scholarly journals Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5104-5110 ◽  
Author(s):  
Robert E. Throm ◽  
Annastasia A. Ouma ◽  
Sheng Zhou ◽  
Anantharaman Chandrasekaran ◽  
Timothy Lockey ◽  
...  
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Cell Clone ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Scale Production ◽  
Producer Cell

AbstractRetroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 107 transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common γ chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 × 107 TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

scholarly journals Packaging Cells Based on Inducible Gene Amplification for the Production of Adeno-Associated Virus Vectors

1998 ◽  
Vol 72 (9) ◽  
pp. 7024-7031 ◽  
Author(s):  
Naoki Inoue ◽  
David W. Russell
Keyword(s):  
Cell Lines ◽  
Gene Amplification ◽  
Large Scale ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Scale Production ◽  
Adeno Associated Virus ◽  
Tetracycline Transactivator ◽  
Producer Cell

ABSTRACT Although vectors based on adeno-associated virus (AAV) offer several unique advantages, their usage has been hampered by the difficulties encountered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replication origin. The packaging and producer cell lines developed express SV40 T antigen under the control of the reverse tetracycline transactivator system, which allows inducible amplification of chromosomal loci linked to the SV40 origin. Culturing these cells in the presence of doxycycline followed by adenovirus infection resulted in helper and vector gene amplification as well as higher vector titers. Clonal producer cell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 104vector particles produced per cell. These stocks were free of detectable replication-competent virus. The lack of a transfection step combined with the reproducibility of stable producer lines makes this packaging method ideally suited for the large-scale production of vector stocks for human gene therapy.

scholarly journals High-Throughput Selection of Retrovirus Producer Cell Lines Leads to Markedly Improved Efficiency of Germ Line-Transmissible Insertions in Zebra Fish

2002 ◽  
Vol 76 (5) ◽  
pp. 2192-2198 ◽  
Author(s):  
Wenbiao Chen ◽  
Shawn Burgess ◽  
Greg Golling ◽  
Adam Amsterdam ◽  
Nancy Hopkins
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Insertional Mutagenesis ◽  
Germ Line ◽  
High Titer ◽  
Retroviral Vectors ◽  
Embryonic Cells ◽  
Zebra Fish ◽  
Glycoprotein G ◽  
Producer Cell

ABSTRACT Vesicular stomatitis virus glycoprotein G-pseudotyped mouse retroviral vectors have been used as mutagens for a large-scale insertional mutagenesis screen in the zebra fish. To reproducibly generate high-titer virus stocks, we devised a method for rapidly selecting cell lines that can yield high-titer viruses and isolated a producer cell line that yields virus at a high titer on zebra fish embryos. Virus produced from this line, designated GT virus, is nontoxic following injection of zebra fish blastulae and efficiently infects embryonic cells that give rise to the future germ line. Using GT virus preparations we generated roughly 500,000 germ line-transmissible proviral insertions in a population of 25,000 founder fish in about 2 months. The GT virus contains a gene trap, and trap events can be detected in the offspring of almost every founder fish. We discuss potential applications of this highly efficient method for generating germ line-transmissible insertions in a vertebrate

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

Abstract 262: Derivation of Duchenne Muscular Dystrophy Disease Specific Cardiomyocytes From Patient Urine

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Xuan Guan ◽  
David L Mack ◽  
Claudia M Moreno ◽  
Fernando Santana ◽  
Charles E Murry ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Urine ◽  
Large Scale ◽  
Lentiviral Vector ◽  
Cellular Reprogramming ◽  
Urine Samples ◽  
Pcr Analysis ◽  
Scale Production ◽  
Mechanism Study

Introduction: Human somatic cells can be reprogrammed into primitive stem cells, termed induced pluripotent stem cells (iPSCs). These iPSCs can be extensively expanded in vitro and differentiated into multiple functional cell types, enabling faithful preservation of individual’s genotype and large scale production of disease targeted cellular components. These unique cellular reagents thus hold tremendous potential in disease mechanism study, drugs screening and cell replacement therapy. Due to the genetic mutation of the protein dystrophin, many DMD patients develop fatal cardiomyopathy with no effective treatment. The underlying pathogenesis has not been fully elucidated. Hypothesis: We tested the hypothesis that iPSCs could be generated from DMD patients’ urine samples and differentiated into cardiomyocytes, recapitulating the dystrophic phenotype. Methods: iPSCs generation was achieved by introducing a lentiviral vector expressing Oct4, Sox2, c-Myc and Klf4 into cells derived from patient’s (n=1) and healthy volunteers’ (n=3) urine. Cardiomyocytes were derived by sequentially treating iPSCs with GSK3 inhibitor CHIR99021 and Wnt inhibitor IWP4. Differentiated cardiomyocytes were subjected to calcium imaging, electrophysiology recording, Polymerase Chain Reaction (PCR) analysis, and immunostaining. Results: iPSCs were efficiently generated from human urine samples and further forced to differentiate into contracting cardiomyocytes. PCR analysis and immunostaining confirmed the expression of a panel of cardiac markers. Both normal and patient iPSC derived cardiomyocytes exhibited spontaneous and field stimulated calcium transients (up to 2Hz), as well as action potentials with ventricular-like and nodal-like characteristics. Anti-dystrophin antibodies stained normal iPSC-derived cardiomyocyte membranes but did not react against DMD iPSC-derived cardiomyocytes. Conclusions: Cardiomyocytes can be efficiently generated from human urine, through the cellular reprogramming technology. DMD cardiomyocytes retained the patient’s genetic information and manifested a dystrophin-null phenotype. Functional assessments are underway to determine differences that may exist between genotypes.

scholarly journals Retroviral-mediated gene correction for X-linked severe combined immunodeficiency

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3097-3102 ◽  
Author(s):  
F Candotti ◽  
JA Johnston ◽  
JM Puck ◽  
K Sugamura ◽  
JJ O'Shea ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Severe Combined Immunodeficiency ◽  
Allogeneic Bone Marrow Transplantation ◽  
Interleukin 2 ◽  
Current Therapy ◽  
Allogeneic Bone ◽  
Combined Immunodeficiency ◽  
Gene Correction

X-linked severe combined immunodeficiency (XSCID) is a lethal disease caused by a defect in the gene encoding the common gamma chain (gamma- c) of the receptor for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL- 15. Allogeneic bone marrow transplantation, the current therapy of choice for this defect, is often complicated by graft-versus-host disease and/or incomplete reconstitution of B-lymphocyte functions. Correction of the gene defect at the level of the autologous lymphohematopoietic progenitors could therefore represent an improvement in the medical management of these patients. To study the feasibility of a gene therapy approach for XSCID, a retroviral vector expressing gamma-c was used to transduce Epstein-Barr virus-transformed B-cell lines derived from patients with XSCID. After transduction, XSCID cells newly expressed gamma-c on the cell surface at levels comparable to those observed on B-cell lines obtained from normal donors. Moreover, the reconstituted gamma-c restored function to the IL- 2 and IL-4 receptors as shown by signal transduction mediated by phosphorylation of the JAK1 and JAK3 members of the Janus family of tyrosine kinases and by restoration of cellular proliferation in response to IL-2.

A new Procedure for Large Scale Production and Freezing of Lymphokine Activated Killer (LAK) Cells to be used in Adoptive Immunotherapy of Cancer

1988 ◽  
Vol 74 (5) ◽  
pp. 523-530 ◽  
Author(s):  
Carlo Gambacorti-Passerini ◽  
Marina Radrizzani ◽  
Licia Rivoltini ◽  
Edoardo Marchesi ◽  
Fernando Ravagnani ◽  
...  
Keyword(s):  
Adoptive Immunotherapy ◽  
Large Scale ◽  
Interleukin 2 ◽  
Peripheral Blood Lymphocytes ◽  
Cell Factory ◽  
Scale Production ◽  
Lymphokine Activated Killer Cells ◽  
Large Scale Production ◽  
Recombinant Interleukin 2 ◽  
Immunotherapy Of Cancer

A new procedure for activation of peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL2) is described. PBL obtained by leukapheresis were subjected to NH4Cl (ACK) treatment to clear erythrocyte contamination; Ficoll separation was not performed. PBL were subsequently seeded in 10-floor multitrays (Cell Factory™, CF), gasified and incubated at 37 °C for 3-4 days in a humidified 5% CO2 atmosphere. This procedure achieved an activation (evaluated as cytotoxicity and proliferation) comparable with that obtained by culturing PBL in small flasks. Optimal activation of PBL was achieved in CF even in the presence of granulocyte contamination of up to 40%. It was also possible to freeze, thaw and recover most of the frozen cells and their cytotoxic activity. With this procedure therefore large quantities of lymphokine activated killer cells (LAK) can be easily produced to be used in adoptive immunotherapy trials.

CD86 and CD54 Co-Expression on VSV-G Pseudotyped HIV-1 Based Vectors Improves Transduction and Activation of Human Primary CD4+ T Lymphocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1754-1754
Author(s):  
Brian Paszkiet ◽  
Andrew Worden ◽  
Yajin Ni ◽  
Saran Bao ◽  
Franck Lemiale ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Large Scale ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Lentiviral Vectors ◽  
Normal Donor ◽  
Activation Markers ◽  
Gene Therapy Trial

Abstract We established the first clinical ex vivo HIV-based vector gene therapy trial in humans with HIV+ CD4+ T-cells. Briefly, this therapy involves modifying patient CD4+ T-cells with our modified lentiviral vector carrying an anti-HIV payload. These cells are then activated and expanded, and re-infused back into the patient. However, cGMP regulations require the use of costly clinical grade reagents (i.e. Retronectin™, CD3/CD28 stimulating paramagnetic beads). In an attempt to reduce ex-vivo processing costs, but not at the expense of transduction levels, we sought to determine a way to directly activate CD4+ T-cells with modified lentiviral vectors. 293FT HEK cell lines, used for producing our lentiviral vectors, were modified to co-express the natural CD28 stimulatory ligand B7.2 (CD86) and ICAM-1 (CD54) proteins on their membrane for co-stimulation and anchoring purposes. When CliniMACS purified normal donor CD4+ T cells were co-cultured with CD54/CD86-expressing cells, in the presence of soluble OKT3 CD3 antibody, CD25 and CD69 activation markers were upregulated, indicating that functional proteins were being expressed at the cell membrane. These CD54 and/or CD86 expressing cells could subsequently be transfected with lentiviral vector plasmid constructs in order to produce host-derived CD54 and/or CD86 bearing HIV-based vectors. EGFP-expressing lentiviral vectors, VRX494, with CD54/CD86-modified envelopes were produced both in these cell lines and by transient transfection of all relevant plasmids, and titers were assayed on Hela-Tat cells by FACS. CD54 modified lentiviral vectors showed increased binding to CD4+ T-cells, as evidenced by significant cell clumping. CD86 (as well as CD54 plus CD86) modified lentiviral vector, with soluble OKT3 CD3 antibody, was shown to activate T-cells, above the levels seen with unmodified lentiviral vectors, as evidenced by the increase in cell surface CD25 and CD69 expression and also the increase in cell size. Cellular expansion of modified lentiviral vector transduced CD4+ T cells reached levels close to CD3:CD28 bead stimulated CD4+ T cell controls over a period of 2 to 3 weeks. The CD3/TCR repertoire was assessed by flow cytometry and, compared to the well-established CD3/CD28 coated M450 Dynabeads stimulatory system as a control, no skewing of the repertoire was observed. CD86 was shown to improve levels of transduction in pre-activated lymphocytes with CD3/CD28 coated M450 Dynabeads. However, CD86 co-expression was crucial for transducing minimally activated CD4+ T cells with only soluble OKT3 CD3 antibody. Levels of transduction and activation were on average 2 to 3 times higher with the modified lentiviral vectors. To our knowledge, we are reporting the first generation of lentiviral particles exhibiting an adhesion property with stimulatory abilities. The development of such a lentiviral vector has valuable implications for clinical application by reducing the number of exogenous reagents in large scale cell processing.

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