Retroviral Particles Produced from a Stable Human-Derived Packaging Cell Line Transduce Target Cells with Very High Efficiencies

Davis, Jennifer L.; Witt, Rochelle M.; Gross, Paul R.; Hokanson, Craig A.; Jungles, Steve; Cohen, Lawrence K.; Danos, Olivier; Spratt, S. Kaye

doi:10.1089/hum.1997.8.12-1459

Cited by 36 publications

(31 citation statements)

References 22 publications

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“…[4][5][6]9 However, the RNA titers thus obtained may overestimate the number of functional vector particles due to the presence of defective interfering particles and packaging cell line DNA. 10,17,20 Also, as shown by our studies above with the amp r PCR assay, carry-over of plasmid DNA is of concern in vectors produced by transient transfection methods.…”

Section: Rrl-cmv-gfp Vector Rna Titers By Real-time Pcrmentioning

confidence: 80%

“…Unfortunately, a RNA titer may not accurately reflect the functional titer due to the presence of defective interfering particles, inhibitors of transduction, and carry over DNA from vector production. 10,17,18 Vector expression in transduced cells, particularly when the vector expresses a transgene such as GFP, is technically simple and can estimate functional titer. 16 The limitations of this method are two-fold: it assumes that the level of expression of all integrated vectors is above the detection threshold of the assay and it may not distinguish cells with multiple copies of vector.…”

Section: -Fold) and Gfp Titers (~10 4 -Fold) To Show That The Lentivmentioning

confidence: 99%

“…Currently a variety of methods for assessing titer are available, but a comprehensive study of lentiviral titer methodology has not been performed. [4][5][6][7][8][9][10][11] Lentiviral vectors possess many characteristics similar to murine-based retroviral vectors, but their production methods often differ significantly (transient transfection versus packaging cell lines, respectively). 12 3 …”

Section: Introductionmentioning

confidence: 99%

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Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods

et al. 2002

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Section: Rrl-cmv-gfp Vector Rna Titers By Real-time Pcrmentioning

confidence: 80%

Section: -Fold) and Gfp Titers (~10 4 -Fold) To Show That The Lentivmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods

et al. 2002

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“…One concern with PCRbased titer methods is an inability to detect defective interfering particles or other inhibitors within the vector supernate. [17][18][19] Nevertheless, titer estimation reflected the biologic titers in this assay, suggesting the contribution of defective particles and inhibitors on titer is similar in the vectors tested in this study. We have noted this to be the case with vector produced in the GP+envAM12 cell line 13 and vector generated in the gibbon ape leukemia virus-derived producer cell line PG13.…”

Section: U/ml Trna Was Used As Carrier During Rna Isolation (A) Ammentioning

confidence: 82%

Rapid titer determination using quantitative real-time PCR

Sanburn

Cornetta

1999

Gene Ther

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Quantitative real-time PCR was utilized to evaluate retrovilated with biologic titers performed on the test material. The ral vector titer. RNA was prepared from vector supernatant 96-well capacity of the machine and 2 h of running time and run in a one-step RT-PCR reaction combining reverse permit titer determinations within 8 h, facilitating the protranscription (RT) and amplification in one tube. Sample cessing of large sample numbers while greatly decreasing analysis was performed in the ABI Prism 7700 Sequence technician time. Real-time PCR improves titer quantifiDetector. PCR was quantitative over a range of 10 1 to cation and the identification of high-titer producer cells. 6 × 10 5 vector particles per reaction (2 × 10 2 to 1 × 10 7 vecThis methodology will help investigators meet the chaltor particles per millilites of supernatant) and closely correlenges of developing vectors which lack selectable markers.Keywords: retroviral vectors; vector production; vector titer; quantitative PCR Estimation of retroviral vector titer and identification of high-titer packaging cell lines have been aided by the incorporation of selectable markers within the vector genome. These markers have typically been drug selection genes 1-4 or molecules expressed on the cell surface. 5 Recent concern about the immunogenic potential of these gene products in animals models and human clinical trials have prompted investigators to remove drug selection genes from vector constructs. 6,7 To simplify titer determination, and facilitate rapid identification of hightiter producer clones, investigators have utilized PCR and RNA dot blot assays to estimate vector RNA in supernatant preparations. [8][9][10] These methods are helpful in separating vectors with large to modest differences in titer but remain semi-quantitative. The development of real-time PCR technology, which combines PCR and fluorescence detection of target sequences, may provide a method for quantitative PCR with increased accuracy over current methods. 11,12 In addition, this technology provides for processing of large number of samples (96 reactions per run), one-step RT-PCR reactions (reverse transcription and PCR amplification in one tube), and real-time read-out (without the need for electrophoresis or dot blot analysis). This article demonstrates how this methodology accurately predicts biologic titers, significantly decreases turn around time, and decreases technician time. It has greatly simplified the screening of vector producer cells for high titer clones. Real-time PCR was performed using the ABI Prism 7700 Sequence Detector (TaqMan; PE Applied Biosystems, Foster City, CA, USA) which combines PCR tech-

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“…The virus titers achieved are comparable to other human based producer cells, and range from 10 6 to 10 7 IP ml À1 (Merten 2004). In addition, it has been reported that particles produced from ProPak and 293-SPA clones have a transduction efficiency that is up to 10 times greater than for vectors produced from NIH 3T3 derived produced cells (Forestell et al 1997;Davis et al 1997). …”

Section: Bioreactor Designmentioning

confidence: 99%

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

2006

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Gene therapy is a promising technology for the treatment of several acquired and inherited diseases. However, for gene therapy to be a commercial and clinical success, scalable cell culture processes must be in place to produce the required amount of viral vectors to meet market demand. Each type of vector has its own distinct characteristics and consequently its own challenges for production. This article reviews the current technology that has been developed for the efficient, large-scale manufacture of retrovirus, lentivirus, adenovirus, adeno-associated virus and herpes simplex virus vectors.

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Retroviral Particles Produced from a Stable Human-Derived Packaging Cell Line Transduce Target Cells with Very High Efficiencies

Cited by 36 publications

References 22 publications

Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods

Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods

Rapid titer determination using quantitative real-time PCR

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

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