Antitumor and Antimetastatic Effect of Small Immunostimulatory RNA against B16 Melanoma in Mice

Small interfering RNAs, depending on their structure, delivery system and sequence, can stimulate innate and adaptive immunity. The aim of this study was to investigate the antitumor and antimetastatic effects of immunostimulatory 19-bp dsRNA with 3’- trinucleotide overhangs (isRNA) on melanoma B16 in C57Bl/6 mice. Recently developed novel cationic liposomes 2X3-DOPE were used for the in vivo delivery of isRNA. Administration of isRNA/2X3-DOPE complexes significantly inhibits melanoma tumor growth and metastasis. Histopathological analysis of spleen cross sections showed hyperplasia of the lymphoid white pulp and formation of large germinal centers after isRNA/2X3-DOPE administration, indicating activation of the immune system. The treatment of melanoma-bearing mice with isRNA/2X3-DOPE decreases the destructive changes in the liver parenchyma. Thus, the developed isRNA displays pronounced immunostimulatory, antitumor and antimetastatic properties against melanoma B16 and may be considered a potential agent in the immunotherapy of melanoma.     

Short double-stranded RNA with immunostimulatory activity: sequence dependence

Small interfering RNAs (siRNA) are able to activate the mammalian innate immune system depending on their structure, sequence, and method of delivery. The immunostimulatory activity of double-stranded RNA can be applied to antiviral and antitumor therapy. Here we identified a set of 19-bp RNA duplexes with 3-nucleotid overhangs in the 3' ends that display immunostimulating activity (here and after immunostimulating RNA, or isRNA) and studied their sequence/activity relationships. It was found that the introduction of substitutions in the middle part of the isRNA sequence (10-16 positions counting from the 5' end of strand 1) does not alter the antiproliferative activity, while substitutions in the 3' end region of isRNA substantially reduce it. isRNAs efficiently inhibit the proliferation of human oral epidermoid carcinoma cells [half-maximal inhibitory concentration (IC(50)) values varied from 10 to 100 nM]. Our research demonstrated that antiproliferative effects of isRNAs are related to cell growth arrest, rather than the induction of apoptosis. These isRNAs strongly stimulate the synthesis of interferon-α (IFN-α), and to a lesser extent the synthesis of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6), in adherent peripheral blood mononuclear cells. An intravenous injection of isRNA/Lipofectamine complexes into C57BL mice increases IFN-α and IL-6 levels in the blood serum up to 15-fold and 3-fold, respectively, compared to the control mice. The results obtained clearly demonstrate the pronounced immunostimulatory and antiproliferative properties of the isRNAs under study. Hence, these short double-stranded RNAs can be considered as potential agents for the therapy of oncological and viral diseases.     

Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7

Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-alpha production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-alpha-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-alpha in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).     

p53 in cytoplasm exerts 3′→5′ exonuclease activity with dsRNA

Double-stranded RNA (dsRNA) is a biologically active molecule that plays important roles in normal cell growth and function. Accordingly, the cell uses multiple mechanisms to control its level. The tumor suppressor protein p53 possesses intrinsic 3′→5′ exonuclease activity. The aim of the present study was to elucidate the degradation of dsRNA by the exonuclease activity of p53. The results show that recombinant, purified wtp53 and endogenous protein in cytoplasmic fractions of cells remove nucleotides from 3’-ends of dsRNA. Several lines of evidence support a connection between p53 and dsRNase activity in cytoplasm: (1) this activity parallels the status of endogenous cytoplasmic p53; (2) the endogenous exonuclease displays a similar dsRNA excision profile characteristic for purified wtp53; (3) cytoplasmic fractions of HCT116(p53+/+) cells exert higher levels of exonuclease activity compared to those of HCT116(p53-/-) cells; (4) transfection of the wtp53, but not exonuclease-deficient mutant p53-R175H, into HCT116 (p53-/-) cells induced high levels of dsRNase activity in cytoplasm; (5) the accumulation of p53 in cytoplasm following the γ-irradiation stress stimuli correlates with the increase in the excision of dsRNA and (6) the dsRNA forms a complex with a protein that can be disrupted by an anti-p53 antibody. Our data suggest that the degradation of dsRNA by p53 protein may direct either the complete degradation of and decrease in the level of dsRNA or incomplete degradation and the generation of short dsRNA products. The possible roles of p53 dsRNase activity in cytoplasm in the inhibition of translation and induction of cell apoptosis, is discussed.     

Cholesterol-modified anti-<Emphasis Type="Italic">MDR1</Emphasis> small interfering RNA: Uptake and biological activity

Small interfering RNAs (siRNA) are considered to be potential agents for specific gene silencing, but low the efficacy of siRNA delivery into cells limits their biomedical application. Accumulation of siRNA coupled with cholesterol residue at the 5′-end of the “sense” strand (chol-siRNA) was studied in HEK293, HepG2, SC1, and KB-8-5 cells. In the absence of a transfection agent, the levels of both carrier-free and chol-siRNAs were very low, whereas transfection agent substantially increased transfection rate in all cell lines; in HEK293, SC1, and KB-8-5 cells transfection efficiency of the chol-siRNA was higher than that of the corresponding unmodified siRNA. Biological activity of anti-MDR1-siRNAs targeted to the 557–577 nt region of the MDR1 gene mRNA was estimated as multiple drug resistance phenotype reverting activity of KB-8-5 cancer cells. The chol-siRNA induced cancer cells’ death in the presence of previously tolerated vinblastine doses more effectively than unmodified siRNA.     

Molecular basis for an attenuated cytoplasmic dsRNA response in human embryonic stem cells

The introduction of double stranded RNA (dsRNA) into the cytoplasm of mammalian cells usually leads to a potent antiviral response resulting in the rapid induction of interferon beta (IFNβ). This response can be mediated by a number of dsRNA sensors, including TLR3, MDA5, RIG-I and PKR. We show here that pluripotent human cells (human embryonic stem (hES) cells and induced pluripotent (iPS) cells) do not induce interferon in response to cytoplasmic dsRNA, and we have used a variety of approaches to learn the underlying basis for this phenomenon. Two major cytoplasmic dsRNA sensors, TLR3 and MDA5, are not expressed in hES cells and iPS cells. PKR is expressed in hES cells, but is not activated by transfected dsRNA. In addition, RIG-I is expressed, but fails to respond to dsRNA because its signaling adapter, MITA/STING, is not expressed. Finally, the interferon-inducible RNAse L and oligoadenylate synthetase enzymes are also expressed at very low levels. Upon differentiation of hES cells into trophoblasts, cells acquire the ability to respond to dsRNA and this correlates with a significant induction of expression of TLR3 and its adaptor protein TICAM-1/TRIF. Taken together, our results reveal that the lack of an interferon response may be a general characteristic of pluripotency and that this results from the systematic downregulation of a number of genes involved in cytoplasmic dsRNA signaling.Key words: dsRNA, interferon, innate immunity, pluripotency, stem cells     

Assembly of pyrene-modified DNA/RNA duplexes incorporating a G-rich single strand region

The structural properties of a DNA/RNA duplex having a pyrene residue at the 5′ end of DNA and a G-rich single strand region at the 3′ end of RNA were studied in detail. Fluorescence and ultracentrifugation analyses indicated the formation of a complex containing four DNA/RNA duplexes, which required a pyrene residue, G-rich sequence, RNA-type backbone, and high salt concentration.     

Stable expression of shRNAs in human CD34+ progenitor cells can avoid induction of interferon responses to siRNAs in vitro

RNA interference occurs when cytoplasmic small interfering RNAs (siRNAs) enter the RNA-induced silencing complex and one strand guides cleavage of the target RNA by the Argonaute 2 protein. A significant concern when applying siRNAs or expressing small hairpin RNAs (shRNAs) in human cells is activation of the interferon (IFN) response. Synthetic siRNAs harboring certain motifs can induce an immune response when delivered to mouse and human immune cells such as peripheral blood mononuclear cells, monocytes, plasmacytoid dendritic cells (pDCs) and nonplasmacytoid dendritic cells (mDCs). In the present study we have tested the immunostimulatory effects of lipid-delivered siRNAs versus Pol III promoter-expressed shRNAs in primary CD34+ progenitor-derived hematopoietic cells. We show that in this system, lipid-delivered siRNAs are potent inducers of IFNalpha and type I IFN gene expression, whereas the same sequences when expressed endogenously are nonimmunostimulatory.     

Chemically modified symmetric and asymmetric duplex RNAs: an enhanced stability to nuclease degradation and gene silencing effect

The present study accents on the relationship between dicing, nuclease stability, and RNAi activity of various types of chemically modified symmetric and asymmetric dsRNAs, covalently bound with amino-groups or cholesterol at one or both terminals. All modified dsRNAs were subjected to cleavage by recombinant Dicer enzyme. They possessed a high resistance to nuclease degradation in cell cultured medium and an excellent RNAi activity in viable cells. The best stability and RNAi activity was detected for 5′-sense amino-modified RNAs. These modifications manifested also a high long-term gene silencing effect within seven days post-transfection, while the RNAi activity of the native 21nt siRNA expired within two days. The conjugation of dsRNA with cholesterol at 5′-sense end resulted in easy intracellular delivery without transfection reagents. After a direct transfection in cells, the cholesterol-conjugated 27nt dsRNA possessed a higher RNAi activity than cholesterol-conjugated 21nt siRNA.     

ATPase‐driven oligomerization of RIG‐I on RNA allows optimal activation of type‐I interferon

The cytosolic pathogen sensor RIG‐I is activated by RNAs with exposed 5′‐triphosphate (5′‐ppp) and terminal double‐stranded structures, such as those that are generated during viral infection. RIG‐I has been shown to translocate on dsRNA in an ATP‐dependent manner. However, the precise role of the ATPase activity in RIG‐I activation remains unclear. Using in vitro‐transcribed Sendai virus defective interfering RNA as a model ligand, we show that RIG‐I oligomerizes on 5′‐ppp dsRNA in an ATP hydrolysis‐dependent and dsRNA length‐dependent manner, which correlates with the strength of type‐I interferon (IFN‐I) activation. These results establish a clear role for the ligand‐induced ATPase activity of RIG‐I in the stimulation of the IFN response.     

Reporters Transiently Transfected into Mammalian Cells Are Highly Sensitive to Translational Repression Induced by dsRNA Expression

In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells—expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.     

Cloning and molecular analysis of double-stranded RNA associated with grapevine leafroll disease*

Eight major dsRNA species ranging from 1.0 to 19.5 kbp were detected in a low-yielding clone of Sultana (Thompson seedless) grape (Vitis vinifera L., cv. Sultana, clone B4L) affected leafroll disease. Using total dsRNA from this Sultana line as template, a number of cDNA clones were produced. The clones were used as probes for northern blot analysis of dsRNA extracted from Sultana B4L, and from six other grapevine leafroll-infected Sultana sources differing in yield performance. Based on the hybridisation of each probe with dsRNA bands from various Sultanas, the cDNA clones could be divided into three groups. One group of cDNA clones hybridised to high molccular weight dsRNA (19.5 kbp) from two low-yielding Sultanas, another group hybridised to high M_r dsRNA from three low-yielding Sultanas and the third group hybridised to a number of smaller dsRNA species ranging in size between 1.15 and 6.5 kbp. Using the latter cDNA clones, the sequence of 965 nucleotides at the 5′-end of a 1.15 kbp dsRNA (dsRNA 6) of B4L Sultana was determined. This RNA contains an open reading frame encoding a putative protein of M, = 33 441 with no homology to known protein sequences. The sequence of dsRNA 6 was found to overlap larger dsRNAs of sizes between 2.2 to 6.5 kbp. This allowed us to determine the sequence upstream of the 5′-end of the positive strand of dsRNA 6. The nucleotide sequence neighbouring the 5′-end of the positive strand of dsRNA 6 conforms to a consensus sequence proposed as a subgenomic promoter element for the coat protein gene of positive strand RNA plant viruses. The results indicate that more than one virus was present in Sultana B4L and that dsRNA 6 may be a subgenomic species of viral origin.     

Structural and Hydrodynamic Characterization of Dimeric Human Oligoadenylate Synthetase 2

Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2′-5′-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2′-5′-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.     

Exploring the mechanism how Marburg virus VP35 recognizes and binds dsRNA by molecular dynamics simulations and free energy calculations

Filoviruses often cause terrible infectious disease which has not been successfully dealt with pharmacologically. All filoviruses encode a unique protein termed VP35 which can mask doubled‐stranded RNA to deactivate interferon. The interface of VP35–dsRNA would be a feasible target for structure‐based antiviral agent design. To explore the essence of VP35–dsRNA interaction, molecular dynamics simulation combined with MM‐GBSA calculations were performed on Marburg virus VP35–dsRNA complex and several mutational complexes. The energetic analysis indicates that nonpolar interactions provide the main driving force for the binding process. Although the intermolecular electrostatic interactions play important roles in VP35–dsRNA interaction, the whole polar interactions are unfavorable for binding which result in a low binding affinity. Compared with wild type VP35, the studied mutants F228A, R271A, and K298A have obviously reduced binding free energies with dsRNA reflecting in the reduction of polar or nonpolar interactions. The results also indicate that the loss of binding affinity for one dsRNA strand would abolish the total binding affinity. Three important residues Arg271, Arg294, and Lys298 which makes the largest contribution for binding in VP35 lose their binding affinity significantly in mutants. The uncovering of VP35–dsRNA recognition mechanism will provide some insights for development of antiviral drug. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 849–860, 2014.     

Tumor-Suppressing,Immunostimulating, and Hepatotoxic Effects of Immunostimulatory RNA in Combination with Dacarbazine in a Murine Melanoma Model

Melanoma is one of the most aggressive tumors and is accompanied by the induction of local and systemic inflammatory responses. Combinations of chemotherapeutic agents with immunotherapy are therefore commonly used for melanoma treatment. A B16 melanoma model was used to study the tumor suppressive, immunostimulating, and hepatotoxic effects of a combination of a small double-stranded immunostimulatory RNA (isRNA) with 3'-trinucleotide overhangs and the cytotoxic drug dacarbazine compared with respective monotherapies. The drugs efficiently suppressed the tumor growth and acted synergistically. Histological and immunohistochemical examinations of tumor nodes showed that the combination of isRNA and dacarbazine significantly decreased mitotic activity and more efficiently increased apoptosis in tumor tissue as compared with either monotherapy. Regardless of the treatment regimen, signs of immune activation were observed in the spleen, including an increase in the number and diameter of lymphoid follicles and the volume density of the white pulp. Destructive changes were detected in the livers of nontreated animals with B16 melanoma. Administration of isRNA in combination with dacarbazine did not cause any additional damage to liver parenchyma, while stimulating regenerative processes in hepatic tissue of tumor-bearing animals.