Retrovirus-mediated transfer and expression of drug resistance genes in human haematopoietic progenitor cells

Hock, Rick S.; Miller, A. Dusty

doi:10.1038/320275a0

Cited by 207 publications

(76 citation statements)

References 21 publications

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“…There was no planned reduction in the dose of chemotherapy owing to chemotherapy-induced cytopenias; however, subsequent cycles were delayed until the absolute neutrophil count was 41000/mm 3 and the platelet count was 4100 000/mm 3 . Stem cell support with peripheral blood progenitor cells was administered on day 9 of each …”

Section: Clinical Trialmentioning

confidence: 99%

“…Expression of these gene products has been demonstrated to reduce the effects of cytotoxic drugs on bone marrow function. This approach has been successful in protecting against the hematopoietic toxicity of methotrexate with dihydrofolate reductase 2,3 ; against placitaxel, doxorubicin, and vinblastine toxicity using the multidrug resistance gene-1 (MDR1) 4,5 ; and toxicity associated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) with O6-methylguanine DNA methyltransferase (MGMT). [6][7][8][9] Retroviral transduction of mobilized peripheral blood progenitor cells has been used in human clinical trials to transfer heterologous genes into hematopoietic cells.…”

Section: Introductionmentioning

confidence: 99%

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A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

et al. 2006

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Administration of chemotherapy is often limited by myelosuppression. Expression of drug-resistance genes in hematopoietic cells has been proposed as a means to decrease the toxicity of cytotoxic agents. In this pilot study, we utilized a retroviral vector expressing methylguanine DNA methyltransferase (MGMT) to transduce hematopoietic progenitors, which were subsequently used in the setting of alkylator therapy (procarbazine, CCNU, vincristine (PCV)) for poor prognosis brain tumors. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells were collected by apheresis and enriched for CD34 þ expression. Nine subjects were infused with CD34 þ -enriched cells treated in a transduction procedure involving a 4-day exposure to cytokines with vector exposure on days 3 and 4. No major adverse event was related to the gene therapy procedure. Importantly, the engraftment kinetics of the treated product was similar to unmanipulated peripheral blood stem cells, suggesting that the ex vivo manipulation did not significantly reduce engrafting progenitor cell function. Gene-transduced cells were detected in all subjects. Although the level and duration was limited, patients receiving cells transduced using fibronectin 'preloaded' with virus supernatant appeared to show improved in vivo marking frequency. These findings demonstrate the feasibility and safety of utilizing MGMT-transduced CD34 þ peripheral blood progenitor cells in the setting of chemotherapy.

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Section: Clinical Trialmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

et al. 2006

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“…There are several reports on the successful use of defective retroviruses which contain neo to confer G418' to mouse and human bone marrow cells in culture (2,8,9,14,17,24,26,49,50). The efficiency of transfer and expression is highly dependent upon the helper-free viral titer, as well as the viral construction.…”

Section: Te and (Iv) Justifiablementioning

confidence: 99%

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

Chang¹,

Wager-Smith²,

Tsao³

et al. 1987

Mol. Cell. Biol.

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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 103 to 5 x 104/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 *2 and amphotropic PA-12 helper cells. Higher viral titers (8 x 105 to 7.5 x 106) were obtained when nonproducer NIH 3T3 cells or I2 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 [6TGF]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418' 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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“…These markers have typically been drug selection genes [1][2][3][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.…”

mentioning

confidence: 99%

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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Retrovirus-mediated transfer and expression of drug resistance genes in human haematopoietic progenitor cells

Cited by 207 publications

References 21 publications

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

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

Rapid titer determination using quantitative real-time PCR

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