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

Ilnaz Rahimmanesh , Mehdi Totonchi, Hossein Khanahmad

Abstract


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/TurboFectTM was 87.2% and 78.9%, respectively, while transfection of T cells by Lipofectamine® 2000 and TurboFectTM 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.


Keywords


Gene transfer techniques; Protamine sulfate; T-Lymphocytes; Transfection.

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References


Jin C, Fotaki G, Ramachandran M, Nilsson B, Essand M, Yu D. Safe engineering of CAR T cells for adoptive cell therapy of cancer using long‐term episomal gene transfer. EMBO Mol Med. 2016;8(7):702-711.

DOI: 10.15252/emmm.201505869.

Hu WS, Pathak VK. Design of retroviral vectors and helper cells for gene therapy. Pharmacol Rev. 2000;52(4):493-511.

Wang GP, Garrigue A, Ciuffi A, Ronen K, Leipzig J, Berry C, et al. DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer. Nucleic Acids Res. 2008;36(9):e49,1-12.

DOI: 10.1093/nar/gkn125.

El-Aneed A. An overview of current delivery systems in cancer gene therapy. J Control Release. 2004;94(1):1-14.

DOI: 10.1016/j.jconrel.2003.09.013.

Darzi L, Boshtam M, Shariati L, Kouhpayeh S, Gheibi A, Mirian M, et al. The silencing effect of miR-30a on ITGA4 gene expression in vitro: an approach for gene therapy. Res Pharm Sci. 2017;12(6):456-464.

DOI: 10.4103/1735-5362.217426.

Schambach A, Baum C. Clinical application of lentiviral vectors-concepts and practice. Curr Gene Ther. 2008;8(6):474-482.

DOI: 10.2174/156652308786848049.

Gresch O, Engel FB, Nesic D, Tran TT, England HM, Hickman ES, et al. New non-viral method for gene transfer into primary cells. Methods. 2004;33(2):151-163.

DOI: 10.1016/j.ymeth.2003.11.009.

Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG. Non-viral vectors for gene-based therapy. Nat Rev Genet. 2014;15(8):541-55

DOI: 10.1038/nrg3763.

Stroh T, Erben U, Kühl AA, Zeitz M, Siegmund B. Combined pulse electroporation-a novel strategy for highly efficient transfection of human and mouse cells. PloS One. 2010;5(3):e9488,1-8.

DOI: 10.1371/journal.pone.0009488.

Zhang Z, Qiu S, Zhang X, Chen W. Optimized DNA electroporation for primary human T cell engineering. BMC Biotechnol. 2018;18(1):4-12.

DOI: 10.1186/s12896-018-0419-0.

Zhao N, Qi J, Zeng Z, Parekh P, Chang CC, Tung CH, et al. Transfecting the hard-to-transfect lymphoma/leukemia cells using a simple cationic polymer nanocomplex. J Control Release. 2012;159(1):104-110.

DOI: 10.1016/j.jconrel.2012.01.007.

Motta S, Brocca P, Del Favero E, Rondelli V, Cantù L, Amici A, et al. Nanoscale structure of protamine/DNA complexes for gene delivery. Appl Phys Lett. 2013;102(5):053703,1-4.

DOI: 10.1063/1.4790588.

Chen J, Yu Z, Chen H, Gao J, Liang W. Transfection efficiency and intracellular fate of polycation liposomes combined with protamine. Biomaterials. 2011;32(5):1412-1418.

DOI: 10.1016/j.biomaterials.2010.09.074.

Tsuchiya Y, Ishii T, Okahata Y, Sato T. Characterization of protamine as a transfection accelerator for gene delivery. J Bioact Compat Polym. 2006;21(6):519-537.

DOI: 10.1177/0883911506070816.

Vighi E, Montanari M, Ruozi B, Iannuccelli V, Leo E. The role of protamine amount in the transfection performance of cationic SLN designed as a gene nanocarrier. Drug Deliv. 2012;19(1):1-10.

DOI: 10.3109/10717544.2011.621989.

Gupta S, Tiwari N, Munde M. A Comprehensive biophysical analysis of the effect of DNA binding drugs on protamine-induced DNA condensation. Sci Rep. 2019;9(1):5891-5901.

DOI: 10.1038/s41598-019-41975-8.

Liu L, Johnson C, Fujimura S, Teque F, Levy JA. Transfection optimization for primary human CD8+ cells. J Immunol Methods. 2011;372(1-2):22-29.

DOI: 10.1016/j.jim.2011.06.026.

Wang W, Li W, Ma N, Steinhoff G. Non-viral gene delivery methods. Curr Pharm Biotechnol. 2013;14(1):46-60.

Mohammadi Z, Shariati L, Khanahmad H, Kolahdouz M, Kianpoor F, Ghanbari JA, et al. A lentiviral vector expressing desired gene only in transduced cells: an approach for suicide gene therapy. Mol Biotechnol. 2015;57(9):793-800.

DOI: 10.1007/s12033-015-9872-3.

Zhao Y, Zheng Z, Cohen CJ, Gattinoni L, Palmer DC, Restifo NP, et al. High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Mol Ther. 2006;13(1):151-159.

DOI: 10.1016/j.ymthe.2005.07.688.

De Ilarduya CT, Arangoa M, Moreno-Aliaga M, Düzgüneş N. Enhanced gene delivery in vitro and in vivo by improved transferrin-lipoplexes. BBA Bioenergetics. 2002;1561(2):209-221.

DOI: 10.1016/S0005-2736(02)00348-6.

Pishavar E, Oroojalian F, Ramezani M, Hashemi M. Cholesterol‐conjugated PEGylated PAMAM as an efficient nanocarrier for plasmid encoding interleukin‐12 immunogene delivery toward colon cancer cells. Biotechnol Prog. 2020;36(3):e2952, 1-8.

DOI: 10.1002/btpr.2952.

Cervia LD, Chang CC, Wang L, Yuan F. Distinct effects of endosomal escape and inhibition of endosomal trafficking on gene delivery via electrotransfection. PloS One. 2017;12(2):e0171699, 1-18.

DOI: 10.1371/journal.pone.0171699.

Hébert E. Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy? Biol Cell. 2003;95(2):59-68.

DOI: 10.1016/s0248-4900(03)00007-8.

Akita H, Kurihara D, Schmeer M, Schleef M, Harashima H. Effect of the compaction and the size of DNA on the nuclear transfer efficiency after microinjection in synchronized cells. Pharmaceutics. 2015;7(2):64-73.

DOI: 10.3390/pharmaceutics7020064.

Masuda T, Akita H, Harashima H. Evaluation of nuclear transfer and transcription of plasmid DNA condensed with protamine by microinjection: the use of a nuclear transfer score. FEBS Lett. 2005;579(10):2143-2148.

DOI: 10.1016/j.febslet.2005.02.071.

Xu X, Li L, Li X, Tao D, Zhang P, Gong J. Aptamer-protamine-siRNA nanoparticles in targeted therapy of ErbB3 positive breast cancer cells. Research Square. 2020:1-40.

DOI: 10.21203/rs.3.rs-42522/v1.

Basiouni S, Fuhrmann H, Schumann J. High-efficiency transfection of suspension cell lines. Biotechniques. 2012;53(2):1-4.

DOI: 10.2144/000113914.


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