigen receptor T cells (CAR Ts) in acute lymphoblastic leukemia and non-Hodgkin lymphoma suggests that CAR Ts may be a promising therapy for hematological malignancies, including multiple myeloma. However, autologous CAR T therapies have limitations that may impact clinical use, including lengthy vein-to-vein time and manufacturing constraints. Allogeneic CAR T (AlloCAR T) therapies may overcome these innate limitations of autologous CAR T therapies. Unlike autologous cell therapies, AlloCAR T therapies employ healthy donor T cells that are isolated in a manufacturing facility, engineered to express CARs with specificity for a tumor-associated antigen, and modified using gene-editing technology to limit T cell receptor (TCR)-mediated immune responses. Here, transcription activator-like effector nuclease (TALEN) gene editing of B cell maturation antigen (BCMA) CAR Ts was used to confer lymphodepletion resistance and reduced graft-versus-host disease (GvHD) potential. The safety profile of allogeneic BCMA CAR Ts was further enhanced by incorporating a CD20 mimotope-based intra-CAR off switch enabling effective CAR T elimination in the presence of rituximab. Allogeneic BCMA CAR Ts induced sustained antitumor responses in mice supplemented with human cytokines, and, most importantly, maintained their phenotype and potency after scale-up manufacturing. This novel off-the-shelf allogeneic BCMA CAR T product is a promising candidate for clinical evaluation.


Subject(s)
B-Cell Maturation Antigen/immunology , Cell Transplantation/methods , Immunotherapy, Adoptive/methods , Multiple Myeloma/therapy , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , T-Lymphocytes/transplantation , Animals , Antineoplastic Agents, Immunological/therapeutic use , B-Cell Maturation Antigen/genetics , Blood Donors , Cell Line, Tumor , Cell Transplantation/adverse effects , Cytotoxicity, Immunologic/genetics , Gene Editing , Genetic Vectors , Graft vs Host Disease/therapy , Humans , Immunotherapy, Adoptive/adverse effects , Mice , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/pathology , Progression-Free Survival , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Rituximab/therapeutic use , T-Lymphocytes/metabolism , Transcription Activator-Like Effector Nucleases/genetics , Transduction, Genetic , Transplantation, Homologous/methods
2.
Hum Gene Ther ; 16(1): 17-25, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15703485

ABSTRACT

This review is intended to exemplify the roles and responsibilities of the two agencies under the Department of Health and Human Services, the National Institutes of Health and the Food and Drug Administration, that have oversight for human gene transfer clinical protocols, as seen through our experience of bringing a first-in-its-class lentiviral vector to clinical trials. In response to the changing circumstances in gene therapy research between 1999 and 2002, the concerns of these agencies regarding gene therapy have been evolving. This review provides an overview of the major safety concerns regarding insertional oncogenesis, the generation of a replication- competent lentivirus (RCL), and vector mobilization thought to be related to lentiviral vectors, which had to be addressed during the regulatory review process before initiating the clinical trial. Specific monitoring assays to address these concerns were established to test for RCL generation, vector mobilization, persistence of vector-modified cells, and abnormal clonal expansion of modified cells. We hope to provide a basic understanding and appreciation of the regulatory process and major safety concerns, toward providing useful insight to those presently embarking on the development of clinical application of lentiviral vectors.


Subject(s)
Genetic Therapy , Genetic Vectors , Lentivirus/genetics , Neoplasms/therapy , Biomedical Research/legislation & jurisprudence , Clinical Trials as Topic , Government Regulation , Human Experimentation/standards , Humans , Neoplasms/genetics
3.
J Gene Med ; 7(6): 818-34, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15693055

ABSTRACT

BACKGROUND: A stable packaging cell line facilitates large-scale lentivirus vector manufacture. However, it has been difficult to produce clinical-scale HIV-1-based lentiviral vectors using a packaging cell line, in part due to toxicity of packaging genes, and gene silencing that occurs during the long culture period necessary for sequential addition of packaging constructs. METHODS: To avoid these problems, we developed a three-level cascade gene regulation system designed to remove tetracycline transactivator (tTA) from cytomegalovirus immediate early promoter (CMV)-controlled expression to reduce cytotoxicity from constitutive expression of tTA and leaky expression of packaging genes. We also performed a one-step integration of the three packaging plasmids to shorten the culture time for clonal selection. RESULTS: Although leaky expression of p24 and vector production still occurred despite the three-level regulation system, little cytotoxicity was observed and producer cells could be expanded for large-scale production. Producer cells yielded remarkably stable vector production over a period greater than 11 days with the highest titer 3.5 x 10(7) transducing units (TU)/ml and p24 300 ng/ml, yielding 2.2 x 10(11) TU and 1.8 milligram (mg) p24 from one cell factory. No replication-competent lentivirus (RCL) was detected. Long-term analysis demonstrated that, although the cells are genetically stable, partial gene silencing occurs after 2-3 months in culture; however, the one-step construct integration allowed prolonged vector production before significant gene silencing. Concentrated vector resulted in 90% transduction in CD4+ lymphocytes at 20 TU per cell. CD34+ progenitor cells were transduced at 41-46% efficiency, and long-term initiating culture (LTC-IC) was transduced at 45-51%. CONCLUSIONS: These results demonstrate for the first time HIV-1-based lentiviral vector production on the large scale using a packaging cell line.


Subject(s)
Genetic Vectors/genetics , HIV-1/genetics , Lentivirus/genetics , Base Sequence , Cell Line , Clone Cells , Cloning, Molecular , Codon , Enzyme-Linked Immunosorbent Assay , Fusion Proteins, gag-pol/chemistry , Fusion Proteins, gag-pol/genetics , Gene Expression Regulation/drug effects , Gene Products, rev/chemistry , Gene Products, rev/genetics , Gene Products, tat/chemistry , Gene Products, tat/genetics , Genetic Engineering , Genetic Vectors/biosynthesis , HeLa Cells , Humans , Kinetics , Membrane Glycoproteins/metabolism , Models, Genetic , Molecular Sequence Data , Plasmids , Tetracycline/pharmacology , Transduction, Genetic , Transfection , Viral Envelope Proteins/metabolism , Virion/metabolism , Virus Replication , rev Gene Products, Human Immunodeficiency Virus , tat Gene Products, Human Immunodeficiency Virus
4.
Blood ; 104(2): 364-73, 2004 Jul 15.
Article in English | MEDLINE | ID: mdl-15059841

ABSTRACT

High gene transfer efficiencies have been difficult to achieve in hematopoietic progenitor cells (HPCs) but are important to therapeutic success of HPC gene therapy. Efficient gene transfer is especially challenging with use of column-purified vector for clinical application, as opposed to centrifuged vector commonly used for research. We investigated novel approaches to increase transduction by using a clinically applicable protocol and quantities of column-purified lentiviral vector. Recognizing the association of adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters with HPC biology, we investigated the effect of transporter inhibitors on transduction. We found the ABC transporter inhibitor verapamil improved transduction efficiency 2- to 6-fold into CD34(+) cells isolated from mobilized peripheral blood, bone marrow, and cord blood. Verapamil also improved transduction in human SCID (severe combined immunodeficient) repopulating cell (SRC) transduction 3- to 4-fold, resulting in 80% to 90% transduction levels in mice receiving primary and secondary transplants without alterations in multilineage reconstitution. Additional ABC transporter substrate inhibitors like quinidine, diltiazem, and ritonavir also enhanced transduction 2- to 3-fold, although ABC transporter inhibitors that are not substrates did not. Enhanced transduction was not observed in mature hematopoietic cells, neurospheres, mesenchymal stem cells, or hepatocytes. Enhancement of transduction in HPCs was observed with vesicular stomatitis virus-G (VSV-G)-pseudotyped lentiviral vector but not with vector pseudotyped with RD114. Thus, we present a new approach for efficient delivery to primitive HPCs by VSV-G-pseudotyped lentiviral vectors.


Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Genetic Vectors , Hematopoietic Stem Cells/physiology , Lentivirus/genetics , Transduction, Genetic/methods , Adult , Animals , Antigens, CD34/analysis , Calcium Channel Blockers/pharmacology , Centrifugation , Diltiazem/pharmacology , Genetic Therapy/methods , HeLa Cells , Hematopoietic Stem Cells/chemistry , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Quinidine/pharmacology , Verapamil/pharmacology
5.
Behav Brain Res ; 138(2): 133-43, 2003 Jan 22.
Article in English | MEDLINE | ID: mdl-12527444

ABSTRACT

The serotonin transporter (5HTT) plays a central role in serotonin neurotransmission. Abnormalities of 5HTT function have been implicated in depression, anxiety and alcohol intake. To better understand the functional role of this important molecule, we have utilized a viral vector approach to overexpress the 5HTT in regions of the rat brain. We have constructed a bicistronic defective herpes virus (HSV-1) vector that expresses both an epitope-tagged 5HTT as well as beta-galactosidase (beta-GAL) as a marker for infected cells. The vector was capable of conferring serotonin uptake activity to Vero cells in culture, indicating transfer of a functional 5HTT. Injection of the 5HTT virus into the rat brain resulted in a dense focus of specific 125I RTI-55 binding at the injection site, indicating that the virally expressed 5HTT can also bind ligand when expressed in the brain. We were also able to overexpress an epitope tagged 5HTT in serotonergic neurons in the dorsal raphe nucleus (DRN) using this approach. These data demonstrate that the levels of the 5HTT in 5HT neurons can be manipulated in the adult rodent brain using an HSV-1 vector.


Subject(s)
Carrier Proteins/genetics , Gene Transfer Techniques , Genetic Vectors/genetics , Herpesvirus 1, Human/genetics , Membrane Glycoproteins/genetics , Membrane Transport Proteins , Nerve Tissue Proteins , Raphe Nuclei/metabolism , Serotonin/metabolism , Animals , Autoradiography , Chlorocebus aethiops , Epitope Mapping , Gene Expression Regulation, Viral/physiology , Male , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Serotonin Plasma Membrane Transport Proteins , Vero Cells , beta-Galactosidase/genetics
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