High-Level Antigen Expression and Sustained Antigen Presentation in Dendritic Cells Nucleofected with Wild-Type Viral mRNA but Not DNA

Abstract

Dendritic cells (DC) are potent antigen-presenting cells that hold promise as cell-based therapeutic vaccines for infectious diseases and cancer. Ideally, DC would be engineered to express autologous viral or tumor antigens to ensure the presentation of relevant antigens to host T cells in vivo; however, expression of wild-type viral genes in primary cell lines can be problematic. Nucleofection is an effective means of delivering transgenes to primary cell lines, but its use in transfecting DNA or mRNA into DC has not been widely investigated. We show that nucleofection is a superior means of transfecting human and monkey monocyte-derived DC with DNA and mRNA compared to lipofection and conventional electroporation. However, the delivery of DNA and mRNA had significantly different outcomes in transfected DC. DC nucleofected with DNA encoding green fluorescent protein (GFP) had poor antigen expression and viability and were refractory to maturation with CD40 ligand. In contrast, >90% of DC expressed uniform and high levels of GFP from 3 h to 96 h postnucleofection with mRNA while maintaining a normal maturation response to CD40 ligation. Monkey DC nucleofected with wild-type, non-codon-optimized mRNA encoding simian immunodeficiency virus Gag stimulated robust antigen-specific effector T-cell responses at 24 h and 48 h postnucleofection, reflecting sustained antigen presentation in transfected DC, whereas no detectable T-cell response was noted when DC were nucleofected with DNA encoding the same Gag sequence. These data indicate that mRNA nucleofection may be an optimal means of transfecting DC with autologous tumor or viral antigen for DC-based immunotherapy.

Functional RNA Delivery Targeted to Dendritic Cells By Synthetic Nanoparticles

“…Recent reports on successful delivery of DNA leading to translation have used a mouse DC-like cell line [40,41], which probably has no relevance to the situation with primary DC, as witnessed with the former controversy over cross-presentation [19]. Indeed, when using human and primate DC, Melhem et al reported that even nucleofection with DNA gave poor results, contrasting with nucleofection of a leukemic cell line [42]. These authors also showed that RNA nucleofection of the DC did show efficient translation of the encoded proteins.…”

Nanoparticle design to induce tumor immunity and challenge the suppressive tumor microenvironment

“…This was demonstrated in a comparative study of Liao et al where a sustained period of antigen presentation was measured after RNA transfection, whereas peptide pulsed DCs rapidly lost their ability to present the antigen [70]. Interestingly, the delivery of mRNA have been shown to generate more rapid and longer lasting antigen presentation compared to DNA, making mRNA an interesting source of antigen [71].…”

A Phagocytotic Inducer from Herbal Constituent, Pentagalloylglucose Enhances Lipoplex-Mediated Gene Transfection in Dendritic Cells

“…11) In particular, it is difficult to transduce naked or plasmid DNA on DCs. 9) The main advantages of lipofection are its ability to transfect all types of nucleic acids in a wide range of cell types, its ease of use, reproducibility and low toxicity, 16) therefore, development of more efficient lipofection must enable DC vaccine therapy, and knowledge of DCs biology should be disseminated.…”

“…Our lipofection efficacy corresponds to previous studies using lipofection in dendritic cells (lipofection efficacy is less than 2% or not detectable.). 9,41) To evalutate the effect of PGG on lipofection, we used PGG in the preceding lipofection during incubation to prepare the pEGFP vector/LPF complex (lipoplex) for lipofection, and determined the efficacy of EGFP for lipofection efficacy by flow cytometry and fluorescent microscopy. PGG expectedly enhanced the expression of EGFP (Fig.…”

“…2,3) Because of these properties, DCs have been considered quite attractive immune cells to achieve gene transduction for DNA-based immunization in tumor immunotherapy [4][5][6][7] and many gene delivery methods have attempted to optimize transduction and transfection to human and murine dendritic cells. [8][9][10] However, despite advances in the understanding of DC biology, the development of genetic immunization strategies using DC-transfected plasmid DNA has been limited by their low transfection efficiencies. 11) Currently, the most efficient method for DC transduction is infection using a viral vector based on poxvirus, lentivirus, and adenovirus, 8,12,13) but viral vectors may be associated with safety concerns and generally require DNA codon optimization to overcome poor gene expression.…”