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Interleukin-10 (IL-10) is a key suppressor of inflammation in chronic infections and in cancer. In mice, the inability of the immune system to clear viral infections or inhibit tumor growth can be reversed by antibody-mediated blockade of IL-10 action. We used a modified selection protocol to isolate RNA-based, nuclease-resistant, aptamers that bind to the murine IL-10 receptor. After 5 rounds of selection high-throughput sequencing (HTS) was used to analyze the library. Using distribution statistics on about 11 million sequences, aptamers were identified which bound to IL-10 receptor in solution with low K(d). After 12 rounds of selection the predominant IL-10 receptor-binding aptamer identified in the earlier rounds remained, whereas other high-affinity aptamers were not detected. Prevalence of certain nucleotide (nt) substitutions in the sequence of a high-affinity aptamer present in round 5 was used to deduce its secondary structure and guide the truncation of the aptamer resulting in a shortened 48-nt long aptamer with increased affinity. The aptamer also bound to IL-10 receptor on the cell surface and blocked IL-10 function in vitro. Systemic administration of the truncated aptamer was capable of inhibiting tumor growth in mice to an extent comparable to that of an anti- IL-10 receptor antibody.

Common tumor vaccination strategies utilizing peptide-pulsed dendritic cells (DC) are limited to targeting antigens with known epitopes in patients expressing a defined restricting allele and can result in the preferential induction of low-avidity T cells that fail to recognize tumor cells. The use of dendritic cells transfected with RNA encoding tumor antigen offers the prospect of antigen-specific immunization without requiring prior knowledge of the immunogenic epitope or restricting allele, since epitopes from the translated protein are processed by the endogenous antigen-presentation machinery. However, its use in vaccine studies has been limited by low RNA transfection efficiency and the use of immature DC as recipient cells. In this study, we report an RNA transfection strategy that routinely achieves expression in 40-50% of mature DC, which are better stimulator cells. Such RNA-transfected mature DC exhibited a prolonged duration of presentation of immunogenic epitopes compared to peptide-pulsed DC, induced greater frequencies of tumor antigen-specific CTL, and generated a CTL population that exhibited higher target avidity and increased tumor lytic capacity. These studies provide compelling in vitro data supporting the evaluation of RNA-transfected mature DC in vaccination protocols as a means to overcome several obstacles to generating anti-tumor responses in vivo.

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type‐specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.

Advances in the optimization of in vitro-transcribed mRNA are bringing mRNA-mediated therapy closer to reality. In cultured cells, we recently achieved high levels of translation with high-performance liquid chromatography (HPLC)-purified, in vitro-transcribed mRNAs containing the modified nucleoside pseudouridine. Importantly, pseudouridine rendered the mRNA non-immunogenic. Here, using erythropoietin (EPO)-encoding mRNA complexed with TransIT-mRNA, we evaluated this new generation of mRNA in vivo. A single injection of 100 ng (0.005 mg/kg) mRNA elevated serum EPO levels in mice significantly by 6 hours and levels were maintained for 4 days. In comparison, mRNA containing uridine produced 10-100-fold lower levels of EPO lasting only 1 day. EPO translated from pseudouridine-mRNA was functional and caused a significant increase of both reticulocyte counts and hematocrits. As little as 10 ng mRNA doubled reticulocyte numbers. Weekly injection of 100 ng of EPO mRNA was sufficient to increase the hematocrit from 43 to 57%, which was maintained with continued treatment. Even when a large amount of pseudouridine-mRNA was injected, no inflammatory cytokines were detectable in plasma. Using macaques, we could also detect significantly-increased serum EPO levels following intraperitoneal injection of rhesus EPO mRNA. These results demonstrate that HPLC-purified, pseudouridine-containing mRNAs encoding therapeutic proteins have great potential for clinical applications.

The dendritic cell (DC) is a newly discovered member of the immune system specialized in presenting antigen to naive T cells, and consequently playing a central role in the induction of T cell, as well as B-cell, immunity. Immunization with DC loaded with tumor antigens could, therefore, represent a powerful method of inducing antitumor immunity. Studies in our laboratory have shown that DC transfected with mRNA encoding tumor antigens are effective inducers of cytotoxic T cells (CTL) and tumor immunity. Tumor RNA can be amplified from microscopic amounts of tumor tissue obtained by microdissection from needle biopsies or fixed tissue sections providing virtually inexhaustible amounts of tumor antigens without the need to identify the relevant antigens in each patient. Phase I/II clinical studies in patients with prostate and renal cancer have established the safety, and feasibility of this approach and despite the advanced nature of the disease, the majority of the patients exhibited specific immunological responses. To enhance the therapeutic impact of this vaccination strategy antisense and siRNA technology are used to induce CD4+ T cell immunity by inhibiting Invariant chain expression in the (mRNA transfected) DC, and CTLA-4 binding RNA aptamers are used to enhance the persistence of vaccine-induced antitumor immunity.