The Design, PEG Modification,and Preliminary Bioactivity Studies Of CpG Containing Oligonucleotides

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides are recognized by immune system via the Toll-like receptor TLR9to exert various functions inducing potent Thl-type immune responses. Synthetic CpG ODN injections were shown to result in significant increase of antigen specific total IgG and IgG2a levels with mild reduction of antigen specific IgGl levels. However, CpG ODN has short half-lives and a high renal clearance in vivo.PEGylation is a method that has been widely used in the modification of biological molecules for improved in vivo stability and half life. We proposed to attempt PEGylation of CpG ODNs to prolong the circulation time and their activity in vivo.We first prepared three CpG ODNs containing PEGs with different molecular weights and evaluated their activities in vitro and in vivo.In vitro immunological activities were assayed by using mouse spleen cells cultured with different CpG ODNs.The levels of IL-6in the supernatants after24h were measured by ELISA. The results indicated that PEGylation interfered with the cell uptake and probably interaction with TLR9. But PEGylated CpG ODNs still retain some immunological stimulation effects.In vivo immunological studies were carried out in mice using ovalbumin (OVA) as the model antigen. The results showed that the PEGylated CpG ODNs were quite effective in stimulating Thl biased immune responses. The effects were more sustained with longer PEG length as compared to the effect of unmodified CpG ODN.In the mean time, we also attempted to design a few shorter CpG ODN sequences and examined their in vitro activitiesIn summary our study provided some insights in the development and application of CpG ODN, and oligonucleotide based therapeutics.