Innate Immune Stimulation Using 3D Wireframe DNA Origami

Abstract

Three-dimensional wireframe DNA origami have programmable structural and sequence features that render them potentially suitable for prophylactic and therapeutic applications. However, their innate immunological properties, which stem from parameters including geometric shape and cytosine-phosphate-guanine dinucleotide (CpG) content, remain largely unknown. Here, we investigate the immunostimulatory properties of 3D wireframe DNA origami on the TLR9 pathway using both reporter cell lines and primary immune cells. Our results suggest that bare 3D polyhedral wireframe DNA origami induce minimal TLR9 activation despite the presence of numerous internal CpG dinucleotides. However, when displaying multivalent CpG-containing ssDNA oligos, wireframe DNA origami induce robust TLR9 pathway activation, along with enhancement of downstream immune response as evidenced by increases in Type I and Type III interferon (IFN) production in peripheral blood mononuclear cells. Further, we find that CpG copy number and spatial organization each contribute to the magnitude of TLR9 signaling and that NANP-attached CpGs do not require phosphorothioate stabilization to elicit signaling. These results suggest key design parameters for wireframe DNA origami that can be programmed to modulate immune pathway activation controllably for prophylactic and therapeutic applications.

Evaluation of Nonmodified Wireframe DNA Origami for Acute Toxicity and Biodistribution in Mice

“…We note that for some applications, such as cancer immunotherapy, the ability to stimulate proinflammatory cytokine induction might be a preferable characteristic of a delivery vehicle. Researchers have demonstrated proof-of-concepts that immune system stimulation can be programmed with NANPs. ,,, We anticipate this capability will be advantageous in future biomedical applications.…”

Evaluation of non-modified wireframe DNA origami for acute toxicity and biodistribution in mice

“…Independent control over the size and geometry of NANPs with site-specific functionalization may improve tissue- and cell-specific targeting 16 , and their sequence-based programmability can be leveraged for logic-gated, controlled cargo release 17, 18 . NANPs have tunable biodegradation profiles 19 , and can be modified chemically and structurally to control immunostimulation 4, 20, 21 . Additionally, NANPs can simply incorporate combinatorial ratios of nucleic acid therapeutics including siRNAs and CRISPR-RNPs through nucleic acid hybridization 15 .…”

“…Initial in vitro cell-based studies revealed context-dependent immunostimulation with examples of rod-like DNA origami being immunologically inert 49 , contrasted by modest immune cell activation by rod-like 20 and wireframe DNA origami 21,50 . Both TLR9 and the cGAS-STING pathways contributed to immune recognition 20, 21 . Additionally, immunostimulation can intentionally be enhanced by the multivalent display of specific immunostimulatory nucleic acids, both in vitro 21, 49 and in vivo 4 .…”

“…Both TLR9 and the cGAS-STING pathways contributed to immune recognition 20, 21 . Additionally, immunostimulation can intentionally be enhanced by the multivalent display of specific immunostimulatory nucleic acids, both in vitro 21, 49 and in vivo 4 . To date, however, no indications of immunotoxicity have been observed in animal models, with studies exploring different NANP geometries and administration routes, namely intravenous (i.v.)…”

DNA origami presenting the receptor binding domain of SARS-CoV-2 elicit robust protective immune response

“…1). In addition, recent studies have shown that DNA origami and particularly the pentagonal bipyramid have negligible immunogenicity, especially in regards to the stimulation of the TLR9 pathway, which is important to determine the specificity of the vaccine response induced by the antigens 43,44 . Therefore, we used the PB structure to present the RBD antigen in a trimeric form, along with multiple CpG ODN 1018 (cytosine-phosphorothioate-guanine oligodeoxynucleotides) adjuvants (Fig.…”