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Mol Ther. 2003 Jun;7(6):827-38.

Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells.

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Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Oncology, Baltimore, Maryland 21231, USA.


Lentiviral vectors (LVs) offer several advantages over traditional oncoretroviral vectors. LVs efficiently transduce slowly dividing cells, including hematopoietic stem-progenitor cells (HSCs), resulting in stable gene transfer and expression. Additionally, recently developed self-inactivating (SIN) LVs allow promoter-specific transgene expression. For many gene transfer applications, transduction of more than one gene is needed. We obtained inconsistent results in our attempts to coexpress two transgenes linked by an internal ribosomal entry site (IRES) element in a single bicistronic LV transcript. In more than six bicistronic LVs we constructed containing a gene of interest followed by an IRES and the GFP reporter gene, GFP fluorescence was undetectable in transduced cells. We therefore investigated how to achieve consistent and efficient coexpression of two transgenes by LVs. In a SIN LV containing the elongation factor 1alpha promoter, we included a second promoter from cytomegalovirus, the phosphoglycerate kinase gene, or the HLA-DRalpha gene. Using a single LV containing two constitutive promoters, we achieved strong and sustained expression of both transgenes in transduced engrafting CD34(+) HSCs and their progeny, as well as in other human cell types. Thus, such dual-promoter LVs can coexpress multiple transgenes efficiently in a single target cell and will enable many gene transfer applications.

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