Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors

Leyva et al. BMC Biotechnology 2011, 11:13 http://www.biomedcentral.com/1472-6750/11/13 RESEARCH ARTICLE Open Access Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors Francisco J Leyva1, Joshua J Anzinger1, J Philip McCoy Jr2, Howard S Kruth1* Abstract Background: Monocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages. Results: Human blood monocytes were transduced using two VSV-G pseudotyped HIV-1 based lentiviral vectors containing EGFP expression driven by either native HIV-LTR (VRX494) or EF1a promoters (VRX1090). Lentiviral vectors were added to cultured macrophages at different times and multiplicities of infection (MOI). Transduction efficiency was assessed using fluorescence microscopy and flow cytometry. Macrophages transduced between 2 and 120 hours after culturing showed the highest transduction efficiency at 2-hours transduction time. Subsequently, cells were transduced 2 hours after culturing at various vector concentrations (MOIs of 5, 10, 25 and 50) to determine the amount of lentiviral vector particles required to maximally transduce human monocytederived macrophages. On day 7, all transduced cultures showed EGFP-positive cells by microscopy. Flow cytometric analysis showed with all MOIs a peak shift corresponding to the presence of EGFP-positive cells. For VRX494, transduction efficiency was maximal at an MOI of 25 to 50 and ranged between 58 and 67%. For VRX1090, transduction efficiency was maximal at an MOI of 10 and ranged between 80 and 90%. Thus, transductions performed with VRX1090 showed a higher number of EGFP-positive cells than VRX494. Conclusions: This report shows that VSV-G pseudotyped HIV-based lentiviral vectors can efficiently transduce human blood monocyte-derived macrophages early during differentiation using low particle numbers that do not interfere with differentiation of monocytes into macrophages. Background Coronary artery disease is the leading cause of death in the United States and other Western societies [1]. Coronary artery disease develops as a consequence of lipid deposition and foam cell formation in the arterial wall leading to the development of atherosclerotic plaques [2]. Atherosclerosis is considered an inflammatory disease in which the major cell types implicated are macrophages, smooth muscle cells, and T lymphocytes [3]. Macrophages are present in virtually every atherosclerotic plaque and have an important role in foam cell and * Correspondence: 1 Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA Full list of author information is available at the end of the article atherosclerotic plaque formation through the uptake and accumulation of cholesterol [4]. Circulating blood monocytes differentiate into macrophages. Monocytes can migrate through the blood vessel wall into surrounding tissue, where they differentiate into macrophages and accumulate in pathological tissue sites in the body. Macrophages produce proinflammatory and proangiogenic mediators, and function as host cells for pathogens. Due to these functions, macrophages can contribute to the initiation and progression of a wide variety of diseases. Therefore, directly manipulating these hematopoietic cell subsets could have significant therapeutic value [5]. Human blood monocyte differentiation into macrophages can be induced in vitro using three different © 2011 Leyva et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Leyva et al. BMC Biotechnology 2011, 11:13 http://www.biomedcentral.com/1472-6750/11/13 methods: culture in 1) human serum (HS), 2) fetal bovine serum (FBS) with granulocyte-macrophage colony-stimulating factor (GM-CSF) or 3) FBS with macrophage colony-stimulating factor (M-CSF). In our experience, macrophages differentiated with HS or GMCSF with FBS show similar phenotype and morphology (i.e., round cells resembling fried eggs) [6,7]. However, the M-CSF differentiated monocyte-derived macrophage has a different phenotype characterized by an elongated shaped macrophage with numerous vacuoles corresponding to macropinosomes, and constitutive uptake of low-density lipoprotein (LDL) [8,9]. Gene transfer methods have been attempted to manipulate gene expression in macrophages. Non-viral methods such as calcium phosphate precipitation, electroporation, and liposomal or polyethylenimine transfection are less time-consuming and allow use of a larger number of constructs per experiment than viral methods. However nonviral gene transfer methods are inefficient at transfecting macrophages [5]. Viral systems generally give higher gene transfer efficiency and longer expression time than nonviral systems, but only allow the transfer of small sizes (less than 8 kb) of foreign DNA [10]. Viral methods using adenoviruses have reported high transduction efficiency, but their lack of integration into the host cell genome makes the expression of any transferred gene transient [11-14]. Retroviruses (except lentivirus) require cell division to integrate their DNA into the host genome to remain stably transduced [15]; therefore, the limited proliferative nature of primary human macrophages does not favor the use of this viral vector. Lentiviruses like HIV have the capacity to infect nondividing and dividing cells and to integrate into the host cell genome [16-20]. Due to these characteristics, HIVbased lentiviral vectors have been proposed as good delivery system candidates for gene therapy, but the attempt to use them in clinical trials has raised concerns about their safety including the risk of genetic recombination leading to the generation of replication-competent retrovirus in humans. Further modifications in the packaging and genetic components of viral genes have been carried out to develop safer HIV-based lentiviral vector systems [21-24]. Due to our interest in gene manipulation of human M-CSF differentiated monocytederived macrophages, the objective of this study was to determine transduction efficiency of primary human blood M-CSF differentiated monocyte-derived macrophages transduced with two VSV-G pseudotyped HIV-1 based lentiviral delivery systems. Results Determination of optimal time to transduce Macrophage cultures from the same donor (donor 1) were transduced with VRX1090 vector using an MOI of Page 2 of 10 50 (fin (...truncated)