2′ fluoro-modified RNAs as immunostimulators

Abstract

Methods of inhibiting the growth of cells or inducing cell death by contacting the cells with or introducing into the cells a composition including a 5 triphosphate, 2 fluoro-modified pyrimidine non-linear single stranded RNA at least 17 nucleotides long with a least 3 base pairings or a 5 triphosphate, 2 fluoro-modified double stranded RNA at least 17 base pairs long in an amount effective to inhibit cell growth, induce cell death or induce cytokine production by the cells. The methods also include administration of the compositions to a subject. The subject may have a proliferative disorder or infectious disease and administration of the compositions provided herein may treat the disorder or disease.

Claims

1. An RNA composition capable of inducing programmed cell death, cytokine production, or chemokine production in cells, the composition comprising a 5 triphosphate, 2 fluoro-modified pyrimidine RNA having at least 17 nucleotides and a double stranded portion of at least 3 base pairings forming a nonlinear structure within 15 nucleotides of the 5 end of the RNA, wherein the RNA comprises a nucleotide sequence having at least 80% sequence identity to a member selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

2. The composition of claim 1, wherein the non-linear structure is a single stem loop structure.

3. The composition of claim 1, wherein the non-linear structure is a multiple stem loop structure.

4. The composition of claim 1, wherein the RNA is a single stranded RNA.

5. The composition of claim 1, wherein the 2 fluoro-modified pyrimidine includes at least one 2 fluoro-modified uridine.

6. The composition of claim 1, wherein at least 10% of the nucleotides contain a 2 fluoro-modification.

7. The composition of claim 1 further comprising a cytoplasmic delivery composition.

8. The composition of claim 7, wherein the cytoplasmic delivery composition is selected from the group consisting of a liposome, synthetic polymer, cell-penetrating peptide, nanoparticle, viral particle, electroporation buffer, and nucleofection reagent.

9. A method for inhibiting growth of cells, inducing programmed cell death, cytokine production, or chemokine production, the method comprising delivering to the cytoplasm of a cell a 5 triphosphate, 2 fluoro-modified pyrimidine RNA composition comprising at least 17 nucleotides and a double stranded portion of at least 3 base pairings forming a nonlinear structure within 15 nucleotides of the 5 end of the RNA, wherein the RNA comprises a nucleotide sequence having at least 80% sequence identity to a member selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

10. The method of claim 9, wherein the RNA is delivered to the cytoplasm via transfection, nucleofection, electroporation, a gene gun, or receptor-mediated endocytosis.

11. The method of claim 9, wherein the composition further comprises a cytoplasmic delivery composition.

12. The method of claim 9, wherein the cell is selected from the group consisting of melanoma, brain cancer, prostate cancer, breast cancer, renal cancer, lung cancer, liver cancer, colorectal cancer, leukemia, lymphoma, and ovarian cancer cells.

13. A method for inhibiting growth of cells, inducing programmed cell death, cytokine production, or chemokine production, the method comprising administering a 5 triphosphate, 2 fluoro-modified pyrimidine RNA composition comprising at least 17 nucleotides and a double stranded portion of at least 3 base pairings forming a nonlinear structure within 15 nucleotides of the 5 end of the RNA to a subject in need of such treatment, wherein the RNA comprises a nucleotide sequence having at least 80% sequence identity to a member selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

14. The method of claim 13, wherein the composition further comprises a cytoplasmic delivery composition.

15. The method of claim 13, wherein the RNA is delivered to the cytoplasm of a cell of the subject.

16. The method of claim 15, wherein the cell is selected from the group consisting of melanoma, brain cancer, prostate cancer, breast cancer, renal cancer, lung cancer, liver cancer, colorectal cancer, leukemia, lymphoma, and ovarian cancer cells.

17. The method of claim 15, wherein the RNA is delivered to the cytoplasm via transfection, nucleofection, electroporation, a gene gun, or receptor-mediated endocytosis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A is a set of schematic structures of RNAs used in this study and FIG. 1B is a graph demonstrating the cytotoxicity of 5ppp 2F RNA aptamers in a sequence independent manner. FIG. 1A is a set of schematic secondary structures of 5ppp 2F RNA aptamers as predicted with the lowest free energy (G) computed by mfold (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form). FIG. 1B is a graph showing the percent growth inhibition of a cell line after transfection with the indicated aptamer. Human melanoma cell line, WM266-4 cells were either treated with transfection reagent alone (mock) or transfected with 0.5 g/ml of RNA aptamers Human melanoma cell line, WM266-4 cells were either treated with transfection reagent alone (mock) or transfected with 0.5 g/ml of RNA aptamers 10F (SEQ ID NO: 4), vWF9.14T10 (SEQ ID NO: 6), vWF9.14T17 (SEQ ID NO: 7), vWF9.14T13 (SEQ ID NO: 9), vWF9.14T14 (SEQ ID NO: 8), FXII-51 (SEQ ID NO: 10), EGFR-E07 (SEQ ID NO: 11) or GPNMB (SEQ ID NO: 5). LO-1 is represented by SEQ ID NO: 12 and Poly(U)30 is represented by SEQ ID NO: 13. All RNAs contain 5ppp and 2F pyrimidine. The growth inhibition was measured by an MTT assay.

(2) FIG. 2A-FIG. 2D demonstrates that cytoplasmic delivery of 5ppp 2F RNA aptamers into human melanoma cells leads to growth inhibition and induction of apoptosis. FIG. 2A is a set of FACS plots showing induction of apoptosis. WM266-4 cells were incubated with either liposome (DF) alone or 5ppp 2F 10F-liposome complex (10F/DF). At 5 h after incubation, cells were replenished with fresh culture medium. Cells were then incubated for an additional 48 hours, harvested and analyzed for cell death. FIG. 2B is a set of Western blots showing activation of caspases and PARP after transfection. WM266-4 cells were either mock-treated, treated with the 5ppp 2F 10F without the transfection reagent, or with the 5ppp 2F 10E-transfection reagent complex at the indicated concentrations (1 g/ml or 0.5 g/ml). The activation of caspase-7, -9, and PARP was assessed by western blot using antibodies specific to the activated (cleaved) proteins (24 h after treatment). Staurosporine-treated cells were used as a positive control. FIG. 2C is a graph showing the percent growth inhibition after the indicated treatment. WM266-4 cells were electroporated with PBS (mock), polyI:C, 5ppp 2F 10F or a 2F pyrimidine nucleoside triphosphate mix (2F NTP). The growth inhibition was assessed at 72 h after electroporation. FIG. 2D is a set of Western blots showing expression of several proteins after the indicated treatments. WM266-4 cells were treated as in (FIG. 2B), and the expression of RIG-I, MDA-5, and PKR was analyzed at 24 hours after treatments. Note that -tubulin levels (loading control) were reproducibly reduced in cells treated with 5ppp 2F 10F/DF complex. The data represent two individual experiments. Error bars are S.D.

(3) FIG. 3A and FIG. 3B are a set of graphs showing that transfection of 5ppp 2F ssRNAs does not stimulate TLR3 and TLR7. FIG. 3A shows that TLR3 is not stimulated. The human TLR3 reporter cell, HEK-hTLR3-NFB/SEAP was purchased from Invivogen. These cells were stably co-transfected with the human TLR3 gene and an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene into HEK293 cells. The SEAP gene is placed under the control of NF-B and AP-1. HEK-hTLR3-NFB/SEAP cells were specifically stimulated with TLR3 ligands including polyI:C and polyI:C-DF complex, but not with DF alone, 5ppp 2F 10F-DF complexes and TLR7 agonist R848. SEAP gene expression was measured using the SEAP detection media QUANTI-BLUE (Invivogen), according to the manufacturer's instructions. FIG. 3B is a graph showing that TLR7 is not stimulated by the ssRNAs. Human TLR7-expressing HEK293 cells were kindly provided by Dr. Todd Brennan (Duke University, Durham, N.C.). Treatment with R848 but not with polyI:C, polyI:C-DF and 5ppp 2F 10F-DF complexes stimulated HEK-TLR7 cells to produce the inflammatory cytokine IL-8. The data represent the mean of two experiments. Error bars represent the S.D.

(4) FIG. 4A-FIG. 4E are a set of data showing that 5ppp 2F ssRNA-induced cell death and growth inhibition of melanoma cells are dependent on cytoplasmic RIG-I and MAVS. FIG. 4A is a set of western blots showing knockdown of the indicated proteins. WM266-4 cells were transfected twice at 2-day intervals with either control 5OH siRNA or 5OH siRNAs against PKR, RIG-I, MDA-5 and MAVS. The knockdown efficiency was assessed 4 days after siRNA transfections by western blot using specific antibodies (as indicated; upper panel). Probing of the same membrane with either actin- or -tubulin-specific antibodies served as loading controls (lower panel). FIG. 4B and FIG. 4C are a set of graphs showing that MAVS and RIG-I are required for cell growth inhibition and cytokine production by ssRNA. WM266-4 cells treated as in (FIG. 4A) were either further treated with transfection reagent alone (mock), or 10F-transfection reagent complex (10F) (0.125 g/ml) at 24 hours after the last siRNA transfection. The growth inhibition (FIG. 4B and IFN production (FIG. 4C) were analyzed at 72 h after 10F treatment. FIG. 4D and FIG. 4E are a set of graphs showing the ability to partially reverse the effect of the ssRNA by treatment with a phosphatase to dephosphorylate the ssRNA. 5ppp 2F 10F (FIG. 4D) and 5ppp 2F 9.14T10 (FIG. 4E) was dephosphorylated by incubation with bacterial alkaline phosphatase (BAP). In addition, 5OH-2F 9.14T10 (Syn9.14T10) was non-enzymatically synthesized to completely remove the 5ppp. The apoptosis, cytotoxic effect and IFN expression of WM266-4 cells treated with mock, 10F. or dephosphorylated 10F (0.125 g/ml each) was determined by flow cytometry after staining cells with Annexin V-PE, MTT assay and IFN ELISA, respectively. The data represent two individual experiments. Error bar represent the S.D.; *P<0.05.

(5) FIG. 5A and FIG. 5B are a set of graphs showing IFN-dependent and independent mechanisms contribute to 5ppp 2F ssRNA-induced growth inhibition of human cancer cells. FIG. 5A is a graph showing the growth inhibition is mediated at least in part by IFN-. The human melanoma cell line, WM266-4 and human dermal fibroblasts were transfected with 10F or mock transfected in the presence (empty bars) and absence (filled bars) of B18R, the vaccinia virus-encoded type I IFN decoy receptor. In parallel, the culture supernatants of cells transfected with either mock or 10F were harvested after 24 h. Fresh melanoma cells and dermal fibroblasts were incubated for 72 h with the culture supernatants in the presence and absence of B18R. The growth inhibition of WM266-4 cells treated with IFN in the presence and absence of B18R was used as an experimental control. FIG. 5B is a graph showing that not all cells are susceptible to ssRNA mediated growth inhibition. A variety of human cancer cell lines and normal human cells were either transfected with 5ppp 2F 10F (0.5 g/ml) or treated with transfection reagent only (mock), and the growth inhibition relative to untreated cells was analyzed at 72 h after transfection by an MTT assay. Error bars represent the S.D. *P<0.05 (5ppp 2F 10F vs Mock).

(6) FIG. 6A-FIG. 6D are a set of graphs showing that 2F modification increases nuclease resistance, cytotoxicity and inflammatory cytokine induction of immunostimulatory 5ppp ssRNAs. FIG. 6A is a graph showing ability of various modified ssRNAs to inhibit the growth of cells over concentration ranges. WM266-4 cells were treated with transfection reagent alone (mock) or the complex of transfection reagent and 5ppp 10F containing 2F pyrimidine, 2OMe pyrimidine or unmodified pyrimidine (2OH) at various concentrations. The viral dsRNA analog PolyI:C was used as an experimental control. Growth inhibition was assessed at 72 h post treatments using the MTT assay. FIG. 6B is a set of graphs showing cytokine induction after treatment with the indicated ssRNAs. WM266-4 cells were either treated with transfection reagent alone (mock) or the complex of transfection reagent and 5ppp 2OH 10F, 5ppp 2F 10F, 5ppp 2OMe 10F or poly I:C (0.5 g/ml each). The secretion of IFN and IL-8 by the melanoma cells was analyzed at 24 h after treatment by ELISA. The data represent the mean of three experiments. Error bars represent the S.D.; *P<0.05 (5ppp 2F 10F vs 5ppp 2OH 10F); P<0.001 (5ppp 2OMe 10F vs 5ppp 2OH 10F). FIG. 6C is a photograph of a gel showing the serum stability of 2F and 2OH ssRNAs. 5ppp 2OH 10F and 5ppp 2F 10F were incubated in 10% fetal bovine serum at 37 C. for 0, 5 min, 10 min, 1 h, 3 h, 6 h, 12 h or 24 h and collected at each time point. RNAs were analyzed on 12% polyacrylamide gels. FIG. 6D is a photograph of a gel showing the cellular stability of 2F and 2OH ssRNAs. WM266.4 cells were transfected with 5ppp 2OH 10F, 5ppp 2F 10F or mock. At 4 h, 8, 12 and 24 h after transfection, total RNAs were isolated and cellular 10F RNA levels were analyzed by RT-PCR. Actin mRNA levels were used as an internal control. The data represent three individual experiments.

(7) FIG. 7 is a set of FACS analysis plots showing the induction of apoptosis by 5ppp-2F small dsRNA after transfection of melanoma cells. 2F modification may improve anti-cancer activity of conventional RIG-I agonist 5ppp dsRNA. Transfection of 5ppp-2F short dsRNA increased apoptosis of human melanoma cells as compared to unmodified 5ppp-2OH dsRNA.

(8) FIG. 8A is a set of structures and FIGS. 8B and 8C are graphs showing that the distance between 5ppp and internal stem structure of 5ppp 2F ssRNAs is inversely correlated to cytotoxic effect and IFN- expression in human melanoma cells. FIG. 8A shows the mfold-predicted structures of 5ppp 2F stem-loop ssRNAs consisting of 5overhang (Motif 1 (SEQ ID NO: 14) and 1A (SEQ ID NO: 15)), blunt end (Motif 2 (SEQ ID NO: 16)) and 3overhang (Motif 3 (SEQ ID NO: 17)). Only one secondary structure was predicted for individual ssRNA by mfold with negative G. FIG. 8B and FIG. 8C are graphs showing the effect of transfection of melanoma cells with each of the indicated ssRNA on growth inhibition and cytokine production. WM266-4 cells were transfected with Motif 1, Motif 1A, Motif 2, Motif 3 or 10F at the indicated concentrations. Cytotoxicity (FIG. 8B) and IFN production (FIG. 8C) by the cells were evaluated by MTT assay and IFN ELISA, respectively. The data represent the mean of three experiments. Error bars represent the S.D.; *P<0.05, compared to 10F; **P<0.05, Motif 1 vs Motif 1A.

DETAILED DESCRIPTION

(9) Compositions capable of inducing programmed cell death, type I interferon (interferon-) and inflammatory cytokine production by cells or activation of Pattern Recognition Receptors (PRR) in cells are provided and described herein. The compositions include a 5 triphosphate, 2 fluoro-modified pyrimidine non-linear single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA) at least 17 nucleotides long. As described in the Examples, multiple 2F RNA aptamers harboring a 5ppp that specifically binds to target molecules in the cells were generated. Surprisingly, intra-cytoplasmic delivery of these RNA aptamers elicits potent cytotoxicity and type I IFN production by human melanoma, prostate cancer and glioblastoma cells. This RNA aptamer-mediated cell death and IFN production is specific to cancer cells and did not inhibit cell growth of various non-cancer cells other than melanocytes. Furthermore, these effects are dependent on 5ppp, 2F modification and the secondary structures of the RNA aptamers. These effects were demonstrated to be sequence independent. Finally, the 5ppp 2F RNA aptamer-induced melanoma cell death is dependent on the activation of cytoplasmic pattern recognition receptors (e.g., RIG-I) and the downstream adaptor molecule, mitochondrial antiviral-signaling protein (MAVS).

(10) As noted the RNAs are suitably at least 17 nucleotides long and are suitably no more than 200 nucleotides long. The RNAs may be between 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 nucleotides and 200, 180, 160, 150, 140, 130, 120, 110, 100 or 90 nucleotides long. In the Examples at least one 2 Fluoro modification in the RNA was needed to inhibit growth of cells. Suitably, the 2 fluoro-modification is present on at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the pyrimidines, suitably the modified pyrimidines are uridines. In one embodiment, all of the uridines in the RNA are 2 fluoro-modified. In another embodiment, all of the pyrimidines in the RNA are 2 fluoro-modified. The 5end of the RNA must be a 5 triphosphate for the composition to function in the methods.

(11) In the Examples, linear RNAs were not effective at inhibiting cell growth, while those with secondary structure were capable of inhibiting cell growth. Thus, suitably the RNAs in the composition form a secondary structure with a double-stranded portion. Suitably, the RNA is capable of forming single or multiple stem loop structures. The double-stranded portion of the RNA should include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 25 or more base pairs. The double stranded portion of the RNA is suitably positioned within 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nucleotides of or at the 5 end of the RNA. Double-stranded RNA with 2fluoro and 5 triphosphate are also useful in the methods of immunostimulation and induction of programmed cell death described herein. The dsRNA should also be between 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 base pairs and 200, 180, 160, 150, 140, 130, 120, 110, 100 or 90 base pairs long.

(12) The compositions described herein may also include a cytoplasmic delivery mechanism. The RNAs in the compositions must be delivered into the cells or produced within the cells to function. As shown in the Examples, extracellular administration of the RNA containing compositions without a delivery mechanism resulted in a lack of effectiveness. Such delivery mechanisms are available to those skilled in the art and include all gene delivery mechanisms including but not limited to synthetic polymers (such as those used for siRNA delivery), cell-penetrating peptides (such as VP16), nanoparticles, viral or liposomal delivery to the cytoplasm of cells (lipofection), delivery via a gene gun, or may include transfection, nucleofection or electroporation. The cytoplasmic delivery mechanisms may be targeted to only deliver the compositions to cells in which cell growth inhibition or induction of programmed cell death is desired. For example, the cellular delivery mechanism may specifically target the RNAs to cancer cells or virally infected cells. The compositions may be targeted to cells for uptake by receptor-mediated endocytosis as well. As shown in the examples, most non-cancerous cells are not susceptible to these RNAs so targeting may not be needed in some systems, but may be useful to obtain sufficient delivery to the target cells. In addition, cells could be genetically engineered to express the RNA compositions described herein. The RNAs could be operably connected to a promoter, such as an inducible promoter, to allow expression of the RNA only upon proper stimulation.

(13) The compositions described herein may also be used to inhibit cancer cell growth or induce programmed cell death of cancer cells which may result in treating cancer in a subject. The compositions may also be useful in treating other non-cancerous proliferative disorders or in treating infected cells, such as cells infected with a virus or other intracellular pathogen. The compositions provided herein may also be administered as an adjuvant to stimulate an immune response to an antigen, pathogen or cancer cell. Subjects that may be administered the compositions described herein include, but are not limited to mammals, domesticated animals and humans and may specifically include dogs, cats, fish, chickens, cows, pigs, sheep, goats.

(14) Along these lines, methods of inhibiting the growth of cells or inducing programmed cell death using the compositions comprising the ssRNAs or dsRNAs described herein are also provided. The methods include contacting cells with the compositions or alternatively administering the compositions to subjects in an amount effective to inhibit the growth of cells, induce programmed cell death of cells or increase inflammatory cytokine production by cells. The inhibition of growth of the cells or induction of cell death is independent of the nucleotide sequence of the RNA, is partially dependent on induction of type I interferons (IFN-), activation of pattern recognition receptors (RIG-I) and induction of MAVS, independently. The type I interferons (IFN-) may have autocrine or paracrine effects on cells. Other cytokines such as IL-8 are also induced in the cells treated with the RNA compositions described herein. The cells may be melanoma cells, or other cancer cells, including but not limited to brain, prostate, ovarian, renal, lung, liver, colorectal and breast cancer cells or leukemia or lymphoma cells.

(15) Suitably, the composition is delivered to the cytoplasm of the cell. The compositions may be delivered through the cytoplasmic delivery mechanisms described above and include but are not limited to liposomes, synthetic polymers, cell penetrating peptides, nanoparticles, viral encapsulation, receptor-mediated endocytosis, electroporation or any other means to deliver the composition to the cytoplasm of the cells.

(16) Cells may be contacted with the composition directly or indirectly in vivo, in vitro, or ex vivo. Contacting encompasses administration to a cell, tissue, mammal, patient, or human. Further, contacting a cell includes adding the composition to a cell culture with a cytoplasmic delivery mechanism or introducing the composition into the cytoplasm of the cell via any available means. Other suitable methods may include introducing or administering the composition to a cell, tissue, mammal, or patient using appropriate procedures and routes of administration as defined below.

(17) Administration of the compositions described herein may inhibit the growth of cancer cells or treat cancer. This effect may be due to a general immunostimulatory effect of administration of the compositions or by programmed cell death initiated by contact with the compositions. The administration of the compositions described herein may inhibit the growth of the cells by 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80% or more as compared to control treated cells. The administration of the compositions described herein may also induce cell death, suitably programmed cell death in 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more than 75% of the treated cells. Programmed cell death includes any means of cell death mediated by an intracellular program and includes, but is not limited of apoptosis, autophagy, necroptosis, anoikis, or other non-apoptotic forms of programmed cell death. Suitably, administration to a subject or contacting cells with the compositions described herein increases the amount of type I interferon (IFN or IFN ) and IL-8 produced by the cells. Suitably the cytokine are increased 2, 3, 4, 5, 7, 10, or more fold as compared to the production of these cytokines in untreated control cells.

(18) The compositions described herein may be administered to a subject to treat cancer in the subject. Treating cancer includes, but is not limited to, reducing the number of cancer cells or the size of a tumor in the subject, reducing progression of a cancer to a more aggressive form, reducing proliferation of cancer cells (inhibiting the growth of) or reducing the speed of tumor growth, killing of cancer cells (via any means), reducing metastasis of cancer cells or reducing the likelihood of recurrence of a cancer in a subject. Treating a subject as used herein refers to any type of treatment that imparts a benefit to a subject afflicted with a disease or at risk of developing the disease, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay the onset of symptoms or slow the progression of symptoms, etc.

(19) The compositions may be used to make pharmaceutical compositions. Pharmaceutical compositions comprising the RNAs and compositions described above and a pharmaceutically acceptable carrier are provided. A pharmaceutically acceptable carrier is any carrier suitable for in vivo administration. Examples of pharmaceutically acceptable carriers suitable for use in the composition include, but are not limited to, water, buffered solutions, glucose solutions, oil-based or bacterial culture fluids. Additional components of the compositions may suitably include, for example, excipients such as stabilizers, preservatives, diluents, emulsifiers and lubricants. Examples of pharmaceutically acceptable carriers or diluents include stabilizers such as carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing agents such as bovine serum or skimmed milk and buffers (e.g., phosphate buffer). Especially when such stabilizers are added to the compositions, the composition is suitable for freeze-drying or spray-drying. The composition may also be emulsified.

(20) The compositions described herein may also be combined with a chemotherapeutic or other therapy for treatment of a disease or condition or in conjunction with an antigen to stimulate a more effective immune response against the antigen or a pathogen comprising the antigen. An antigen includes any peptide, carbohydrate or lipid to which an immune response can be generated. In particular the antigen is capable of stimulating a B cell or T cell immune response such that a memory response is generated. The compositions may be administered in any order, at the same time or as part of a unitary composition. The composition described herein and the therapeutic may be administered such that one is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks or more.

(21) An effective amount or a therapeutically effective amount as used herein means the amount of the composition that, when administered to a subject for treating a state, disorder or condition, such as cancer, is sufficient to effect a treatment (as defined above). The therapeutically effective amount will vary depending on the composition, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated.

(22) The compositions described herein may be administered by any means known to those skilled in the art, including, but not limited to, oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intracranial, intratumoral, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, or transmucosal absorption. Thus the compositions may be formulated as an ingestable, injectable, topical or suppository formulation. The compositions may also be delivered with in a lipoplex, polyplex, target-specific nanoparticle or time-release vehicle. Administration of the compositions to a subject in accordance with the invention appears to exhibit beneficial effects in a dose-dependent manner. Thus, within broad limits, administration of larger quantities of the composition is expected to achieve increased beneficial biological effects than administration of a smaller amount. Moreover, efficacy is also contemplated at dosages below the level at which toxicity is seen.

(23) It will be appreciated that the specific dosage administered in any given case will be adjusted in accordance with the compositions being administered, the disease to be treated or inhibited, the condition of the subject, and other relevant medical factors that may modify the activity of the composition or the response of the subject, as is well known by those skilled in the art. For example, the specific dose for a particular subject depends on age, body weight, general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given patient can be determined using conventional considerations, such as by means of an appropriate conventional pharmacological or prophylactic protocol.

(24) The maximal dosage for a subject is the highest dosage that does not cause undesirable or intolerable side effects. The number of variables in regard to an individual prophylactic or treatment regimen is large, and a considerable range of doses is expected. The route of administration will also impact the dosage requirements. It is anticipated that dosages of the composition will reduce symptoms of the condition at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to pre-treatment symptoms or symptoms is left untreated. It is specifically contemplated that pharmaceutical preparations and compositions may palliate or alleviate symptoms of the disease without providing a cure, or, in some embodiments, may be used to cure the disease or disorder.

(25) Suitable effective dosage amounts for administering the compositions may be determined by those of skill in the art, but typically range from about 1 microgram to about 100,000 micrograms per kilogram of body weight weekly, although they are typically about 1,000 micrograms or less per kilogram of body weight weekly. In some embodiments, the effective dosage amount ranges from about 10 to about 10,000 micrograms per kilogram of body weight weekly. In another embodiment, the effective dosage amount ranges from about 50 to about 5,000 micrograms per kilogram of body weight weekly. In another embodiment, the effective dosage amount ranges from about 75 to about 1,000 micrograms per kilogram of body weight weekly. The effective dosage amounts described herein refer to total amounts administered, that is, if more than one composition is administered, the effective dosage amounts correspond to the total amount administered. The composition can be administered as a single dose or as divided doses. For example, the composition may be administered two or more times separated by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days, three days, four days, one week, two weeks, or by three or more weeks.

(26) The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms including, comprising, or having, and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as including, comprising, or having certain elements are also contemplated as consisting essentially of and consisting of those certain elements.

(27) Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word about to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

(28) No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.

(29) The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

EXAMPLES

(30) Materials and Methods

(31) Cell Culture

(32) WM266-4, WM115, SK-MEL-2 and normal human colon epithelial cells (all from ATCC, Manassas, Va.) were maintained in Eagle's Minimum Essential Medium supplemented with 10% FBS, 1 Non-essential Amino Acid Solution, and 1 mM sodium pyruvate (all from Invitrogen, Carlsbad, Calif.). MALME-3M, DU145, LNCaP, HeLa, Hs578T, HT1080, LN-229, A-172, human lung fibroblast and human bone marrow stromal cell (all from ATCC), Panc-1 (gift from Dr. Rebekah White at Duke University, Durham, N.C.), normal human dermal fibroblast (Invitrogen) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 10% FBS. SK-OV-3 (ATCC) was maintained in McCoy's 5A medium (Invitrogen) with 10% FBS. Xeno-43 (gift from Dr. Darell D. Bigner, Duke University) was maintained in Zinc Option Media (Invitrogen). Normal human epidermal melanocytes (ATCC) were maintained in Dermal Cell Basal Medium (ATCC) supplemented with the Adult Melanocyte Growth Kit (ATCC). HPDE cells (gift from Dr. Rebekah White at Duke University) were maintained in Keratinocyte-SFM (Invitrogen). PC3, NCI-H1838 and peripheral blood mononuclear cells (PBMCs) were maintained in RPMI 1640 (Invitrogen) with 10% FBS, HEPES and 1 mM sodium pyruvate. Cells were incubated at 37 C. in a humidified atmosphere with 5% CO.sub.2.

(33) Generation of Immunostimulatory and Cytotoxic RNAs

(34) All 2F-modified and unmodified ssRNAs used in this study were established by in vitro transcription from DNA templates using the Y639F mutant T7 RNA polymerase, as previously described (Layzer and Sullenger (2007) Oligonucleotides 17: 1-11). Unmodified and 2F-modified ribonucleoside triphosphates were used at a 1 mM and 3 mM final concentration, respectively, in the in vitro transcription reaction. 2OMe-modified RNA was transcribed from DNA templates using the Y639F/H784A double mutant T7 RNA polymerase (Padilla and Sousa (2002) Nucleic Acids Res 30: e138). Unmodified and 2OMe-modified ribonucleoside triphosphates were used at 3 mM in the in vitro transcription reaction. The von Willebrand Factor-specific aptamer vWF 9.14 derivatives, 9.14T10 and 9.14T17, were previously reported (Nimjee et al. (2012) Mol Ther 20: 391-397). Aptamer E07 specifically binds to human epidermal growth factor receptor (EGFR) (Li et al. (2011) PLoS One 6: e20299). The human melanocytic cell-targeting aptamer 10F has been selected by ex vivo cell-based SELEX. The transmembrane glycoprotein NMB-specific aptamer GPNMB has been selected for affinity binding to human GPNMB by in vitro SELEX. The coagulation factor XII-specific aptamer FXII-51 was provided by Dr. Becky Smock (Duke University). PolyI:C was purchased from Invivogen, San Diego, Calif. 5ppp-2F Poly(U)30 and 5OH-2F 9.14T10 were non-enzymatically synthesized by TriLink, San Diego, Calif. siRNAs with 3 TT overhangs for knockdown studies were purchased from Invitrogen and had the guide strand sequences 5-CCACCUUGAUGCCUGUGAA-3 (for MAVS; SEQ ID NO: 1), 5-AUCACGGAUUAGCGACAAA-3 (for RIG-I; SEQ ID NO: 2), and 5-GUAUCGUGUUAUUGGAUUA-3 (for MDAS; SEQ ID NO: 3). The anti-PKR siRNA was purchased as a pool of three target specific 19-25 nt siRNAs from Santa Cruz Biotechnology (Dallas, Tex.). The sequences of the aptamers used in this study are provided in Table 1.

(35) TABLE-US-00001 TABLE1 5ppp-2FssRNAsequences Aptamer Length Target Sequence(SEQIDNO:) 10F 71 Melanoma/ GGGAGGACGAUGCGGUACCUGACAGCAUCUU melanocyte GAUAAUGGUCCUACGGAGCCGUUCCAGACGA CUCGCCCGA(SEQIDNO:4) GPNMB 71 Glioblastoma GGGAGGACGAUGCGGGGAAGUACCCAAGGU CUGUGAACCCGUAACCAUGCGGCCCCAGACG ACUCGCCCGA(SEQIDNO:5) 9.14T10 60 von GGGAGGUGGACGAACUGCCCUCAGCUACUUU Willebrand CAUGUUGCUGACGCACAGACGACUCGCUG Factor(vWF) (SEQIDNO:6) 9.14T17 40 vWF GGGAGGUGGACGAACUGCCCUACGCACAGAC GACUCGCUG(SEQIDNO:7) 9.14T14 28 vWF GGGAGGUCAGCUACUUUCAUGUUGCUGA (SEQIDNO:8) 9.14T13 51 vWF GGGAGGUGCCCUCAGCUACUUUCAUGUUGCU GACGCACAGACGACUCGCUG(SEQIDNO:9) FX11-51 51 Coagulation GGCUCGGCUGCCAGCAGGUCACGAGUCGCAG Factor11 CGACUCGCUGAGGAUCCGAG(SEQIDNO:10) E07 93 EGFR GGCGCUCCGACCUUAGUCUCUGUGCCGCUAU AAUGCACGGAUUUAAUCGCCGUAGAAAAGC AUGUCAAAGCCGGAACCGUGUAGCACAGCAG A(SEQIDNO:11) LO-1 40 None GGGGAAGUGAAUGGGUGAGGUGGAAGUGAG UGAGUGAAAU(SEQIDNO:12) Poly(U)30 30 None UUUUUUUUUUUUUUUUUUUUUUUUUUUUUU (SEQIDNO:13) Motif1 29 None GGGAGGACGAUGCGGUACCUGACAGCAUC (SEQIDNO:14) Motif1A 37 None GGGUAAGUGGGAGGACGAUGCGGUACCUGA CAGCAUC(SEQIDNO:15) Motif2 23 None GGAUGCGGUACCUGACAGCAUCU(SEQIDNO: 16) Motif3 30 None GGAUGCGGUACCUGACAGCAUCUUGAAAUA (SEQIDNO:17)
Induction of Innate Immune Responses and Apoptosis by Intracellular Delivery of RNAs

(36) For the intracellular delivery of immune stimulatory RNAs, RNAs were transfected with the DharmaFECT Duo liposomal transfection reagent (Thermo Scientific, Waltham, Mass.) at a transfection reagent (l):RNA (g) ratio of 3.75:1, according to the manufacturer's instructions. Unless otherwise stated, for the induction of innate immune responses and apoptosis, immune stimulatory RNAs (500 ng/ml) were transfected into 50 to 70% confluent cells. Cells were incubated with an RNA-transfection agent complex for 5 h, followed by replenishment with fresh culture medium. To electroporate cells, 110.sup.5 cells suspended in 200 l Opti-MEM I (Invitrogen) were mixed with 1 g of immune stimulatory RNAs or 5 g of 2Fluoro pyrimidine ribonucleoside triphosphate in 2-mm cuvettes and were electroporated at 300 V for 500 s using an Electro Square Porator ECM 830 (BTX, San Diego, Calif.). For the knockdown of RNA-sensing PRRs, cells were transfected with PRR-specific siRNAs (25 nM) twice at 2-day intervals using DharmaFECT-1 (Thermo Scientific). At 5 h after the second siRNA transfection, cells were harvested, replated into a 96-well plate and incubated overnight. Cells were then treated with immune stimulatory RNA transfections. Staurosporine (2 M) (Sigma, St Louis, Mo.) and recombinant human IFN (20 ng/ml) (PeproTech, Rocky Hill, N.J.) were used as positive controls of anti-melanoma cytotoxicity. B18R (100 ng/ml) (eBioscience, San Diego, Calif.) was added directly to the growth media of cells to neutralize IFN.

(37) Quantification of Growth Inhibition and Apoptosis

(38) Growth inhibition relative to untreated cells was quantified at 72 h after RNA transfection using an MTT Cell Proliferation Assay Kit (Cayman Chemicals, Ann Arbor, Mich.), according to the manufacturer's instructions. The percent growth inhibition was calculated by using the following equation: % growth inhibition=([O.D.].sub.untreated[O.D.].sub.treated)/[O.D.].sub.untreated100. The induction of cell death was measured 48 h after RNA transfection by flow cytometry after staining with Annexin V-PE and 7-Aminoactinomycin D (7-AAD) using the PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, Calif.) and analyzed using the CellQuest software (BD Biosciences).

(39) Immunoblot Analysis and Antibodies

(40) Cell lysates were prepared in 1RIPA buffer (Sigma) in the presence of the complete protease inhibitor cocktail (Roche). Twelve to 20 g of protein lysates were electrophoretically separated on 4-20% Mini-PROTEAN TGX polyacrylamide gels (Bio-Rad, Hercules, Calif.) and transferred to polyvinylidene fluoride (PVDF) membranes (PolyScreen, PerkinElmer). After rinsing in TBST.sub.20, membranes were blocked for 1 h in 10% dry milk in TBTS.sub.20, followed by overnight incubation with primary antibodies anti-MAVS (1:200) (E-3; Santa Cruz), anti-RIG-I (1:500) (D14G6; Cell Signaling, Danvers, Mass.), anti-MDAS (1:500) (D74E4; Cell Signaling), anti-PKR (1:350) (Catalog No 3072; Cell Signaling), anti-cleaved caspase-9 (1:500) (D2D4; Cell Signaling), anti-cleaved caspase-7 (1:1,000) (D6H1; Cell Signaling), and anti-cleaved PARP (1:1,000) (D64E10; Cell Signaling). When different proteins were sequentially detected on the same membrane, membranes were treated for 8 minutes with Restore Western Blot Stripping Buffer (Thermo Scientific), washed, blocked and probed again, as described above. Primary antibodies were detected using horseradish peroxidase (HRP)-conjugated anti-rabbit (1:4,000) (NA934; GE Healthcare, Pittsburgh, Pa.) or anti-mouse (1:4,000) (NA931; GE Healthcare) secondary antibodies. Anti--tubulin-HRP (1:2,000) (9F3; Cell Signaling) and anti--actin (1:2000) (13E5; Cell Signaling) were used as loading controls. HRP activity was visualized using the Immun-Star WesternC Chemiluminescence Kit (Bio-Rad). In order to determine caspase activation and PRR expression, cell lysates were harvested at 24 h after RNA transfection. In order to confirm siRNA-mediated knockdown of MAVS and PRRs, cell lysates were harvested 6 days after the first transfection of siRNA.

(41) Enzyme-Linked Immunosorbent Assay (ELISA)

(42) Cells were transfected with RNAs as described above. At 24 hours after RNA transfection, culture supernatants were collected and stored at 80 C. for later analyses. The production of human IL-8 was analyzed with a human IL-8 ELISA kit (eBioscience). IFN- production was determined with a human IFN- ELISA kit (PBL Biomedical Laboratories, Piscataway, N.J.) by following the manufacturer's instructions.

(43) Statistical Analysis

(44) Two-tailed, paired Student's t test was applied for determining statistical significance. Confidence intervals equal to or less than 0.05 constitute statistical significance.

(45) Results

(46) Intracytoplasmic Delivery of 5ppp-2F RNA Aptamers Inhibits Melanoma Cell Growth and Induces Programmed Cell Death in a Sequence-Independent and Structure-Dependent Manner

(47) Short, structured 5ppp ssRNAs can be recognized by various RNA-sensing PRRs including RIG-I, PKR and TLR7 and induce type I IFN production by multiple human cells (Diebold et al. (2004) Science 303: 1529-1531; Heil et al. (2004) Science 303: 1526-1529; Besch et al. (2009) J Clin Invest 119: 2399-2411; Toroney et al. (2012) RNA 18: 1862-1874; and Schmidt et al. (2009) Proc Natl Acad Sci USA 106: 12067-12072). Interestingly, introduction of these 5ppp ssRNAs into the cytoplasm could selectively kill human melanoma cells in a RIG-I-dependent and IFN-independent manner (Besch et al. (2009) J Clin Invest 119: 2399-2411). RNA aptamers are short ssRNAs (30-100 bases) that are often designed to bind to proteins. Most of the in vitro transcribed (IVT) RNA aptamers contain 2 ribose modifications, 5ppp and a high degree of secondary structure, similar to PRR-activating ssRNAs except with nucleotide modifications. To test whether IVT RNA aptamers have anti-melanoma activity, we generated diverse RNA aptamers bearing 5ppp and 2F pyrimidines that can recognize different protein receptors (FIG. 1A, Table 1). Surprisingly, all IVT RNA aptamers except short RNA aptamer 9.14 T14 tested in this study readily induced more than 80% growth inhibition of human melanoma cells within 72 hours after liposomal transfection, at least in part by the induction of apoptosis (FIG. 1B and FIG. 2A). Because this anti-melanoma effect was also seen with the various IVT RNA aptamers, but not with 5ppp-2F ssRNAs predicted to have minimal secondary structures, such as Poly(U)30 and LO-1, we concluded that the induction of anti-melanoma responses by 5ppp-2F RNA aptamers is largely dependent on the presence of folded secondary structure(s) rather than sequence.

(48) Transfection with 5ppp-2F RNA Aptamers can Induce Caspase-Mediated Programmed Cell Death and Innate Immune Activation of Human Melanoma Cells

(49) 5ppp-2F RNA aptamer-induced apoptosis is accompanied by the activation of caspase-9, an initiator caspase of the intrinsic apoptosis pathway and its downstream effector molecules, including caspase-7 and poly(ADP-ribose) polymerase (PARP) (FIG. 2B). Certain cationic liposomes used as non-viral vectors have been shown to induce apoptosis (Iwaoka et al. (2006) J Leukoc Biol 79: 184-191). RNA aptamer-mediated cell death, however, is not dependent on non-specific cytotoxicity associated with liposomal transfection because direct intra-cytoplasmic delivery of RNA aptamers by electroporation also inhibited melanoma cell growth (FIG. 2C). 5-Fluorouracil (5-FU) is a pyrimidine analog and broadly used as an anti-metabolic agent that induces apoptosis by inhibiting DNA and RNA synthesis (Longley et al. (2003) Nat Rev Cancer 3: 330-338). One can argue that, unlike PRR-activating 5ppp ssRNAs, RNA aptamers containing 2F pyrimidines may interfere with DNA and RNA synthesis within melanoma cells, resulting in non-specific inhibition of melanoma cell proliferation. To test this possibility, human melanoma cells were electroporated with the 5ppp-2F RNA aptamer, 2F pyrimidine ribonucleoside triphosphate (2F NTP) or PBS. The electroporation of RNA aptamers induced apoptosis of melanoma cells as efficiently as the liposomal transfection of these RNAs, however, the 2F NTP transfection did not alter melanoma cell viability (FIG. 2C). Moreover, transfection of 5ppp-2F RNA aptamers led to a strong induction of innate immune RNA-sensing PRRs, including RIG-I, MDA-5, and PKR, which was not observed upon treatment of the cells with staurosporine, a conventional apoptosis-inducing drug (FIG. 2D). These data suggest that the 5ppp-2F RNA aptamer employs a unique mechanism to induce melanoma growth inhibition, apoptosis and innate immune activation as compared to a conventional pro-apoptotic agent.

(50) The RIG-I-MAVS Signaling Pathway Regulates the Induction of Anti-Melanoma Responses by 5ppp-2F RNA Aptamers

(51) Foreign, non-self RNAs are recognized by multiple RNA-sensing PRRs, including endosomal TLR3 and TLR7 and cytoplasmic RIG-I, MDA-5 and PKR. Direct cytoplasmic delivery of 5ppp-2F RNA aptamers by electroporation results in similar growth inhibition of melanoma cells as compared to liposomal transfection (FIG. 2C). Furthermore, liposomal transfection of 5ppp-2F RNA aptamers did not activate human TLR3 and TLR7 (FIGS. 3A and 3B, respectively). These data suggest that 5ppp-2F RNA aptamers activate cytoplasmic RNA-sensing PRRs rather than endosomal TLRs to induce cell death of melanoma cells. To determine the cytoplasmic PRR responsible for the recognition of 5ppp-2F RNA aptamers, we transiently knocked-down the expression of cytoplasmic RNA-sensing PRRs, including RIG-I, MDA-5 and PKR and an essential mitochondrial adaptor for RIG-I and MDA-5 signaling-induced apoptosis, MAVS, using specific 5OH siRNAs. Consecutive knockdown of PRRs and MAVS in WM266-4 cells sustained knockdown effect at the protein level until day 6 post-transfection (FIG. 4A), which provided sufficient time for studying the cytotoxic effect of 5ppp-2F RNA transfection. Depletion of either RIG-I or MAVS significantly rescued the cells from 5ppp-2F RNA-induced cytotoxicity (FIG. 4B) and reduced IFN production by human melanoma cells transfected with 5ppp-2F RNA aptamers (FIG. 4C). Thus, RIG-I and MAVS are required for the recognition of 5ppp-2F RNA aptamers and the downstream signal transduction, respectively, ultimately inducing cell death and IFN expression. In contrast, knockdown of MDA-5 had no effect on cell viability, whereas depletion of PKR may even enhance the cytotoxic effect of 5ppp-2F RNA aptamers. An essential component of substrates that are recognized by RIG-I is a 5ppp. Dephosphorylation of 5ppp-2F RNA aptamers, 10F. and 9.14T10, partially prevented apoptosis and IFN production by human melanoma cells but did not completely eliminate it, most likely because of inefficient dephosphorylation (FIGS. 4D and E). Therefore, we evaluated a chemically synthesized version of the 2F 9.14T10 aptamer without 5ppp (Syn9.14T10), and observed that this synthetic aptamer completely lacked cytotoxic and IFN induction activities. These observations further support the hypothesis that RIG-I is the prominent cytoplasmic PRR recognizing 5ppp-2F RNA aptamers.

(52) IFN-Dependent and Independent Mechanisms of 5ppp-2F RNA-Induced Cell Death and Growth Inhibition of Human Melanoma Cells

(53) IFN is a pleiotropic cytokine that can inhibit proliferation and induce cell death (Trinchieri (2010) J Exp Med 207: 2053-2063). It has a wide range of immune stimulatory activities including augmentation of T helper type 1 (Thl) cell responses, upregulation of MHC class I molecules, generation of natural killer (NK) cell- and T cell-mediated cytotoxicity and anti-tumor activities including anti-proliferative, anti-angiogenic and pro-apoptotic effects (Trinchieri (2010) J Exp Med 207: 2053-2063). To determine the contribution of IFN to 5ppp-2F RNA-induced melanoma cell death, cells were co-treated with B18R, a vaccinia-virus-encoded decoy receptor for type I IFN to neutralize IFN in the media and to abrogate autocrine/paracrine IFN signaling. While co-treatment with B18R only partially rescued 5ppp-2F 10F-transfected melanoma cells from cell death, it completely abolished the apoptosis of melanoma cells treated with either recombinant IFN or culture supernatants isolated from melanoma cells transfected with 5ppp-2F 10F (FIG. 5A). The IFN-containing culture supernatants isolated from 5ppp-2F 10F-transfected melanoma cells minimally affected the growth of normal non-melanocytic cells, such as human dermal fibroblasts (FIG. 5A). Our data suggest that transfection of 5ppp-2F RNAs can induce growth inhibition and apoptosis of human melanoma cells through both type 1 IFN-dependent and -independent mechanisms, and autocrine/paracrine IFN can induce collateral growth inhibition of otherwise untreated melanoma cells.

(54) Melanoma, Prostate Cancer and Glioblastoma Cells are Highly Sensitive to 5ppp-2F RNA-Induced Cytotoxicity when Compared to Other Types of Cells

(55) Next, we investigated whether 5ppp-2F RNA-induced cytotoxicity is specific for human melanoma cells. While 5ppp-2F RNAs inhibited the growth of human melanoma cell lines (WM266-4, WM115, SK-MEL2, and MALME-3M), human prostate cancer cell lines (PC-3, DU145 and LNCaP) and human glioblastoma cell lines (LN-229, Xeno-43 and A-172) no significant cytotoxic effects of 5ppp-2F RNAs were observed after transfection of other types of human cancer cells, including pancreatic cancer (Panc-1), cervical cancer (HeLa), breast cancer (Hs578T), fibrosarcoma (HT1080), ovarian cancer (SK-OV-3) and non-small cell lung cancer (NCI-H1838) (FIG. 5B). Furthermore, 5ppp-2F RNA transfection did not cause significant cytotoxic effects in a variety of human non-malignant cells including dermal fibroblasts, lung fibroblasts, colon epithelia cells, pancreatic ductal epithelial cells (HPDE), bone marrow stromal cells and PBMCs, whereas the growth of primary human melanocytes was inhibited by 5ppp-2F RNA aptamer transfection. This result suggests that melanocytic cells and prostate cancer cells are much more sensitive to 5ppp-2F RNA-induced cytotoxicity as compared to other types of cells.

(56) The 2F Modification Increases Anti-Cancer Activity, Innate Immune Activation and Nuclease Resistance of RIG-I-Activating 5ppp RNAs

(57) 2OMe-modified RNAs have been widely described as being able to evade innate immune recognition by RNA-sensing PRRs (Robbins et al. (2007) Mol Ther 15: 1663-1669 and Sioud (2010) Methods Mol Biol 629: 387-394). However, the influence of 2F modifications on PRR sensing is much less clear; indeed, examples of both reduced and enhanced innate immune recognition have been reported (Uzri and Gehrke (2009) J Virol 83: 4174-418; Nallagatla and Bevilacqua (2008) RNA 14: 1201-1213; Hwang et al. (2012) Nucleic Acids Res 40: 2724-2733). In order to better dissect the contribution of 2-ribose modifications to 5ppp-2F RNA-induced cytotoxicity, we established 5ppp 10F RNAs with 2OMe pyrimidine, 2F pyrimidine or unmodified pyrimidine (2OH). Transfection of these 5ppp RNA variants into melanoma cells revealed that substituting 2OH with 2OMe completely abolished the cytotoxicity and cytokine production, whereas 2F-modified 5ppp RNAs significantly increased the cytotoxicity and IFN and IL-8 production over 2-fold compared to unmodified 5ppp RNAs (FIGS. 6A and B). Transfection of 5ppp-2F 10F RNAs into human melanoma cells led to strong induction of cytokine production and growth inhibition, comparable to levels observed for the gold standard PRR-activating anti-cancer agent, polyI:C. Finally, unmodified 5ppp 10F RNAs instantly degraded after exposure to serum, whereas 2F modifications could render the 5ppp 10F RNAs resistant to serum degradation (FIG. 6C). Furthermore, 2F modification improve cellular stability of 5ppp ssRNAs compared to unmodified RNAs (FIG. 6D). Although 2OMe modification could augment serum stability of 5ppp RNAs, this RNA modification eliminated the anti-cancer activity and innate immune activation of 5ppp RNAs. Unlike the 2OMe modification, the 2F modification not only enhanced serum stability but also cancer cell death and innate immune activation. Therefore, increasing nuclease resistance of RNAs by the 2F modification cannot be the sole mechanism of action for the enhancement of anti-cancer and innate immune activation of modified 5ppp RNAs.

(58) The ability of a dsRNA to induce cell cytotoxicity was also tested. Melanoma cells were transfected with 5ppp-2F dsRNA or 5ppp-2OH dsRNA and induction of apoptosis determined by Annexin V staining of the cells. As shown in FIG. 7, 5ppp-2F short dsRNA induced Annexin V and is killing the cells via apoptosis as demonstrated by increased 7-amino actinomycin D staining as well. In contrast, the 5ppp-2OH short dsDNA was not inducing apoptosis in the transfected cells.

(59) Distance Between 5ppp and Stem Structure is Inversely Correlated with Induction of Growth Inhibition and IFN Expression by 5ppp-2F ssRNAs in Human Cancer Cells

(60) The structure of RIG-I bound to 5ppp RNA ligands suggested that both the 5ppp and dsRNA motif are required for the initial recognition of RNA ligands by RIG-I (Jiang et al. (2011) Nature 479: 423-427). Schmidt et al. demonstrated that the minimal requirements of RIG-I-activating 5ppp-2OH ssRNAs for IFN production by human monocytes were 5ppp and a 5- to 10-base-pair stem structure (Schmidt et al. (2009) Proc Natl Acad Sci USA 106: 12067-12072). Therefore, we hypothesized that short 5ppp-2F ssRNAs containing a single stem structure would induce IFN expression and cytotoxic effects in human melanoma cells. To test this hypothesis, we generated three different types of 5ppp-2F ssRNAs shorter than 30 nucleotides and containing only a single predicted stem-loop structure, including 1) Motif 1: 29-mer ssRNA with a 5overhang stem-loop structure derived from the first 29 nucleotides of 5ppp-2F 10F, 2) Motif 2: truncated variant of Motif 1 (23-mer) with a blunt-end stem-loop structure and 3) Motif 3: extended variant of Motif 2 (30-mer) with an extended 3 end to generate a 3overhang stem-loop structure (FIG. 8A and Table 1). Motif 1 treatment resulted in significantly lower cytotoxicity (P=0.0392) and IFN induction (P=0.0172) in human melanoma cells compared to 10F. treatment, while Motif 2 treatment had 1.5- to 3-fold greater cytotoxicity (P=0.0039) and 3- to 10-fold greater IFN induction (P=0.0329) in the cells compared to 10F. (FIGS. 8B and C). The difference between the cytotoxicity and IFN induction of Motif 3 and 10F treatments was not statistically significant. These data suggest that the distance between the 5ppp and the internal stem structure is inversely correlated with recognition of 5ppp 2F ssRNAs by RIG-I. To support this finding, we generated an extended variant of Motif 1 with a longer 5overhang (Motif 1A; FIG. 8A). As expected, treatment with Motif 1A resulted in a significantly decreased cytotoxic effect (P=0.0409) and IFN production (P=0.0415) in human melanoma cells compared to treatment with Motif 1. Therefore, we concluded that a short blunt-ended stem-loop ssRNA containing 5ppp and 2F is a particularly potent immunostimulatory and anti-melanoma therapeutic RNA.