Self-nanoparticle forming immunostimulatory DNA : structure-function relationship studies

Abstract

Toll-like receptors (TLRs) are one the most critical and widely studied members of the family of pattern recognition receptors expressed on innate immune cells. They recognize microbial signatures, such as bacterial/viral DNA, LPS, from gram negative bacteria, peptidoglycan from gram positive bacteria, zymosan from yeast, lipopeptides or profilin protein from parasites, and even single or double stranded RNA of viruses. Among several members of TLR family, TLR9, that recognizes microbial unmethylated dinucleotide motifs on DNA initiate a robust Th1- biased inflammatory response. Synthetic oligodeoxynucleotides expressing unmethylated CpG motifs, mimic bacterial DNA effect and can be harnessed for the treatment of health problems ranging from infectious diseases to cancer, or to allergy/asthma as well as stand alone immunoprotective agents and also as a vaccine adjuvants that improve protection against pathogens. To date, various classes of CpG ODNs have been identified and were shown to induce differential immune activation in mice and man. Distinct structure-function relationship analyses revealed that these single-stranded linear ODNs alter the immune milieu as they are formulated to form complex multimeric DNA aggregates. Recently, Guanosine-rich D type CpG ODNs has been reported to form complex aggregates, that are differentially regulating immune cells to mount an anti-viral immunity. However, the clinical trials of this type are hampered mainly due to batch to batch variation during large-scale synthesis. To the best of our knowledge, there is no report on self-nanoparticle forming DNA except G-rich sequences. This thesis project was designed to generate stable, selfnanoparticle forming, G-run free, CpG expressing ODNs. In this thesis, we designed a new generation CpG ODN, then characterized their structural and immunological properties. Our results suggest that dendrimeric structure confers higher immunostimulatory potential unparallel to conventional ODNs. Following four hours of in vivo ODN administration into mice indicated that nanoparticle-forming CpG ODNs initiated substantially high spleen and peritoneal exudate cell activation as evidenced by IFNγ and IL-12 production from culture medium. In order to shed light on the uptake and binding mechanisms, blocking experiments revealed that at least one type of scavenger receptor is critical for nanoparticle ODN internalization. Collectively, these data suggested that the improved stability to nucleases along with significantly higher binding to immune cells (no additional ODN formulation is required) seem to be the critical factors contributing to the nanoparticle CpG ODN mediated immune activation. The in vitro and in vivo performances implicated that these next generation immune stimulatory DNA molecules are promising candidates for various clinical applications.