A bicistronic SIN‐lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines

Richard, Emmanuel; Geronimi, Fabien; Lalanne, Magalie; Ged, Cécile; Redonnet‐Vernhet, Isabelle; Lamrissi-Garcia, Isabelle; Gerson, Stanton L.; Verneuil, Hubert de; Moreau‐Gaudry, François

doi:10.1002/jgm.407

Cited by 24 publications

(20 citation statements)

References 42 publications

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“…To avoid this problem, we recommend using the same promoter to drive both the selection marker and the transgene. An alternative approach is to use the bicistronic lentivector, which allows the expression of the target gene and the selection marker under the control of one common promoter (37)(38)(39).…”

Section: Discussionmentioning

confidence: 99%

Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner

Xia

Zhang

Zieth

et al. 2007

Stem Cells and Development

151

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Lentiviruses have been increasingly used for genetic modification of human cells including embryonic stem (ES) cells. Using four ubiquitous promoters-cytomegalovirus (CMV), cytomegalovirus immediate-early enhancer/chicken β-actin hybrid (CAG), phosphoglycerate kinase (PGK), and human elongation factor-1α (EF1α)-in a lentiviral vector to drive the expression of the enhanced green fluorescent protein (EGFP) gene in human ES cells and mouse ES cells, we determined the extent of EGFP suppression by assessing the percentage of cells that were transduced with the EGFP gene but did not fluoresce green. A much higher level of transgene suppression was observed in human ES cells as compared to mouse ES cells. The suppression was also highly promoter dependent, leading to inactivation of more than 95% of the EGFP genes under the CMV or CAG promoter while only 55% under the PGK promoter. No promoter-dependent suppression was observed in transient transfection of human ES cells. Thus, the common phenomenon of poor transgene expression in human ES cells may be caused mainly by suppression of the transgene right after transduction and integration. Cautions should be taken to choose the optimal promoter when lentiviruses are used for genetic modification of human ES cells.

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Section: Discussionmentioning

confidence: 99%

Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner

Xia

Zhang

Zieth

et al. 2007

Stem Cells and Development

151

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“…This approach could have additional therapeutic applications in genetic diseases, as demonstrated in mouse models of thalassemia [ 65 ]; protoporphyria [ 66 ]; HIV therapy [ 67,68 ]; and graft modulation following allogeneic transplant. Importantly, it has recently been demonstrated that the effective chemoresistance and in vivo selection seen in murine models can be robustly recapitulated in large animal canine allograft and autograft models [ 39,62 ].…”

Section: Engineering Chemoresistancementioning

confidence: 99%

Live and let die: In vivo selection of gene-modified hematopoietic stem cells via MGMT-mediated chemoprotection

Milsom

Williams

2007

DNA Repair

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Gene transfer into hematopoietic stem cells (HSC) provides a potential means of correcting monogenic defects and altering drug sensitivity of normal bone marrow to cytotoxic agents. These applications have significant therapeutic potential but the translation of successful murine studies into human therapies has been hindered by low gene transfer in large animals (including humans), and recent serious side effects in a human immunodeficiency trial related to insertional mutagenesis. The latter trial, along with other subsequent trials, while bringing into focus the potential risks of integrating vector systems, also clearly demonstrate the potential usefulness of in vivo selection as it relates to inefficient stem cell transduction. Developing from initial studies by our group and other investigators in which drug resistance was utilized to demonstrate the feasibility of using gene transfer to effect protection from myelotoxicity of chemotherapeutic agents, expression of mutant forms of O 6 -methyguanine-DNA-methytransferase (MGMT) coupled with the simultaneous use of pharmacologic inhibitors and chemotherapeutic agents has been shown to provide a powerful method to select HSC in vivo. While stem and progenitor cell protection and resulting selection in vivo has potential applications for the treatment of selected cancers (allowing dose escalation) andor correction of monogenic disease (allowing an iatrogenic survival advantage of transduced cells in vivo), such an in vivo selection may have untoward effects on stem cell behavior. These deleterious effects may include stem cell exhaustion; lineage skewing; accumulation of genotoxic lesions; and clonal dominance driven towards a pro-leukemic phenotype. Knowledge of the likelihood of such deleterious events occurring as well as their potential implications will be critical to future clinical applications and may also enhance our understanding of both normal stem cell behavior and the evolution of hematopoietic malignancies.

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“…For erythropoietic protoporphiria ex vivo applications, Richard et al [148] have co-expressed the transgene of interest, i.e. the human FECH cDNA, with a human alkylating drug resistance mutant O(6)-methylguanine DNA methyltransferase.…”

Section: Multiple Gene Expression Designmentioning

confidence: 99%

Lentiviral vectors: optimization of packaging, transduction and gene expression

Delenda

2004

J. Gene Med.

112

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SummaryGene transfer vectors based on retroviruses including oncogenic retroviruses and lentiviruses provide effective means for the delivery, integration and expression of exogenous genes in mammalian cells. Lentiviral (LV) vectors provide attractive gene delivery vehicles in the context of non-dividing cells. This review summarizes the different optimized LV genetic systems that have been developed to date. In all cases, the production of LV-derived vectors consists of a genetically split gene expression design. The viral elements that are specifically required are (i) the LV packaging helper proteins consisting of at least the gag-pol genes, (ii) the LV transfer vector RNA containing the transgene expression cassette, and (iii) an heterologous glycoprotein. While the genetic requirements and performances of the two former viral elements will be treated herein, the latter element relative to the envelope pseudotyping of LV vectors will not be further described (cf. review by Cosset in this issue).

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A bicistronic SIN‐lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines

Cited by 24 publications

References 42 publications

Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner

Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner

Live and let die: In vivo selection of gene-modified hematopoietic stem cells via MGMT-mediated chemoprotection

Lentiviral vectors: optimization of packaging, transduction and gene expression

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