TransfeX and TransIT-LT1 mediated gene delivery to cervical and oral squamous cell carcinoma cells
Nejat Düzgüneş: 0000-0001-6159-1391
18th Annual Meeting of the American Society of Gene & Cell Therapy
New Orleans, LA
May 13-16, 2015
Date of Presentation
OBJECTIVES: Cancer gene therapy involves the introduction of therapeutic genes into malignant cells. The use of lipid-based nonviral vectors is a promising approach to gene delivery since it avoids potential immune effects presented with the use of viral vectors.. However, the transfection efficiency of non-viral vectors needs to be improved to achieve adequate transgene expression in most cancer cells. In particular, oral squamous cell carcinoma cells are highly resistant to transfection by non-viral vectors. We investigated the ability of two novel reagents to deliver the gene for firefly luciferase into cervical and oral squamous cell carcinoma cells. METHODS: HeLa cervical carcinoma, and HSC-3 and H357 human oral squamous cell carcinoma cells were seeded in 48-well culture plates one day prior to transfection, and used at ~85% confluence. The cells were transfected with either TransfeX (ATCC, Manassas, VA) or TransIT-LT1 (Mirus, Madison, WI). Each transfection reagent was used at ratios of 1, 2, 4, or 8 μl per 1 μg plasmid DNA (pCMV.Luc). Toxicities of the reagents were measured with the Alamar Blue cell viability assay. Transfection activity was evaluated by measuring luciferase expression 48 h after transfection, using the Luciferase Assay System (Promega) and a Turner Designs TD-20/20 luminometer. Data were expressed as relative light units (RLU) per ml of cell lysate. RESULTS: In HeLa cells, luciferase expression with 4 μl TransfeX/1 μg DNA reached 50,620,00±13,195,000 RLU/ml, the highest activity ever seen in our laboratory. TransIT-LT1 at 4 μl/1 μg DNA achieved 10,221,000±1,616,000 RLU/ml. In HSC-3 cells, luciferase expression obtained with TransfeX was 1,035,000±37,000 RLU/ml, and 35,000±6,000 RLU/ml with TransIT-LT1. In H357 cells that are highly resistant to transfection with previously available reagents, TransfeX showed maximum transfection efficiency at 2 μl TransfeX/1 μg DNA, with 2,514,000±250,000 RLU/ml. CONCLUSIONS: While TransIT-LT1 is not quite as effective, TransfeX facilitates unusually high levels of transgene expression at levels comparable to that achieved by adenoviral transduction (our unpublished data). Transfection of HeLa cells with TransfeX exhibited the highest transfection activity obtained in our laboratory, and was almost 5 times higher than that achieved with TransIT-LT1. In HSC-3 cells, transfection efficiency of TransfeX was 29 times higher than that of TransIT-LT1. Very high levels of luciferase activity were obtained in H357 cells with TransfeX. TransfeX is likely to be beneficial in various cancer gene therapy approaches and other gene therapies involving non-viral vectors.
TransfeX and TransIT-LT1 mediated gene delivery to cervical and oral squamous cell carcinoma cells.
Paper presented at 18th Annual Meeting of the American Society of Gene & Cell Therapy in New Orleans, LA.