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Hum Gene Ther. 2010 Nov;21(11):1555-67. doi: 10.1089/hum.2010.050. Epub 2010 Oct 6.

Generation of human induced pluripotent stem cells bearing an anti-HIV transgene by a lentiviral vector carrying an internal murine leukemia virus promoter.

Author information

1
Department of Microbiology, Immunology and Molecular Genetics, University of California at Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA.

Abstract

The recent development of induced pluripotent stem cells (iPSCs) by ectopic expression of defined reprogramming factors offers enormous therapeutic opportunity. To deliver these factors, murine leukemia virus (MLV)-based vectors have been broadly used in the setting of hematopoietic stem cell transplantation. However, MLV vectors have been implicated in malignancy induced by insertional mutagenesis, whereas lentiviral vectors have not. Furthermore, the infectivity of MLV vectors is limited to dividing cells, whereas lentiviral vectors can also transduce nondividing cells. One important characteristic of MLV vectors is a self-silencing property of the promoter element in pluripotent stem cells, allowing temporal transgene expression in a nonpluripotent state before iPSC derivation. Here we test iPSC generation using a novel chimeric vector carrying a mutant MLV promoter internal to a lentiviral vector backbone, thereby containing the useful properties of both types of vectors. Transgene expression of this chimeric vector was highly efficient compared with that of MLV vectors and was silenced specifically in human embryonic stem cells. Human fetal fibroblasts transduced with the vector encoding each factor were efficiently reprogrammed into a pluripotent state, and these iPSCs had potential to differentiate into a variety of cell types. To explore the possibility of iPSCs for gene therapy, we established iPSC clones expressing a short hairpin RNA (shRNA) targeting chemokine receptor 5 (CCR5), the main coreceptor for HIV-1. Using a reporter construct for CCR5 expression, we confirmed that CCR5 shRNA was expressed and specifically knocked down the reporter expression in iPSCs. These data indicate that our chimeric lentiviral vector is a valuable tool for generation of iPSCs and the combination with vectors encoding transgenes allows for rapid establishment of desired genetically engineered iPSC lines.

PMID:
20524893
PMCID:
PMC2978547
DOI:
10.1089/hum.2010.050
[Indexed for MEDLINE]
Free PMC Article
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