Comparison of the Efficiency and Safety of Non-viral Vector-Mediated Gene Transfer into a Wide Range of Human Cells.

Abstract

Non-viral vectors for gene therapy, although less efficient for gene delivery, offer several advantages over viral vectors. Non-viral vectors are particularly suitable with respect to simplicity of use and ease of large-scale production, and they are relatively inexpensive to produce, easier to quality control, and generate little or no specific immune response. Nonviral DNA delivery has also become a powerful and popular research tool for elucidating gene structure, regulation, and function. Various types of synthetic vectors such as cationic lipids and polymers have been developed for gene transfer with high transfection efficiency. 1-3)It is necessary to evaluate the efficiency and safety of gene transfer with gene therapy vectors. Many factors may affect the efficacy and safety of non-viral vector-mediated gene transfer, including the structure, charge, and formulation of the vector, the preparation of DNA/vector complexes, 4,5) DNA/vector ratio, charge and size of complexes, [6][7][8] time of exposure, and interaction with serum and blood cells.9,10) As optimal conditions might well differ for different cell types, careful optimization is required to evaluate the efficacy and safety of vectors for the target cells and target organs. [11][12][13][14] In this study, we compared the transfection efficiency and safety of commercially available non-viral vectors with a variety of human cells including primary cells, blood cell lines, and adherent cell lines. We also clarified important factors that affected the evaluation of these vectors. The transfection efficiency and cytotoxicity of all non-viral vectors used was dependent on the type of vector, vector concentration, presence of serum, and type of cell. These results also provided useful information for understanding the characteristics of each vector and selecting the most suitable vector for gene transfer into target cells. (293); 45% MEM and 45% RPMI1640 medium supplemented with 10% FCS (NB-1); RPMI1640 medium supplemented with 10% FCS (SBC-1, K-562, U937, HL60, IM-9 and Jurkat cells); and 199 medium supplemented with 20% FBS containing 5 U/ml of heparin and 15 mg/ml Endothelial Cell Growth Supplement (Technoclone) on collagen type I-coated dishes (HUVEC, HUVSMC and HSFB). In the case of human hepatocytes, frozen cells were thawed and plated on collagen-type-Icoated 96-well plates the day before the experiments in Hepatocyte Culture Medium (Clonetics Co., Walkersville, MD, U.S.A.). MATERIALS AND METHODS CellsNon-viral Vectors Six commercially available transfection reagents were used as non-viral vectors: Lipofectin, LipofectAMINE PLUS, and DMRIE-C (GIBCO-BRL, Gaithersburg, MD), SuperFect and Effectene (QIAGEN, Hilden, Germany), and DOTAP (Boehringer Mannheim, Mannheim, Germany). The composition of each of the vectors was as follows: Lipofectin 15) : 1 : 1 (w/w) liposome formulation of the cationic lipid N- [1-(2,3-dioleyloxy)propyl]-N,N,Ntrimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) (1 mg/ml); LipofectAMINE PLUS: 3 : ...

Gene delivery through cell culture substrate adsorbed DNA complexes

“…Transfection of primary cells or cell lines can have significant variations due to numerous cell and vector properties, such as proliferation, metabolism, internalization, and intracellular trafficking. Transfection of primary cells can involve further obstacles, such as the differential expression of cell surface proteoglycans or decreased viability caused by the transfection reagent (Uchida et al, 2002). Substrate-mediated delivery may decrease the amount of DNA, or the quantity of transfection reagent, which may enhance either transfection or viability for primary cells.…”

“…The ability of substrate-mediated delivery to transfect primary cells was assessed using human dermal fibroblasts (HDF), a primary cell type that has been difficult to transfect due to cytotoxicity (Uchida et al, 2002). Lipoplexes delivered as a bolus resulted in large amounts of cellular debris, poor cell morphology, and reduced cellular viability ( Figure 9A).…”

Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo

“…5 The positive charge significantly increases the uptake of the liposomes by the endothelial cells of the blood vessels in tumor tissues 6 and this has made the cationic liposomes useful for tumor targeted drug delivery. Although cationic liposomes have been used to transfect nucleic acids in cells for several years, their application in gene therapy is limited because of the variable transfection 7,8 and systemic toxicity when injected i.v. [9][10][11] To circumvent the problems associated with systemic administration, cationic liposomes have been made to deliver genes locally.…”

Non‐viral gene therapy for myocardial engineering

“…However, non-viral transfection reagents have begun to address the problem of the extremely low transfection efficiencies observed with naked plasmids. Gebhart et al have reported on a relatively thorough comparison of a number of non-viral vectors in a variety of cell lines in terms of luciferase activity [10], and Uchida et al present a similar comparison in terms of percentage LacZ positive cells [11]. Efficiencies as high as 35% have been observed with polymer based non-viral gene therapies in 10% serum [11].…”

“…Gebhart et al have reported on a relatively thorough comparison of a number of non-viral vectors in a variety of cell lines in terms of luciferase activity [10], and Uchida et al present a similar comparison in terms of percentage LacZ positive cells [11]. Efficiencies as high as 35% have been observed with polymer based non-viral gene therapies in 10% serum [11]. Viruses, in contrast, generally have efficiencies between 40 and 90% depending on the type of virus and the cell line in question [12].…”