scholarly journals kat: a high-efficiency retroviral transduction system for primary human T lymphocytes

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 43-50 ◽  
Author(s):  
MH Finer ◽  
TJ Dull ◽  
L Qin ◽  
D Farson ◽  
MR Roberts
Keyword(s):  
T Lymphocytes ◽  
High Efficiency ◽  
High Titer ◽  
Virus Production ◽  
Retroviral Vectors ◽  
Expression Vectors ◽  
Retroviral Transduction ◽  
Human T Lymphocytes ◽  
293 Cells ◽  
Viral Titers

Abstract We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was produced without the need to generate stable producer clones. kat expression vectors, which produce high levels of retroviral vector transcripts and retroviral packaging functions, were transfected into 293 cells followed by virus harvest 48 hours posttransfection. Viral titers as high as 3.8 proviral copies/cell/mL of frozen supernatant in 3T3 cells were obtained, 10- to 50-fold greater than transient viral titers reported using 3T3-based retroviral packaging lines. Cocultivation of primary human CD8+ T lymphocytes after transient transfection of 293 cells with kat plasmids resulted in transduction efficiencies of 10% to 40%, 5- to 10-fold greater compared to cocultivation with a high titer PA317 producer clone and significantly greater than previously reported results for transduction of primary human T lymphocytes with retroviral vectors. Virus produced using the kat system was shown to be free of detectable replication competent retrovirus by an extended provirus mobilization assay, demonstrating that this system is as safe as currently available stable packaging lines. The kat virus production system should be of general use for the rapid production of high titer viral supernatants, as well as for high-efficiency transduction hematopoietic cell types refractory to retroviral transduction.

scholarly journals kat: a high-efficiency retroviral transduction system for primary human T lymphocytes

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 43-50 ◽  
Author(s):  
MH Finer ◽  
TJ Dull ◽  
L Qin ◽  
D Farson ◽  
MR Roberts
Keyword(s):  
T Lymphocytes ◽  
High Efficiency ◽  
High Titer ◽  
Virus Production ◽  
Retroviral Vectors ◽  
Expression Vectors ◽  
Retroviral Transduction ◽  
Human T Lymphocytes ◽  
293 Cells ◽  
Viral Titers

We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was produced without the need to generate stable producer clones. kat expression vectors, which produce high levels of retroviral vector transcripts and retroviral packaging functions, were transfected into 293 cells followed by virus harvest 48 hours posttransfection. Viral titers as high as 3.8 proviral copies/cell/mL of frozen supernatant in 3T3 cells were obtained, 10- to 50-fold greater than transient viral titers reported using 3T3-based retroviral packaging lines. Cocultivation of primary human CD8+ T lymphocytes after transient transfection of 293 cells with kat plasmids resulted in transduction efficiencies of 10% to 40%, 5- to 10-fold greater compared to cocultivation with a high titer PA317 producer clone and significantly greater than previously reported results for transduction of primary human T lymphocytes with retroviral vectors. Virus produced using the kat system was shown to be free of detectable replication competent retrovirus by an extended provirus mobilization assay, demonstrating that this system is as safe as currently available stable packaging lines. The kat virus production system should be of general use for the rapid production of high titer viral supernatants, as well as for high-efficiency transduction hematopoietic cell types refractory to retroviral transduction.

scholarly journals Highly-efficient gene transfer with retroviral vectors into human T lymphocytes on fibronectin

1998 ◽  
Vol 102 (2) ◽  
pp. 566-574 ◽  
Author(s):  
Boris Fehse ◽  
Ulrika M Schade ◽  
Zhixiong LI ◽  
Almut Uhde ◽  
Stefan Koch ◽  
...  
Keyword(s):  
Gene Transfer ◽  
T Lymphocytes ◽  
Retroviral Vectors ◽  
Highly Efficient ◽  
Human T Lymphocytes ◽  
Efficient Gene

scholarly journals Engineering the Rapid Adenovirus Production and Amplification (RAPA) Cell Line to Expedite the Generation of Recombinant Adenoviruses

2017 ◽  
Vol 41 (6) ◽  
pp. 2383-2398 ◽  
Author(s):  
Qiang Wei ◽  
Jiaming Fan ◽  
Junyi Liao ◽  
Yulong Zou ◽  
Dongzhe Song ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Production Efficiency ◽  
High Efficiency ◽  
Luciferase Activity ◽  
High Titer ◽  
Hek 293 ◽  
Production Methods ◽  
Fluorescent Markers ◽  
Gene Delivery Vectors ◽  
293 Cells

Background/Aims: While recombinant adenoviruses are among the most widely-used gene delivery vectors and usually propagated in HEK-293 cells, generating recombinant adenoviruses remains time-consuming and labor-intense. We sought to develop a rapid adenovirus production and amplification (RAPA) line by assessing human Ad5 genes (E1A, E1B19K/55K, pTP, DBP, and DNA Pol) and OCT1 for their contributions to adenovirus production. Methods: Stable transgene expression in 293T cells was accomplished by using piggyBac system. Transgene expression was determined by qPCR. Adenoviral production was assessed with titering, fluorescent markers and/or luciferase activity. Osteogenic activity was assessed by measuring alkaline phosphatase activity. Results: Overexpression of both E1A and pTP led to a significant increase in adenovirus amplification, whereas other transgene combinations did not significantly affect adenovirus amplification. When E1A and pTP were stably expressed in 293T cells, the resultant RAPA line showed high efficiency in adenovirus amplification and production. The produced AdBMP9 infected mesenchymal stem cells with highest efficiency and induced most effective osteogenic differentiation. Furthermore, adenovirus production efficiency in RAPA cells was dependent on the amount of transfected DNA. Under optimal transfection conditions high-titer adenoviruses were obtained within 5 days of transfection. Conclusion: The RAPA cells are highly efficient for adenovirus production and amplification.

PML-RAR induces promyelocytic leukemias with high efficiency following retroviral gene transfer into purified murine hematopoietic progenitors

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2989-2995 ◽  
Author(s):  
Saverio Minucci ◽  
Silvia Monestiroli ◽  
Sabrina Giavara ◽  
Simona Ronzoni ◽  
Francesco Marchesi ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
High Efficiency ◽  
High Titer ◽  
Retroviral Vectors ◽  
Model System ◽  
Promyelocytic Leukemia ◽  
Myeloid Differentiation ◽  
Proliferative Potential ◽  
Hematopoietic Progenitors

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations resulting in fusion proteins of the retinoic acid receptor (RAR). Here, we report a novel murine model system for APL, based on the transduction of purified murine hematopoietic progenitors (lin−) using high-titer retroviral vectors encoding promyelocytic leukemia–RAR (PML-RAR), and the green fluorescent protein (GFP) as a marker. PML-RAR–expressing lin− cells were impaired in their ability to undergo terminal myeloid differentiation and showed increased proliferative potential in vitro. Inoculation of transduced lin− cells into syngeneic, irradiated mice resulted in the development of retinoic acid-sensitive promyelocytic leukemias at high frequency (> 80%) and short latency (approximately 4 months). Morphologic and immunophenotypic analysis revealed no gross abnormalities of the preleukemic bone marrows. However, hematopoietic progenitors from PML-RAR preleukemic mice showed a severe impairment in their ability to undergo myeloid differentiation in vitro. This result, together with the monoclonality or oligoclonality of the leukemic blasts, supports a “multiple-hit” model, where the fusion protein causes a “preleukemic” phase, and leukemia occurs after additional genetic lesions. This model system faithfully reproduces the main characteristics of human APL and represents a versatile tool for the in vitro and in vivo study of mechanisms of leukemogenesis and the design of protocols for differentiation treatment.

Expansion and Fibronectin-Enhanced Retroviral Transduction of Primary Human T Lymphocytes for Adoptive Immunotherapy

1999 ◽  
Vol 8 (4) ◽  
pp. 401-410 ◽  
Author(s):  
Marcus Stockschlader ◽  
Monika Haiss ◽  
Sebastian Exner ◽  
Oliver Schmah ◽  
Hendrik Veelken ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Adoptive Immunotherapy ◽  
Retroviral Transduction ◽  
Human T Lymphocytes

scholarly journals Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy

Gene Therapy ◽  
2002 ◽  
Vol 9 (20) ◽  
pp. 1359-1368 ◽  
Author(s):  
R F Duarte ◽  
F E Chen ◽  
M W Lowdell ◽  
M N Potter ◽  
M L Lamana ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Lymphocytes ◽  
Functional Impairment ◽  
Retroviral Transduction ◽  
Human T Lymphocytes

scholarly journals High-Efficiency Gene Transfer into Normal and Adenosine Deaminase-Deficient T Lymphocytes Is Mediated by Transduction on Recombinant Fibronectin Fragments

1998 ◽  
Vol 72 (6) ◽  
pp. 4882-4892 ◽  
Author(s):  
Karen E. Pollok ◽  
Helmut Hanenberg ◽  
Timothy W. Noblitt ◽  
Wendy L. Schroeder ◽  
Ikunoshin Kato ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
T Lymphocytes ◽  
Adenosine Deaminase ◽  
High Efficiency ◽  
Human Gene ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Human Gene Therapy ◽  
Human T Lymphocytes

ABSTRACT Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3+lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow

1987 ◽  
Vol 7 (2) ◽  
pp. 854-863
Author(s):  
S M Chang ◽  
K Wager-Smith ◽  
T Y Tsao ◽  
J Henkel-Tigges ◽  
S Vaishnav ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Cultured Cells ◽  
High Titer ◽  
Retroviral Vectors ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Mouse Bone Marrow ◽  
Cellular Expression ◽  
Viral Titers

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.

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