Development of a suspension packaging cell line for production of high titre, serum-resistant murine leukemia virus vectors

Pizzato, Massimo; Merten, Otto‐Wilhelm; Blair, Edward D.; Takeuchi, Yasuhiro

doi:10.1038/sj.gt.3301457

Cited by 30 publications

(36 citation statements)

References 37 publications

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“…CEMFLYA19 cells cultured in 250 ml spinner flasks reached a density of 1.5-3.0 · 10 7 cells ml À1 , and produced an average of 1.6 · 10 7 IP ml À1 . This was reported to be 2.5-6 fold higher than T-flask cultures (Pizzato et al 2001). The authors also reported that preliminary results showed that this cell line could produce 5 · 10 6 IP ml À1 in a 1.4 l perfused reactor.…”

Section: Bioreactor Designmentioning

confidence: 83%

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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Section: Bioreactor Designmentioning

confidence: 83%

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

2006

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“…GFP-retroviruses produced in Db-Jurkat packaging cell lines revealed half-lives of 6.5 h for the MLV-10A1 pseudotype and 9 h for the GALV pseudotype, whereas half-lives of the same pseudotypes generated in 293 packaging cells were higher (GALV and MLV-10A1 pseudotypes each 11 h) at 371C. Pizzato et al 12 and our earlier studies reported a half-life of the amphotropic MLV pseudotype produced in CEM and PA317 packaging cells, respectively, of approximately 4 h, whereas the same pseudotype produced in FLYA13 and CCRIP packaging cells showed a slightly higher stability. 17,15 GALV and MLV-10A1 pseudotypes produced in the NIH3T3-derived packaging cell lines PG13 and PT67 have half-lives of 13 h and 14 h, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, it was demonstrated that suspension cells can be used to construct retrovirus packaging cell lines. Chan et al 11 used a human lymphoblastoid B-cell line (WIL-2), whereas Pizzato et al 12 used a lymphoblastoid B-cell line (Namalwa) and a T-cell line (CEM) to generate T-cell line-based packaging cells S Reu et al packaging cell lines. In these situations, vector particles were pseudotyped with the ecotropic or amphotropic MLV envelope.…”

Section: Discussionmentioning

confidence: 99%

“…12 Briefly, 1 ml (1 Â 10 7 cells) of a pre-cooled Db-Jurkat cell suspension was mixed with 10 mg of the pCeB plasmid DNA and transferred into a pre-cooled 0.4-cm cuvette. Electroporation was performed using a Gene Pulser II electroporater (Bio-Rad Laboratories GmbH, Munich, Germany) at a voltage of 250 mV and a capacitance of 960 mF.…”

Section: Transfection Of Packaging Constructs and Selection Of Cell Cmentioning

confidence: 99%

“…To date, most packaging cell lines are derived from adherent cells of human or mouse origin and only a few suspension cell-based producer cells have been described. 11,12 Here, we report on the generation of stable suspension packing cell lines based on the human lymphoblastoid T-cell line Db-Jurkat for the simplified generation of TCRretrovirus particles. Db-Jurkat cells lack CD3 surface protein expression owing to the deficiency of endogenous TCRb-chains.…”

Section: Introductionmentioning

confidence: 99%

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Suspension packaging cell lines for the simplified generation of T-cell receptor encoding retrovirus vector particles

et al. 2007

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A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography

Segura

Kamen

Trudel

et al. 2005

Biotechnol. Bioeng.

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Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000.

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Development of a suspension packaging cell line for production of high titre, serum-resistant murine leukemia virus vectors

Cited by 30 publications

References 37 publications

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

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

Suspension packaging cell lines for the simplified generation of T-cell receptor encoding retrovirus vector particles

A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography

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