Membrane Anchored Immunostimulatory Oligonucleotides for In Vivo Cell Modification and Localized Immunotherapy**

Locally delivered immunomodulators are utilized to treat unresectable tumors and solid tumor resection sites to prevent local recurrence. Synthetic immunostimulatory oligonucleotides such as double-stranded RNA or unmethylated cytosine–guanosine motifs (CpG-ODNs) mimic molecular signatures of pathogens (viruses or bacteria, respectively) and trigger an immunostimulatory cascade including maturation, differentiation and proliferation of multiple host immune cells through pattern recognition receptors. As a result, these synthetic ODNs have been extensively studied as therapeutic agents for cancer and as vaccine adjuvants. However, a key element for the effectiveness of immunostimulatory ODNs is the close association of oligonucleotides with tumor antigen or tumor cells. For example, intratumoral/ peritumoral CpG-ODN injections can lead to tumor regression in settings where intravenous CpG treatment has no effect. Also to this end, several CpG adjuvant studies indicated that co-delivery of CpG and antigens to the same antigen presenting cells (APC) significantly enhances antitumor responses. Two fundamental limitations of directly injecting ODNs into tumors are 1) relatively rapid loss of ODNs from the injection site due to their relatively low molecular weights and 2) lack of physical association between tumor cells and ODNs. We hypothesized that a membraneinteractive ODN that could spontaneously insert into cell membranes would in principle overcome both of these limitations, by prolonging ODN retention at tumor sites and more importantly, by providing a physical connection between tumor cells and ODNs. In vitro anchoring of oligonucleotides to cell surfaces or lipid membranes has been achieved by chemical conjugation between ODNs and cell surfaces or by spontaneous insertion of lipophilic ODN conjugates into membranes (Figure 1a). We expected the latter approach would be