Development of potent unmethylated CpG DNA hydrogel by introducing i-motifs into long single-stranded DNA

Abstract

Unmethylated cytosine-phosphate-guanine (CpG) DNA is recognized by Toll-like receptor 9, expressed in the endosomes of immune cells, and induces the secretion of proinflammatory cytokines. CpG DNA is, therefore, expected to be used as vaccine adjuvants, but there are many obstacles for its therapeutic application, such as poor cellular uptake and biostability. Long single-stranded DNA (lssDNA) synthesized by rolling circle amplification can be a useful delivery carrier for CpG DNA because of its cellular uptake efficiency, but the immunostimulatory effect is transient because it is easily degraded in endosomes. To improve its stability, we constructed lssDNA which forms hydrogel by i-motifs in an acidic environment mimicking endosome, and incorporated CpG DNA into lssDNA (i-CpG-lssDNA). We synthesized lssDNA containing the optimized i-motif sequence, and confirmed the formation of a DNA hydrogel in an acidic environment. The i-CpG-lssDNA elicited a potent proinflammatory cytokine production in murine macrophages, compared to CpG DNA-containing lssDNA without i-motifs. Consistently, its intradermal administration induced potent inflammatory cytokines at the regional lymph nodes. These results suggested that i-CpG-lssDNA could serve as a novel type of adjuvant for the induction of a potent immune response.