Lentiviral Vectors for Enhanced Gene Expression in Human Hematopoietic Cells

Abstract

Accumulated data indicate that current generation lentiviral vectors, which generally utilize an internal human cytomegalovirus (CMV) immediate early region enhancer-promoter to transcribe the gene of interest, are not yet optimized for efficient expression in human hematopoietic stem/progenitor cells (HSPCs). As a first step toward this goal, we constructed self-inactivating derivatives of the HIV-1-based transfer vector pHR' containing the enhanced green fluorescent protein (GFP) gene as reporter and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). GFP expression was driven by a variety of strong viral and cellular promoters, including the murine stem cell virus (MSCV) long terminal repeat (LTR), a Gibbon ape leukemia virus (GALV) LTR, the human elongation factor 1alpha (EF1alpha) promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), and the human phosphoglycerate kinase 1 (PGK) promoter. In contrast to results obtained in human embryonic kidney 293T cells and fibrosarcoma HT1080 cells, in which the CMV promoter expressed GFP at the highest levels, significantly higher levels of GFP expression (3- to 5-fold) were achieved with the MSCV LTR, the EF1alpha promoter, and the CAG promoter in the human HSPC line KG1a. Removal of the WPRE indicated that it stimulated GFP expression from all of the vectors in KG1a cells (up to 3-fold), although it only marginally improved the performance of the intron-containing EF1alpha and CAG promoters (<1.5-fold stimulation). The vectors using the MSCV LTR, the GALV LTR, and the PGK promoter were the most efficient at transducing primary human CD34+ cord blood progenitors under the conditions employed. High-level GFP expression in the NOD/SCID xenograft model was demonstrated with the pHR' derivative bearing the MSCV LTR. These new lentiviral vector backbones provide a basis for the rational design of improved delivery vehicles for human HSPC gene transfer applications.

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities

“…Currently, a few systematic examinations of promoter activities have been carried out in gene transfer experiments in vivo 28-30 and Optimization 19,29,31 however, information regarding the promoter activities in HSCs is limited and controversial. The aim of this study was to identify an Ad35 vector platform for efficient transgene expression in human HSCs by optimizing a promoter that directs transgene expression.…”

“…A variety of promoters have been used for transduction into human CD34 + cells, including the phosphoglycerate kinase 1 promoter (PGK promoter), 16 the human cytomegalovirus immediateearly region promoter/enhancer (CMV promoter), 17,18 and the CMV immediate-early enhancer/the chicken b-actin promoter with the b-actin intron sequence (CA promoter). 8 However, few studies have simultaneously compared the relative strength of various types of promoters in human CD34 + cells, 19 and information regarding promoter activities in human CD34 + cells is controversial. In addition, the promoter activities have not been fully evaluated in immature CD34 + subpopulations.…”

Functional transfer of CD40L gene in human B-cell precursor ALL blasts by second-generation SIN lentivectors

“…18 The best available results so far for gene transduction have been obtained with second-generation lentiviral vectors monitoring the expression of the CD80 transgene up to 48 h. 5 More recently, second-generation lentiviral vectors have been further modified by deleting a portion of the U3 LTR region in the so-called self-inactivating vector (SIN) with improved safety, 8,9,15,19,20 and by the introduction of two cis-acting elements, one derived from the pol sequence, called central polypurine tract sequence (cPPT), 11,21,22 and the other, termed Wpre, obtained from the genome of the woodchuck hepatitis virus, both believed to increase transgene expression. [23][24][25] As previously demonstrated by our group 26 and others, [27][28][29][30][31][32] CD40 triggering by the CD40L molecule is capable of inducing a number of morphological and functional changes of BCP-ALL blasts, including the upregulation of the surface markers CD40, CD86, CD80, MHC class I and II, CD54 and CD58, and the secretion of chemoattractants MDC and TARC. Furthermore, data indicate that the transgenic expression of CD40L alone or in concomitance with other molecules in several hematological malignancies can elicit an antitumor immune response both in vitro and in vivo.…”

“…15,19,[62][63][64] Moreover, our vectors contain two cis-acting elements (cPPT and Wpre) which should have optimized the expression of the transgene in the transduced B-ALL cells. [8][9][10][11]24,25 Their direct role, however, cannot be implied by these data, even if our previous data suggest an improved activity of the cPPT element in B-cell lines. 12 Finally, only few reports to date have addressed the issue of transducing leukemic BCP-ALL cells.…”

Vectors for the treatment of autoimmune disease

“…102 Use of wild-type HIV LTR promoter gave rise to higher expression, but demanded the production of Tat protein, which proved to be toxic. 103 Among others, human elongation factor-1a (EF-1a) promoter was proposed recently for gene expression in HSCs and DCs 104 and provided the highest stable expression in cells of haematopoietic lineages. 105 SFFV promoter was shown to enhance the long-term expression of a transgene in primary human haematopoietic cells in vivo.…”