Clinical scale production of an improved retroviral vector expressing the human multidrug resistance 1 gene (MDR1)

Eckert, Hans-Georg; Kühlcke, Klaus; Schilz, Andrea; Lindemann, Carsten; Basara, Nadežda; Fauser, Axel A.; Baum, Christopher

doi:10.1038/sj.bmt.1702368

Cited by 27 publications

(18 citation statements)

References 18 publications

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“…Supernatants of SF1m were also produced from cloned PG13 cells containing a single proviral integration and had a similar titer. 22 Gene transfer in primary human hematopoietic cells PG13 cells produce replication-incompetent murine Ctype retroviruses pseudotyped with the envelope protein of the gibbon ape leukemia virus (GALV). 23 GALVpseudotyped retroviruses were previously shown to allow efficient transduction of human and primate hematopoietic cells, including primitive NOD/SCID-repopulating cells, provided that self-renewal divisions of hematopoietic cells occur and virus-cell interactions are supported using recombinant fibronectin and/or centrifugation.…”

Section: Resultsmentioning

confidence: 99%

“…RS4 was subsequently expanded to a master cell bank (100 vials of 1 × 10 6 cells) and used for production of defined batches of cell-free supernatants in two tray cell-factories. 22 Serum-free supernatants were harvested from these cultures and stored at −70°C. Titers of thawed aliquots were in the range of 1-3 × 10 6 infectious particles per ml (Figure 2b).…”

Section: Resultsmentioning

confidence: 99%

“…For harvest of supernatants, the growth medium of PG13 cells was shifted from serum-containing to serum-free medium (X-vivo 10; BioWhittacker, Verviers, Belgium), as described. 22 Cell culture supernatants were harvested 8-16 h after medium exchange, filtered (0.45 m filter; Gelman Sciences, Dreieich, Germany) and kept frozen in aliquots at −70°C. Samples of defined vector batches were titrated on predefined numbers of HT1080 target cells.…”

Section: Cloning Of Psf91m3mentioning

confidence: 99%

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Improved post-transcriptional processing of an MDR1 retrovirus elevates expression of multidrug resistance in primary human hematopoietic cells

et al. 2001

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We describe the functional analysis of a novel retroviral vector, SF91m3, which was designed for improved expression of the in vivo selectable marker, multidrug resistance 1 gene (MDR1), in hematopoietic cells. SF91m3 combines several promising features. The vector backbone lacks viral coding sequences and AUG-start codons 5Ј of the MDR1 cDNA. A point mutation of a cryptic splice acceptor of the MDR1 cDNA increases the probability of transferring an intact provirus. The titer of a PG13 packaging cell clone containing a single proviral integration is high (Ͼ2 × 10 6 particles/ml from frozen stocks of serum-free vector harvests). Human hematopoietic cells transduced with SF91m3 reliably express MDR1 before and after passage through NOD/SCID mice,

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Cloning Of Psf91m3mentioning

confidence: 99%

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Improved post-transcriptional processing of an MDR1 retrovirus elevates expression of multidrug resistance in primary human hematopoietic cells

et al. 2001

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“…Retroviral vector stocks were produced and stored as described. 23 Retroviral transduction was performed as described. 22 In brief, CD34 + cells were prestimulated for 16-20 h at a density of 1 Â 10 6 cells/ml X-VIVO-10 medium (BioWhittaker, Verviers, Belgiun), supplemented with interleukin 3 (20 ng/ml), interleukin 6 (10 ng/ml), stem cell factor (50 ng/ml), Flt-3 ligand (100 ng/ml) and thrombopoietin (20 ng/ml) (R & D Systems, Weisbaden, Germany).…”

Section: Detection Of Retroviral Vector Integration Sites B Gentner Ementioning

confidence: 99%

Rapid detection of retroviral vector integration sites in colony-forming human peripheral blood progenitor cells using PCR with arbitrary primers

et al. 2003

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We have developed a highly sensitive polymerase chain reaction (PCR)-based technique termed two-step PCR, which uses arbitrary primers to identify proviral integration sites in retrovirally marked human colony-forming cells. The two-step PCR was established on cell line clones transduced with the SF1m retroviral vector and independently validated by demonstrating identical integration sites with ligationmediated PCR, a different technique requiring restriction enzyme digestion and adapter ligation for amplifying unknown DNA flanking the provirus. Two-step PCR was performed on peripheral blood progenitor cell (PBPC) colonies that contained as few as 75 cells, which was estimated by quantitative real-time PCR. We were able to amplify and directly sequence proviral integration sites in 35 % of PBPC colonies (25/72, five donors). Identity to the vector long-terminal repeat was confirmed and flanking DNA was found to match with human database sequences, reaffirming specificity. Two-step PCR is a valuable new tool for rapid analysis of genomic target sites for viral vectors, and will aid significantly in understanding clonal development of hematopoiesis and other cell types. Our protocol has the potential for general applicability as the arbitrary primers described here bind to genomic DNA and are thus independent of the vector backbone used.

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“…[2][3][4][5][6][7] With the exception of roller bottles and Cell Factories, all other large-scale systems utilize at least some nondisposable parts that need to be reused, thus necessitating the performance of extensive cleaning and sterilization validation studies.…”

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confidence: 99%

Production scale-up and validation of packaging cell clearance of clinical-grade retroviral vector stocks produced in Cell Factories

et al. 2005

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The clinical implementation of gene therapy requires large-scale production of viral vector stocks (VS) derived from packaging cell lines. Upon scaling-up, maintenance of high viral titers and filtration of the VS become significantly challenging. Thus, production schemes amenable to straightforward validation must be developed. To this end, we have established a semi-closed process to manufacture batches of 7 l or more of clinical-grade oncoretroviral VS using 10-tray Cell Factories. Using a peristaltic pump, the VS are collected on 3 consecutive days, filtered, pooled and stored frozen. To ensure the absence of viable vectorproducing cells (VPCs) from each VS unit-dose, we undertook an orthogonal log-removal validation study to demonstrate the ability of both the filtration system to remove viable cells and the VS freezing process to inactivate them. We demonstrate a total VPC-reduction of 11.6 log, thus insuring the absence of contaminating VPCs in transduced clinical samples. We also show that this production process generates stable VS that can be stored at À801C for more than 3 years. Importantly, this relatively simple and affordable process can be customized to generating large volume of VS for small animal or nonhuman primate studies. This methodology is not limited to the generation of cell-free clinical oncoretroviral VS, and can be applied to other types of vectors produced in packaging cell lines, such as lentiviral vectors. Gene Therapy (2006) 13, 95-100.

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Clinical scale production of an improved retroviral vector expressing the human multidrug resistance 1 gene (MDR1)

Cited by 27 publications

References 18 publications

Improved post-transcriptional processing of an MDR1 retrovirus elevates expression of multidrug resistance in primary human hematopoietic cells

Improved post-transcriptional processing of an MDR1 retrovirus elevates expression of multidrug resistance in primary human hematopoietic cells

Rapid detection of retroviral vector integration sites in colony-forming human peripheral blood progenitor cells using PCR with arbitrary primers

Production scale-up and validation of packaging cell clearance of clinical-grade retroviral vector stocks produced in Cell Factories

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