Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells.
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.
Yu
Xiaobing
X
Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Oncology, Baltimore, Maryland 21231, USA.
Zhan
Xiangcan
X
D'Costa
Jenice
J
Tanavde
Vivek M
VM
Ye
Zhaohui
Z
Peng
Tien
T
Malehorn
Matthew T
MT
Yang
Xiaoming
X
Civin
Curt I
CI
Cheng
Linzhao
L
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000276:immunology; D005822:Genetic Vectors / Q000378:metabolism; D006085:Graft Survival; D049452:Green Fluorescent Proteins; D006684:HLA-DR Antigens / Q000235:genetics / Q000378:metabolism; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D018345:Mice, Knockout; D016513:Mice, SCID; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014161:Transduction, Genetic; D019076:Transgenes; D001613:beta 2-Microglobulin / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
D018952:Antigens, CD34; D006684:HLA-DR Antigens; D008164:Luminescent Proteins; D020648:Peptide Elongation Factor 1; D001613:beta 2-Microglobulin; D049452:Green Fluorescent Proteins; D010735:Phosphoglycerate Kinase