A Polysaccharide Carrier to Effectively Deliver Native Phosphodiester CpG DNA to Antigen-Presenting Cells

Abstract

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) activate the vertebrate innate immune system via toll-like receptor 9 (TLR-9). Although CpG DNA is a promising immunotherapeutic agent, its short circulation time in biological fluids due to nuclease is the major drawback. This paper proposes that a natural polysaccharide called schizophyllan (SPG) can be used as an effective CpG DNA carrier because SPG can complex with CpG DNA and the resultant complex shows the nuclease resistance of the bound DNA. In order to increase cellular uptake in vitro, we chemically attached spermine, cholesterol, arginine octamer, or RGD peptide to SPG. The complexes made of the chemically modified SPG and CpG DNA having a phosphorothioate (PS) or phosphodiester (PO) backbone led to increased secretion of cytokines of about 4- to 15-fold, compared with the uncomplexed dose. Furthermore, when PO CpG DNA was complexed with unmodified SPG, the IL-12 level increased by almost 3- to 11-fold compared with the naked dose. The PO CpG DNA/unmodified SPG complex data suggested that unmodified SPG might effectively deliver PO in vivo due to the electrically neutral nature of unmodified SPG. When the complexed CpG DNAs were injected intraperitoneally, a large amount of IL-12 production was observed compared with the uncomplexed material. Both in vivo and vitro assays indicated that the SPG complex may be of use for CpG DNA therapy.

Nonagonistic Dectin-1 ligand transforms CpG into a multitask nanoparticulate TLR9 agonist

“…In this study, we generated a novel K CpG ODN that we designated K3-SPG. Although it had been reported that there are molecular interactions between single-stranded nucleic acids and β-glucan (37) and that murine and humanized CpG ODNs can be wrapped by SPG to increase their original TLR9-agonistic activities (20), our report demonstrates that a rod-shaped nano-sized K3-SPG particle exhibits dual characteristics of K and D CpG ODNs (Fig. 1).…”

“…To make a complex between CpG ODNs and schizophyllan (SPG), CpG ODNs need additional sequences of phosphorothioate backbone of 40-mer polydeoxyadenylic acid (dA 40 ) at the 5′ or 3′ end (20,22). Fig.…”

“…It has been shown to form a complex with polydeoxyadenylic acid (dA) as a triple-helical structure (19). Although we previously demonstrated that mouse and humanized CpG ODN with PO poly(dA) at the 5′ end complexed with SPG enhanced cytokine production and acted as an influenza vaccine adjuvant (20,21), it has been difficult to achieve high yields of the CpG-SPG complex toward its more efficient and cost-effective preclinical as well as clinical development. Recently, when the PS backbone of the dA sequence was linked to CpG ODN, the efficacy of complex formation was elevated by nearly 100% (22).…”

Immunostimulatory activity of polysaccharide–poly(I:C) nanoparticles

“…Polysaccharides used in this study are; i) naturally occurring, readily available, easily extracted in bulk ii) cheap iii) does not require sophisticated formulation technology as it is water soluble iv) readily undergoes stable interaction with charged Recent studies showed that a natural b-glucan polysaccharide isolated from Schizophyllan modified with peptides and cholesterol formed a complex with TLR9 ligand, CpG ODNs and significantly elevated secretion of cytokines (IL12, IL6 and TNFa) from BMDCs, and murine macrophages [27,28]. These compounds were proposed to trigger several immune cells through various receptors including complement receptor (CR3), scavenger receptors, as well as TLR4 or TLR2/6 [29e32].…”

A Novel Polysaccharide/Polynucleotide Complex and its Application to Bio-functional DNA Delivery System

“…When the complex of native SPG and CpG DNA (s-SPG/CpG DNA) was added, its secretion was increased by about 20-40% compared with that of the naked dose. 67 This increment can be ascribed to the SPG-mediated protection from the nucleasemediated hydrolysis. The CpG DNA complexed with the chemically modified SPG-derivatives increases the cytokine secretion remarkably, i.e., five-to ten-fold compared with that of the naked dose.…”