High Levels of Transduction of Human Dendritic Cells with Optimized SIV Vectors

Abstract

As major antigen-presenting cells and effectors in the maintenance of tolerance, dendritic cells (DCs) are key cells of the immune system and can thus be envisioned to have roles in immunotherapy strategies. We, and others, previously showed that simian immunodeficiency virus (SIV)-derived lentiviral vectors were able to deliver a gene into human differentiated DCs. We describe here the upgrading of the SIV vector system and the improvements of the transduction protocol, which allowed us to transduce more than 90% of human monocyte-derived DCs. We developed new SIV lentiviral vectors carrying SIV splice regulatory elements and either the woodchuck hepatitis virus regulatory element (WPRE) or the murine phosphoglycerate-kinase 1 (PGK) promoter. We show that insertion of the WPRE in the SIV vector is detrimental to gene transfer in DCs, while this sequence increases transgene expression in 293T cells. Using an optimized SIV vector, high levels of transgene expression were obtained in more than 30% of human DCs at a multiplicity of infection (MOI) of 1, and close to 100% using a MOI of 20. VSV-G pseudotyped vectors generated with only gag, pol, tat, and rev helper functions failed to transduce DCs. This defect was completely rescued when the SIV accessory gene vpx was provided in trans in vector-producing cells. Genetically modified DCs were shown to behave as bona fide DCs in both allogenic and autologous mixed leukocyte reactions. These findings allow us to propose an optimal system for efficient and safe DC transduction with improved SIV vectors.

Optimization of equine infectious anemia derived vectors for hematopoietic cell lineage gene transfer

“…Additionally, the incorporation of the WPRE element in EIAV vectors had no effect in primary human hematopoietic cells and was slightly inhibitory in mouse muscle in vivo (data not shown). 16 Mangeot et al 21 reported that WPRE significantly reduced gene transfer to primary human dendritic cells in the context of SIV vectors. These data suggest the use of the WPRE elements should be investigated in a cell type and vector specific manner.…”

“…21 Therefore, we investigated the viral constitutive transport elements (CTE) from the Mason-Pfizer monkey virus; the direct repeat 1 element (DR1) from the Rous sarcoma virus and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) in the context of EIAV vectors. Transduction of the T-cell lines with either the CTE or DR1 containing vectors increased the number of EGFP positive cells between three-and approximately five-fold (Table 1).…”

Lentiviral vectors: optimization of packaging, transduction and gene expression

“…Nevertheless, the transduction of human dendritic cells (DCs) may require the presence of vpx [25]. In the context of FIV-derived LV vectors, Johnston et al [26] have removed the accessory genes encoding vif and orf2 from the packaging construct without observing any decrease in the particle production.…”

“…Interestingly, they have shown that the WPRE was significantly more active than its HBV counterpart; this increased activity may correlate with the presence of an additional cis-acting sequence in the WPRE which is not found in the HPRE. Surprisingly, while the insertion of the WPRE in SIV-derived vectors has also been shown to increase transgene expression in 293T cells, its presence seemed to be detrimental to gene transfer in human DCs [25]. Relative to the EIAV-based system, the addition of the WPRE cis-acting sequence to vectors markedly increased gene expression in mouse skeletal muscle cells [57].…”

“…The ESE elements are described as purine-rich sequences recruiting arginine-rich proteins responsible for exon recognition [78]. Such purine-rich regions which have been described downstream of the tat/rev splice acceptor sites of the HIV-1 [79] and SIV [25] genomes have been found to increase the transduction efficiency of LV-derived transfer vectors.…”