Cells were collected and cultured for 2 h at 37C in serum-free RPMI 1640 media supplemented with sodiumpyruvate (2mM), non-essential amino acids, sodium bicarbonate (1

Cells were collected and cultured for 2 h at 37C in serum-free RPMI 1640 media supplemented with sodiumpyruvate (2mM), non-essential amino acids, sodium bicarbonate (1.5 g/l), penicillin (100 U/ml) and streptomycin (100mg/l). provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populationsin vivousing lentiviral vectors. Keywords:Macrophage, promoter, lentivirus == INTRODUCTION == In gene therapy, it is advantageous to use selective targeting strategies to control expression of therapeutic genes. Transgene expression driven by tissue-specific promoters may help reduce the effects of off-target expression and improve the efficiency and safety of lentiviral vectors for gene therapy. Lentivirus-derived vectors are efficient delivery vehicles for genes as they can target and permanently integrate into hematopoietic stem cells and non-dividing cells,e.g.macrophages, with high efficiency. Using gene therapy with a tissue specific promoter, macrophages (or other cell types) can be targeted to either overexpress or silence a specific protein. The use of tissue specific promoters can limit both ARRY-520 R enantiomer genotoxicity (e.g.by limitation of enhancer activity in nontarget cells which leads to reduced activation of oncogenes near the vector integration site) and cytotoxicity (ectopic transgene expression in nontarget cells).1 Macrophages are key players in several common ARRY-520 R enantiomer diseasese.g.infections,2arthritis,3atherosclerosis4and cancer.5,6Macrophage-specific promoters (MSPs) can be used for gene delivery approaches as valuable tools to study the function of the macrophageper se, or to treat diseases in a macrophage-dependent fashion. Several different MSPs have earlier been published in different settings.716Among these, previous studies indicate that the CD68 promoter7,8and a synthetic promoter (generated by randomly ligating myeloid-specificcispromoter elements for several different transcription factors and inserting them upstream of the p47phoxbasal promoter)16result in the highest transgene expression levels and macrophage specificity. However, these promoters exist in different lengths and orientations and further characterization is required to assess which promoter is superior. In this study we sought to find an MSP with high specificity resulting in high protein expression in macrophages and low leakage in non-target cells using lentiviral delivery. To achieve an appropriate comparison between promoters, we constructed lentiviral vectors containing promoters fused with GFP. We compared three variants of the CD68 promoter (full length, the 343 base pair (bp) proximal region and the 150 bp proximal region) and two variants of the synthetic ARRY-520 R enantiomer promoter ARRY-520 R enantiomer (in forward and reverse orientation)in vitroandin vivo. We show that MSPs can efficiently be used for lentiviral gene delivery and that the 150 bp proximal region of the CD68 promoter results in significant ARRY-520 R enantiomer protein expression and is currently the best available promoter for macrophage specific expression of transgenes. == RESULTS == == The constructs == We generated lentiviral constructs with expression cassettes containing promoters, GFP and woodchuck post-transcriptional regulatory element (WPRE). The following promoters were used: (i) the spleen focus forming virus long terminal repeat promoter (SFFV), used as a positive control; (ii) full length human CD68 promoter in reverse orientation (CD68FL(r)); (iii) the proximal 343 bp of the CD68 promoter (CD68/343(f)); (iv) the proximal 150 bp of the CD68 promoter (CD68/150(r)); (v) the synthetic promoter in the forward orientation (SP(f)); and (vi) the synthetic promoter in the reverse orientation (SP(r)). All constructs are shown inFigure 1. == Figure 1. Lentiviral contructs used in this study. == LTR, long terminal repeat; SFFV, spleen focus forming virus; GFP, green fluorescent protein; WPRE, woodchuck post-transcriptional regulatory element; cPPT, central polypurine tract; bGHpA, bovine growth hormone poly adenylation signal; SP, synthetic promoter. == Expression of GFP in cell line and primary macrophages == In order to determine if lentiviral particles encoding GFP driven by MSPs could transduce macrophages and lead to protein expression, we transduced various cell lines and primary cells. FACS analysis was performed to determine the GFP expression in different cell populations and typical gating is shown inFigure 2. To compare the MSP driven GFP expression in macrophages, a mouse Mouse monoclonal to BID macrophage cell line (RAW 264.7) was initially used. We found that all MSPs except CD68FL(r) were functional and lead to GFP expression.