The challenging nature of primary T lymphocytes for transfection: Effect of protamine sulfate on the transfection efficiency of chemical transfection reagents.

BACKGROUND AND PURPOSE

The optimization of an effective non-viral gene delivery method for genetic manipulation of primary human T cells has been a major challenge in immunotherapy researches. Due to the poor transfection efficiency of conventional methods in T cells, there has been an effort to increase the transfection rate in these cells. Protamine is an FDA-approved compound with a documented safety profile that enhances DNA condensation for gene delivery.

EXPERIMENTAL APPROACH

In this study, the effect of protamine sulfate on the transfection efficiency of standard transfection reagents, was evaluated to transfect primary human T cells. In this regard, pre-condensation of DNA was applied using protamine, and the value of the zeta potential of DNA/protamine/cargo complexes was determined. T cells were transfected with DNA/protamine/cargo complexes. The transfection efficiency rate was evaluated by flow cytometry. Also, the green fluorescent protein expression level and cytotoxicity of each complex were identified using real-time polymerase chain reaction and MTT assay, respectively.

FINDINGS/RESULTS: Our results demonstrated that protamine efficiently increases the positive charge of DNA/cargo complex without any cytotoxic effect on the primary human T cells. We observed that the transfection efficiency in DNA/protamine/ Lipofectamine 2000 and DNA/protamine/TurboFect™ was 87.2% and 78.9%, respectively, while transfection of T cells by Lipofectamine 2000 and TurboFect™ would not result in sufficient transfection.

CONCLUSION AND IMPLICATIONS

Protamine sulfate enhanced the transfection rate of T cells; and could be a promising non-viral gene delivery method to achieve a safe, rapid, cost-effective, and efficient system which will be further applied in gene therapy and T cells manipulation methods.