Enhanced Immunostimulatory Activity of Covalent DNA Dendrons

Abstract

The present study focused on the design and synthesis of covalent DNA dendrons bearing multivalent cytosine‐phosphate‐guanine oligodeoxynucleotides (CpG ODNs) that can stimulate the immune system through the activation of TLR9. These dendrons were synthesized using branching trebler phosphoramidite containing three identical protecting groups that enabled the simultaneous synthesis of multiple strands on a single molecule. Compared with linear ODNs, covalent DNA dendrons were found to be more resistant to nuclease degradation and were more efficiently taken up by macrophage‐like RAW264.7 cells. Cellular uptake was suggested to be mediated by macrophage scavenger receptors. The covalent DNA dendrons composed of multivalent immunostimulatory branches enhanced the secretion of proinflammatory cytokines TNF‐α and IL‐6 from RAW264.7 cells, and 9‐branched DNA dendrons showed the highest enhancement. Given their enhanced efficacy, we expect covalent DNA dendrons to be useful structures of oligonucleotide medicines.

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“…Branched covalently bound DNA structures, termed DNA dendrons, achieved more efficient uptake as a direct consequence of valency. 68,69,172 In particular, dendrons with six branches carrying peptide antigen exhibited the best uptake, especially compared to a free DNA and peptide mixture (Fig. 2D).…”

“…68 Dendrons that incorporate agonist sequences into the branches or stem can be efficiently used to deliver CpG agonist oligonucleotides in both DCs and macrophages. 69,172 Nine-branched structures promoted the greatest enhancement in inflammatory cytokine production in macrophages. 172 The authors hypothesized that this may be due to multivalent activation of TLR9 receptors by a single DNA structure.…”

“…69,172 Nine-branched structures promoted the greatest enhancement in inflammatory cytokine production in macrophages. 172 The authors hypothesized that this may be due to multivalent activation of TLR9 receptors by a single DNA structure. 172 However, other literature proposes a minimum key distance between TLR9 substrates that is necessary to achieve multivalency.…”

“…Previous studies have implicated the key role of agonist spatial distribution when interacting with TLR9. 173,242,243 DNA-based biomaterials allow for precise control over spacing of both adjuvant and antigen through covalent or chemical linkages, 69,172,244 hybridization, [245][246][247][248] folding, 249,250 and self-assembled presentation. 75,166,251 Prior work has highlighted a range of optimal spacing for CpG agonist sequences to achieve multivalent TLR9 binding in bone marrow-derived dendritic cells (BMDCs), human pDCs, MoDCs, and mouse RAW 264.7 macrophages.…”

“…172 The authors hypothesized that this may be due to multivalent activation of TLR9 receptors by a single DNA structure. 172 However, other literature proposes a minimum key distance between TLR9 substrates that is necessary to achieve multivalency. 173 Therefore, further exploration will elevate the ability to harness multivalency.…”