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

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.

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T Lymphocytes as Targets of Gene Transfer with Moloney-Type Retroviral Vectors

Current Gene Therapy ◽

10.2174/1566523013348274 ◽

2001 ◽

Vol 1 (4) ◽

pp. 325-337 ◽

Cited By ~ 6

Author(s):

F. Ayuk ◽

A. Zander ◽

B. Fehse

Keyword(s):

Gene Transfer ◽

T Lymphocytes ◽

Retroviral Vectors

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Mobilization of Full-Length Semliki Forest Virus Replicon by Retrovirus Particles

Journal of Virology ◽

10.1128/jvi.00664-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9889-9895 ◽

Cited By ~ 6

Author(s):

Eric Piver ◽

Christine Collin ◽

Noémie Renault ◽

Thierry Bru ◽

Jean-Christophe Pagès

Keyword(s):

Gene Transfer ◽

Semliki Forest Virus ◽

Direct Consequence ◽

Retroviral Vectors ◽

Retroviral Vector ◽

Full Length ◽

Efficient Gene ◽

Retroviral Transfer

ABSTRACT Conciliating biosafety with efficient gene transfer remains a constant concern in the development of retroviral vectors. Semliki Forest virus (SFV) replicons allow important retroviral vector production with interesting features. It is noteworthy that retroviruses have the ability to package Ψ+ and, to some extent, Ψ− cellular RNAs. Therefore, it was important to study the retroviral transfer of highly abundant SFV genomes expressing retroviral proteins. Here, we show that full-length SFV-vector replicons, with or without Ψ, are efficiently packaged into retrovirus particles. Mechanistically, our data suggest that SFV packaging is the sum of its retroviral nucleocapsid-dependent recruitment together with a passive hijacking of membrane-anchored SFV replicon. A direct consequence of this phenomenon is the formation of particles harboring autonomous replicative abilities and contaminating vector preparations. Importantly, we confirm that retroviral SFV mobilization is not an exclusive feature of murine gamma retroviruses, since it is also observed using lentivectors.

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Local Gene Delivery System by Bubble Liposomes and Ultrasound Exposure into Joint Synovium

Journal of Drug Delivery ◽

10.1155/2011/203986 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 13

Author(s):

Yoichi Negishi ◽

Yuka Tsunoda ◽

Yoko Endo-Takahashi ◽

Yusuke Oda ◽

Ryo Suzuki ◽

...

Keyword(s):

Gene Expression ◽

Gene Transfer ◽

Gene Delivery ◽

Delivery System ◽

Imaging System ◽

Effective Means ◽

Gene Delivery System ◽

Highly Efficient ◽

Efficient Gene

Recently, we have developed novel polyethylene glycol modified liposomes (bubble liposomes; BL) entrapping an ultrasound (US) imaging gas, which can work as a gene delivery tool with US exposure. In this study, we investigated the usefulness of US-mediated gene transfer systems with BL into synoviocytes in vitro and joint synovium in vivo. Highly efficient gene transfer could be achieved in the cultured primary synoviocytes transfected with the combination of BL and US exposure, compared to treatment with plasmid DNA (pDNA) alone, pDNA plus BL, or pDNA plus US. When BL was injected into the knee joints of mice, and US exposure was applied transcutaneously to the injection site, highly efficient gene expression could be observed in the knee joint transfected with the combination of BL and US exposure, compared to treatment with pDNA alone, pDNA plus BL, or pDNA plus US. The localized and prolonged gene expression was also shown by an in vivo luciferase imaging system. Thus, this local gene delivery system into joint synovium using the combination of BL and US exposure may be an effective means for gene therapy in joint disorders.

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