Use of human peripheral blood mononuclear cells to define immunological properties of nucleic acid nanoparticles

Abstract

This protocol assesses proinflammatory properties of nucleic acid nanoparticles (NANPs) using a validated preclinical model, peripheral blood mononuclear cells (PBMCs), that is highly predictive of cytokine responses. The experimental procedure details the preparation of pyrogen-free NANPs, isolation of PBMCs from freshly collected human blood, and analysis of characteristic biomarkers (type I and III interferons) produced by PBMCs transfected with NANPs. Although representative NANPs with high and low immunostimulatory potential are used as standards throughout the procedure, this protocol can be adapted to any NANPs or therapeutic nucleic acids, irrespective of whether they are carrier based or carrier free; additional cytokine biomarkers can also be included. We test several commercial platforms and controls broadly accessible to the research community to quantify all biomarkers in either single- or multiplex format. The continuous execution of this protocol takes <48 h; when immediate analysis is not feasible, single-use aliquots of the supernatants can be frozen and stored (-20 °C; 12 months).

Fig. 1 |. The main strategies for NANP design.

a, Schematics showing the workflow for NANP production. b,c, Two main design strategies: the first relies on intramolecular bonds within each monomer that promote the formation of magnesium-dependent interacting motifs required for intermolecular interactions (b), and the second is based on assembly of NANPs and individual monomers designed to form intermolecular bonds only with their cognate partner strands (c). Scale bars, 500 nm (main); 100 nm (insets). 3WJ, three-way junction; PK, pseudoknot; SELEX, systematic evolution of ligands by exponential enrichment; Us, uracils.

Fig. 2 |. Schematic representation of the experimental design required to assess the immunological properties of NANPs and their interactions with PBMCs.

Corresponding Procedure steps are indicated in blue. Steps 54–57, which describe mechanistic studies, are not shown.

Fig. 3 |. Verification that NANPs retain structural integrity upon complexation with Lipofectamine 2000, and of their cellular uptake.

a, Schematic representation of NANPs’ association with L2K, followed by their release upon detergent (Triton X-100) treatment. b, Ethidium bromide (EtBr) total staining native PAGE, indicating formation of NANPs, their complexation with L2K, and successful release upon treatment with Triton X-100. c, NANPs labeled with Alexa Fluor 488 (AF488) form complexes with L2K and retain their structural integrity upon detergent-mediated release. Note that in b and c, NANPs’ complexation with L2K prevents their entering the gel, whereas treatment with Triton X-100 restores NANPs’ electrophoretic mobility. d, Inhibition of NANPs’ inflammatory response due to TLR recognition. ODN2088 is the oligonucleotide that blocks the function of all endosomal TLRs. Each bar shows a mean response of three independent samples (N) and a standard deviation (N = 3) for each of three donors. e, Inhibition of the inflammatory response due to NANP uptake via scavenger receptor. ODN2216 is an oligonucleotide known to stimulate the IFN response and is used as a positive control; fucoidan, poly-I and dextran are known inhibitors of the scavenger receptor, whereas poly-C and chondroitin are non-specific (i.e., non-inhibitory) controls for poly-I and dextran, respectively. Each bar shows a mean response of three independent samples and a standard deviation (N = 3) for each of three donors. Each sample was tested in duplicate on an ELISA plate (%CV <25). The data used in d and e are reproduced with permission from ref., American Chemical Society. ODN2208 is a CpG DNA oligonucleotide with mixed backbone; ODN 2216 is a CpG oligonucleotide and TLR9 agonist; fucoidan is a complex polysaccharide; poly-I is polyinosinic acid; poly-C is polycytidylic acid; chondroitin is a glycosaminoglycan; dextran is a complex branched glucan. D, donor number; ULOQ, upper limit of quantification.