COMPOSITIONS AND METHODS FOR IMMUNOSTIMULATORY RNA OLIGONUCLEOTIDES

Abstract

The present invention provides 4-nucleotide (4mer) RNA motifs that confer immunostimulatory activity, in particular, IFN-α-inducing activity to a RNA oligonucleotide. The present invention also provides RNA oligonucleotides, including siRNA, with high or low immunostimulatory activity. The present invention further provides the use of the RNA oligonucleotides of the invention for therapeutic purposes.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to the field of immunotherapy and drug discovery. The present invention provides a method for determining the immunostimulatory activity of a RNA oligonucleotide. The present invention also provides a method for predicting the immunostimulatory activity of a RNA oligonucleotide. The present invention further provides a method for preparing RNA oligonucleotides with high or low immunostimulatory activity. Moreover, the present invention provides RNA oligonucleotides with immunostimulatory activity and the therapeutic uses thereof. In addition, the present invention provides RNA oligonucleotides with gene silencing activity and with either high or low immunostimulatory activity, the methods of their preparation, and their therapeutic uses.

BACKGROUND OF THE INVENTION

[0002] The vertebrate immune system established different ways to detect invading pathogens based on certain characteristics of their microbial nucleic acids. Detection of microbial nucleic acids alerts the immune system to mount the appropriate type of immune response that is required for the defense against the respective type of pathogen detected. Detection of viral nucleic acids leads to the production of type I interferon (IFN), the key cytokine for anti-viral defence. An understanding of how nucleic acids interact with the vertebrate immune system is important for developing different nucleic acid-based therapeutic strategies for the immunotherapy of diseases (Rothenfusser S et al. 2003, Curr Opin Mol Ther 5:98-106) and for developing gene-specific therapeutic agents (Tuschl T et al. 2002, Mol Interv 2: 158-167).

[0003] For the recognition of long dsRNA, two detection modes are known, the serine threonine kinase PKR (Williams B R, 2001, Sci Signal Transduction Knowledge Environment 89: RE2; Meurs E F et al. 1992, J Virol 66: 5805-5814; Katze M G et al. 1991, Mol Cell Biol 11: 5497-5505) and Toll-like receptor (TLR) 3 (Alexopoulou L et al. 2001, Nature 413: 732-738). Whereas PKR is located in the cytosol, TLR3 is present in the endosomal compartment (Matsumoto M et al. 2003, J Immunol 171: 3154-3162). TLR3 is a member of the Toll-like receptor family that has evolved to detect pathogen-specific molecules (Takeda K et al. 2003, Annu Rev Immunol 21: 335-376).

[0004] A second characteristic feature of viral nucleic acids used by the immune system to recognize viral infection are CpG motifs found in viral DNA, which are detected via TLR9 (Lund J et al. 2003, J Exp Med 198: 513-520; Krug A et al. 2004, Blood 103: 1433-1437). CpG motifs are unmethylated CG dinucleotides with certain flanking bases. The frequency of CpG motifs is suppressed in vertebrates, allowing the vertebrate immune system to detect microbial DNA based on such CpG motifs (Krieg A M et al. 1995, Nature 374: 546-549; Bauer S et al. 2001, Proc Natl Acad Sci USA 98: 9237-9242; Wagner H et al. 2002, Curr Opin Microbiol 5: 62-69). Like TLR3, TLR9 is located in the endosomal compartment where it directly binds to CpG motifs (Latz E et al. 2004, Nat Immunol 5: 190-198).

[0005] In addition to long dsRNA and CpG DNA, two recent publications suggest a third mechanism by which viral nucleic acids are recognized. These studies demonstrate that single-stranded RNA (ssRNA) of ssRNA viruses is detected via TLR7 (mouse and human) and TLR8 (only human) (Diebold S S et al. 2004, Science 303: 1529-1531; Heil F et al. 2004, Science 303: 1526-1529). Guanine analogues have been identified earlier as specific ligands for TLR7 and TLR8 (Lee J et al. 2003, Proc Natl Acad Sci USA 100: 6646-6651; Heil F et al. 2003, Eur J Immunol 33: 2987-2997). Like TLR9 (receptor for CpG DNA) (Latz E et al. 2004, Nat Immunol 5: 190-198), TLR7 and TLR8 are located in the endosomal membrane (Heil F et al. 2003, Eur J Immunol 33: 2987-2997).

[0006] Detection of viral nucleic acids leads to the production of type I IFN (IFN-α and IFN-β). The major producer of type I IFN in humans is the plasmacytoid dendritic cell (PDC, also called interferon producing cell, IPC). The plasmacytoid dendritic cell (PDC) is a highly specialized subset of dendritic cells that is thought to function as a sentinel for viral infection and is responsible for the vast amount of type I IFN during viral infection (Asselin-Paturel C et al. 2001, Nat Immunol 2: 1144-1150). There is increasing evidence that PDC preferentially use nucleic acid-based molecular patterns to detect viral infection. TLR expression of human and mouse PDC is limited to TLR7 and TLR9 (Krug A et al. 2001, Eur J Immunol 31: 3026-3037; Hornung V et al. 2002, J Immunol 168: 4531-4537; Edwards A D et al. 2003, Eur J Immunol 33: 827-833).

[0007] IFN-α was the first type of interferon to be identified and commercialized; it is widely used clinically in the treatment of a variety of tumors (e.g., hairy cell leukemia, cutaneous T cell leukemia, chronic myeloid leukemia, non-Hodgkin's lymphoma, AIDS-related Kaposi's sarcoma, malignant melanoma, multiple myeloma, renal cell carcinoma, bladder cell carcinoma, colon carcinoma, cervical dysplasia) and viral diseases (e.g., chronic hepatitis B, chronic hepatitis C). IFN-α products that are currently in clinical use include the recombinant protein and the highly purified natural protein, both of which have high production costs. Therefore, there is a need for more economical ways of providing IFN-α to patients in need. Furthermore, IFN-α is currently administrated systematically and causes a broad spectrum of side effects (e.g. fatigue, flu-like symptoms, diarrhea). Most alarmingly, IFN-α causes a decrease in bone marrow function which leads to increased susceptibility to life-threatening infections, anemia and bleeding problems. Therefore, there is a need for ways of providing IFN-α in a more localized (i.e., target-specific) matter to reduce the occurrence of side effects.

[0008] In addition to inducing an anti-viral interferon response, dsRNA also induces post-transcription gene silencing, a highly conserved anti-viral mechanism known as RNA interference (RNAi). Briefly, the RNA III Dicer enzyme processes dsRNA into short interfering RNA (siRNA) of approximately 22 nucleotides. The antisense strand of the siRNA binds a target mRNA via base pairing and serves as a guide sequence to induce cleavage of the target mRNA by an RNA-induced silencing complex RISC. dsRNA has been an extremely powerful tool in studying gene functions in C. elegence and Drosophila via gene silencing. However, its use in mammalian cells has been limited because the interferon response it elicits is detrimental to most mammalian cells.

[0009] Subsequently, it was found that siRNA was also capable of inducing RNAi, causing degradation of the target mRNA in a sequence-specific manner and it was thought to be short enough to bypass dsRNA-induced nonspecific effects in mammalian cells (Elbashri S M et al. 2001, Nature 411:494-498). Since then, siRNA has been widely used as a gene silencing tool in deciphering mammalian gene functions in research and drug discovery, and there has been great interest in its potential in therapeutic applications.

[0010] siRNA can be used to reduce or even abolish the expression of disease/disorder-related genes for preventing or treating diseases caused by the expression or overexpression of the disease-related genes. Such diseases include, but are not limited to, infections, metabolic diseases, autoimmune diseases and cancer. However, concern has been raised recently about the potential for siRNA to activate immune responses which may be undesirable for certain indications and thus limit the use of siRNA as a gene silencing agent for therapeutic purposes (Sioud M et al. 2003, Biochem. Biophys. Res. Commun. 312:1220-1225). Therefore, there is a need for methods for predicting the potential of a given siRNA to induce an interferon response and for methods for designing and preparing siRNAs for gene silencing which are devoid of unwanted immunostimulatory activities.

[0011] On the other hand, for certain therapeutic applications, for example, the prevention or treatment of cancer and viral infections, immunostimulatory activity may be desirable as an additional functional activity of the siRNA.

[0012] In an effort to apply siRNA for the specific downregulation of TLR9 in PDC in our previous publication (Hornung V et al. 2005, Nat Med 11: 263-270), we made the surprising observation that, despite the inability of PDC to detect long dsRNA, certain siRNA sequences were potent in vitro inducers of IFN-α in PDC. We found that i) short interfering RNA (siRNA) induces IFN-α in human plasmacytoid dendritic cells when transfected with cationic lipids, ii) this activity of siRNA is sequence-dependent but independent of the G or U content of the siRNA, iii) the immunostimuatory activity of siRNA and the antisense activity are two independent functional activities of siRNA, iv) the immune recognition of siRNA occurs on the single strand level, v) siRNAs containing the 9mer sequence motif 5''-GUCCUUCAA-3'' show potent immunostimulatory activity, and vi) such siRNAs induce systemic immune responses in mice, and vii) the induction of immune responses by siRNA requires the presence of TLR7 in mice. Our findings suggest that the 9mer sequence motif 5''-GUCCUUCAA-3'' may be a ligand for TLR7.

[0013] The natural ligand for TLR7 has not been well defined to date. Guanine analogues have been identified earlier as specific ligands for TLR7 and TLR8 (Lee J et al. 2003, Proc Natl Acad Sci USA 100: 6646-6651; Heil F et al. 2003, Eur J Immunol 33: 2987-2997), whereas guanosine ribonucleoside or a derivative thereof has been identified as TLR7 ligand in WO03086280.

[0014] It is an object of the present invention to identify RNA oligonucleotide motifs for stimulating an immune response, in particular, IFN-α induction. It is also an object of the present invention to identify ligands for activating TLR7 and TLR8. It is another object of the present invention to develop a method for determining the immunostimulatory activity, in particular, the IFN-α-inducing activity, of a RNA oligonucleotide. It is yet another object of the present invention to develop a method for predicting the immunostimulatory activity, in particular, IFN-α-inducing activity, of a RNA oligonucleotide. It is a further object of the invention to develop a method for designing and preparing RNA oligonucleotide having or lacking immunostimulatory activity, in particular, IFN-α-inducing activity. It is also an object of the invention to provide RNA oligonucleotides having high immunostimulatory activity which can be used to induce an immune response, in particular, IFN-α production, in patients in need thereof. It is yet another object of the present invention to provide siRNA molecules that either have or lack immunostimulatory activity which can be used to treat disorders caused by the expression or overexpression of disorder-related genes.

SUMMARY OF THE INVENTION

[0015] The present invention provides a method for determining the immunostimulatory activity of a RNA oligonucleotide, a method for predicting the immunostimulatory activity of a RNA oligonucleotide, a method for preparing a RNA oligonucleotide with high or low immunostimulatory activity, and a method for preparing a RNA oligoncleotide with gene silencing activity and with high or low immunostimulatory activity.

[0016] The present application also provides an in vitro method for inducing IFN-α production from a mammalian cell, and an in vitro method for activating a dendritic cell.

[0017] The present invention further provides a RNA oligonucleotide with immunostimulatory activity, a RNA oligonucleotide with gene silencing activity and with high or low immunostimulatory activity, and the therapeutic uses thereof.

[0018] In addition, the present invention provides a pharmaceutical composition comprising one or more of the RNA oligonucleotides of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1: PBMC of three individual donors were isolated and stimulated with ssRNA oligonucleotides 9.2sense (5'-AGCUUAACCUGUCCUUCAA-3') and 9.2antisense (5'-UUGAAGGACAGGUUAAGCU-3') that were complexed with either Lipofectamine, poly-L-arginine, poly-L-histidine or poly-L-lysine in duplicates. 24 hours after stimulation supernatants were harvested and IFN-α was assessed by ELISA. Data are presented as mean values±SEM.

[0020] FIG. 2: PBMC of three different healthy donors were isolated and stimulated with poly-L-arginine complexed ssRNA oligonucleotides in duplicates. 44 hours after stimulation IFN-a production was assessed in supernatant via ELISA. For all tested ssRNA oligonucleotides, the mean values of the measured duplicates were normalized to the positive control ssRNA oligonucleotide 9.2sense (5'-AGCUUAACCUGUCCUUCAA) by dividing the mean value of tested oligonucleotide by the mean value of 9.2sense (=100%). Data from three different donors were summarized and are presented as mean values±SEM.

[0021] FIG. 3: PBMC of six different healthy donors were isolated and stimulated with poly-L-arginine complexed ssRNA oligonucleotides in duplicates. 44 hours after stimulation IFN-a production was assessed in supernatant via ELISA. For all tested ssRNA oligonucleotides, the mean values of the measured duplicates were normalized to the positive control ssRNA oligonucleotide 9.2sense (5'-AGCUUAACCUGUCCUUCAA) by dividing the mean value of tested oligonucleotide by the mean value of 9.2sense (=IFN-a index of a given oligonucleotide). Next, all individual IFN-a indices were adjusted to the mean value of all IFN-a indices by subtracting the mean value of all IFN-a indices from the individual IFN-a index of a given oligonucleotide (=adjusted IFN-a index). Data from six individual donors were summarized and were assorted in ascending order displaying the corresponding SEM. In addition, a statistical analysis was performed to assess a putative significant difference for the adjusted IFN-α indices of all top thirty ssRNA oligonucleotides. A two-tailed Student's t-test was employed to calculate the p-value off all possible ssRNA oligonucleotide combinations. A p-value>0.01 and <0.05 is depicted by a black box, whereas a p-value<0.01 is depicted as a grey box.

[0022] FIG. 4: The occurrence of 1mer motifs (5'-X-3'), 2mer motifs (5'-XX-3',5'-X*X-3',5'-X**X-3') and 3mer motifs (5'-XXX-3',5'-XX*X-3',5'-X*XX-3') in ssRNA oligonucleotides with an IFN-a index below the mean IFN-a index (group 1) or above the mean IFN-α index (group 2) was analyzed. The relative occurrence of a given motif within a group of ssRNA oligonucleotides was calculated by dividing the absolute number of occurrences of a given motif within a group through the absolute number of occurrences of all possible motifs within this group. A significant overrepresentation or underrepresentation of a given motif was analyzed using a chi-square test. The null hypothesis of equal distribution within both groups was rejected when the calculated p-value was below 0.05 (significant differences in distribution are indicated by "*"). For all motifs analyzed, relative occurrences are depicted in FIG. 4 for group 1 oligonucleotides (black bars) and for group 2 oligonucleotides (white bars): 1mer motifs 5'-X-3' (FIG. 4A); 2mer motifs 5'-XX-3' (FIG. 4B1), 5'-X*X-3' Figure (FIG. 4B2), 5'-X**X-3' (FIG. 4B3) and 3mer motifs 5'-XXX-3' (FIG. 4C1-4C4), 5'-XX*X-3' (FIG. 4C5-4C8), 5'-X*XX-3' (FIG. 4C9-4C12).

[0023] FIG. 5: For all possible 1 mer motifs (5'-X-3'), 2mer motifs (5'-XX-3',5'-X*X-3',5'-X**X-3') or 3mer motifs (5'-XXX-3',5'-XX*X-3',5'-X*XX-3') a mean IFN-a index was assigned by calculating a mean IFN-α index of all ssRNA oligonucleotides containing the corresponding motifs (=IFN-a score of a given motif). The IFN-α score of all possible motifs is depicted in FIG. 5±SEM: 1mer motifs 5'-X-3' (FIG. 5A); 2mer motifs 5'-XX-3' (FIG. 5B1), 5'-X*X-3' Figure (FIG. 5B2), 5'-X**X-3' (FIG. 5B3) and 3mer motifs 5'-XXX-3' (FIG. 5C1-5C4), 5'-XX*X-3' (FIG. 5C5-5C8), 5'-X*XX-3' (FIG. 5C9-5C12).

[0024] FIG. 6: A calculated IFN-α index was assigned to each oligonucleotide by using the obtained motif-IFN-a scores. For each set of motifs [1mer motifs (5'-X-3'), 2mer motifs (5'-XX-3',5'-X*X-3',5'-X**X-3') or 3mer motifs (5'-XXX-3',5'-XX*X-3',5'-X*XX-3] a predicted IFN-a index was calculated for each ssRNA oligonucleotide. Next, the obtained predicted IFN-a indices were compared to the actual adjusted IFN-α indices. Data are depicted the following way: For all ssRNA oligonucleotides the predicted IFN-α indices are shown as a black bars, whereas data are sorted in ascending order according to the actual IFN-α score that is depicted as a red index line. The y-axis on the left side depicts the scale for the predicted IFN-α score, while the y-axis on the right side depicts the scale for the actual IFN-α score.

[0025] FIG. 7: PBMC from healthy donors were isolated and stimulated with poly-L-arginine complexed ssRNA oligonucleotides in duplicates. 44 hours after stimulation IFN-a production was assessed in supernatant via ELISA. For all tested ssRNA oligonucleotides, the mean values of the measured duplicates were normalized to the positive control ssRNA oligonucleotide 9.2sense (5'-AGCUUAACCUGUCCUUCAA) by dividing the mean value of tested oligonucleotide by the mean value of 9.2sense (=1). A: A panel of ssRNA oligonucleotides was tested with different positions of the 5'-GUCA-3'-motif within the 19mer ssRNA oligonucleotide (see table 4). The 5'-GUCA-3'-motif is indicated by bold letters. Data from two independent donors were summarized and are depicted as mean values±SEM. B/C: 16 ssRNA oligonucleotides, which included all possible oligonucleotides with permutated bases at the flanking positions to the 5'- and the 3'-end of the central 5'-GUCA-3'-motif (table 5), were complexed with poly-L-arginine and used to stimulate PBMC. 44 hours after stimulation IFN-a production was assessed in supernatant via ELISA. Data for all 16 oligonucleotides from three independent donors were summarized as mean values±SEM (B). In addition all 16 oligonucleotides were assorted into groups according to the base preceding or following the central 5'-GUCA-3'-motif (C). On the left side oligonucleotides with a common base preceding the central 5'-GUCA-3'-motif were grouped, whereas on the right side oligonucleotides with a common base following the central 5'-GUCA-3'-motif were grouped. Individual ssRNA oligonucleotide IFN-a data were summarized according to the respective group and are depicted as mean values±SEM. A two-tailed Student's t-test was used to calculate a statistically significant difference between the various groups (p<0.05 is indicated by a "*").

[0026] FIG. 8: A: PBMC from two healthy donors were isolated and stimulated with poly-L-arginine complexed ssRNA oligonucleotides (Table 6) in duplicates. 44 hours after stimulation IFN-α production was assessed in supernatant via ELISA. IFN-α data were summarized as mean values and subsequently normalized to the positive control RNA9.2sense (5'-AGCUUAACCUGUCCUUCAA-3'). In addition, respective sequences were analyzed using the IFN-α point score matrix (Table 7) and subsequently normalized to RNA9.2sense. A correlation coefficient of 0.84 was calculated for these two sets of data. Measured IFN-α levels are depicted in white bars, whereas predicted IFN-α scores are shown in black bars (A). Next, IFN-α point score matrix was employed to analyze IFN-α-inducing RNA oligonucleotides that have been described in the literature. Given the fact that in the study performed by Judge et al. (2005, Nat Biotechnol 23:457-462) double-stranded RNA oligonucleotides were tested, a mean value for the individually analyzed single-stranded components was calculated. Data were normalized to the most potent RNA oligonucleotide (=100%) within the respective panel of oligonucleotides (B). For the prediction of single-stranded RNA oligonucleotides reported by Heil et al. (2004 Science 303:1526-1529), the predicted IFN-α point scores are depicted (C).

[0027] FIG. 9: PBMC from individual healthy donors were isolated and stimulated with poly-L-arginine complexed ssRNA-oligonucleotides in duplicates. For a detailed list of all tested oligonucleotides see Table 1. 44 hours after stimulation IFN-α production was assessed in supernatant via ELISA. For all tested ssRNA-oligonucleotides, the mean values of the measured duplicates were normalized to the positive control ssRNA-oligonucleotide 9.2sense (5'-AGCUUAACCUGUCCUUCAA) by dividing the mean value of tested oligonucleotide by the mean value of 9.2sense (=100%). Data from nine different (A-D) donors or three different donors (E) were summarized and are presented as mean values±SEM.

[0028] FIG. 10: PBMC from individual healthy donors were isolated and stimulated with poly-L-arginine complexed ssRNA-oligonucleotides in duplicates. For a detailed list of all tested oligonucleotides see Table 13. 44 hours after stimulation IFN-α production was assessed in supernatant via ELISA. For all tested ssRNA-oligonucleotides, the mean values of the measured duplicates were normalized to the positive control ssRNA-oligonucleotide 9.2sense (5'-AGCUUAACCUGUCCUUCAA) by dividing the mean value of tested oligonucleotide by the mean value of 9.2sense (=100%). Data from two different donors were summarized and are presented as mean values±SEM.

[0029] FIG. 11: PBMC of six different healthy donors were isolated and stimulated with poly-L-arginine complexed ssRNA-oligonucleotides in duplicates. 44 hours after stimulation IFN-α production was assessed in supernatant via ELISA. For all tested ssRNA-oligonucleotides, the mean values of the measured duplicates were normalized to the positive control ssRNA-oligonucleotide 9.2sense (5'-AGCUUAACCUGUCCUUCAA) by dividing the mean value of tested oligonucleotide by the mean value of 9.2sense. Next, for each individual donor a global normalization to the mean was performed by subtracting the mean of all data from a particular donor from the individual raw data. Data from six individual donors were visualized using tree view and are depicted in ascending order (A). In addition all individual data were summarized as mean values±SEM and are depicted in ascending order (B).

[0030] FIG. 12: The occurrence of 3mer motifs in all ssRNA-oligonucleotides was analyzed. The mean level of IFN-α induction was calculated by grouping all oligonucleotides that contained a respective 3mer motif. For example the 3mer motif 5'-GUC-3' was contained in ssRNA oligonucleotides ANP 35, 83, 131, 137, 138, 139 and 179 with respective IFN-α induction levels of 1.33, 0.68, 0.93, 0.79, 0.44, 0.84 and 0.73. The mean IFN-α induction level of the 3mer motif 5'-GUC-3' was thus calculated to be 0.82 with a standard error of mean of 0.10. 3mer motifs that were gapped by one nucleotide between either the first and the second nucleotide position (5'-N-NN-3') or the second and third nucleotide position (5'-NN-N-3') were also included in the analysis. A two-tailed T-Test was used to identify motifs that were either significantly higher or lower in IFN-α induction than the residual motifs. For all motifs analyzed, the mean IFN-α induction level was visualized using tree view. The data were assorted according to the first nucleotide position of the motif in four groups. (p-value<0.05 is indicated by *).

[0031] FIG. 13: The top 15 percent of all ssRNA oligonucleotides and the respective mean IFN-α induction levels are shown in ascending order. The presence of the identified potent 3mer motifs 5'GUY-3' (5'-GUC-3',5'-GUU-3'), 5'-GUNY-3' (5'-GUNC-3',5'-GUNU-3') and 5'-GNUY-3' (5'-GNUC-3',5'-GNUU-3') is indicated by a grey box. All ssRNA oligonucleotides that contain any of the above motifs are indicated by a black box.

[0032] FIG. 14: PBMC of four different healthy donors were isolated and stimulated with the following poly-L-arginine complexed ssRNA-oligonucleotides: ANP143 (5'-AAAAAAAGUUCAAAAAAAA-3'), RNA40 (5'-GCCCGUCUGUUGUGUGACUC-3'), p-Gal control sense (5'-UUGAUGUGUUUAGUCGCUA-3') and 9.2sense (5'-AGCUUAACCUGUCCUUCAA-3'). 44 hours after stimulation IFN-α production was assessed in supernatant via ELISA. All tested ssRNA-oligonucleotides were normalized to the positive control ssRNA-oligonucleotide 9.2sense by dividing the mean value of tested oligonucleotide by the mean value of 9.2sense. Significant differences were analyzed using a two-tailed T-Test.

[0033] FIG. 15: The occurrence of 3mer motifs was analyzed in the 193 oligonucleotide library. For each oligonucleotide the 3mer motif with the highest calculated mean IFN-α induction level was identified and assigned to the respective oligonucleotide. The predicted data are depicted in ascending order (black bars) according to the corresponding measured IFN-α induction levels (black line). In addition, the correlation coefficient for the two data sets was determined.

[0034] FIG. 16: The occurrence of 3mer motifs was analyzed in the 193 oligonucleotide library. For each oligonucleotide the 3mer motif with the highest calculated mean IFN-α induction level was identified and assigned to the respective oligonucleotide. The predicted data are depicted in ascending order. Various threshold levels (dotted lines) were tested for both for the positive predictive value and the sensitivity to identify oligonucleotides below the IFN-α induction level of 0 (A). For each threshold level, data were then regrouped according to the predicted IFN-α induction level (selected oligonucleotides: left group, eliminated oligonucleotides: right group). For each group, the mean level of IFN-α induction±SEM is depicted in the lower panel. The positive predictive value and the sensitivity for each threshold is indicated in the upper left.

[0035] FIG. 17: The prediction algorithm was used to analyze all possible siRNA duplexes targeting the mRNA of human TLR9 (NM_--017442). For the 3868 bp long mRNA of TLR9 all possible 19mer siRNA duplexes were considered and the IFN-α prediction algorithm was applied on both the sense and the antisense strand of each siRNA duplex. The predicted IFN-α induction levels are depicted in stacked columns for the sense (upper columns in black) and the antisense strand (lower column in grey). The relative targeting position of the siRNA duplex is given on the y-axis, whereas the predicted IFN-α induction is depicted on the x-axis (A). In addition, six selected regions of the TLR9 mRNA and the respective predicted IFN-α induction levels are depicted in detail in B.

[0036] FIG. 18: HEK 293 cells were transfected with an expression plasmid coding for human TLR9 with a C-terminal YFP-tag. Various siRNA-duplices targeting human TLR9 mRNA were cotransfected. The starting base of the individual siRNA is given in the lower panel. 20 hours after transfection, TLR9 expression was analyzed by flow cytometry. Data are depicted as percentage of TLR9-expression referring to an irrelevant control siRNA as 100% and siRNA_sb1647 as 0%. Results are shown as mean values±SEM (n=3) (A). In addition, above siRNA duplexes and the respective single stranded components were used to transfect human PBMC from five individual donors. 40 hours after transfection IFN-α induction was measured via ELISA. Data are depicted as mean values±SEM (B).

[0037] FIG. 19: Based on the algorithm described in example 17, a computer program was written that applies the algorithm to all possible siRNA duplexes targeting all human RNA transcripts (50421 as of 09/2006) as published by the National Center for Biotechnology Information (NCBI). Each entry into the NCBI database (ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/human.rna.fna.gz) of all listed human RNA transcripts was analyzed the following way: A list of all possible 19mer siRNA duplexes targeting a given RNA transcript was generated. Of all siRNA duplexes the IFN-α induction of both the sense and the antisense strand was predicted using the method described in example 17. The obtained data is stored in a database (CD-ROM) and can be retrieved by a search engine. Using the search interface, the user can pick the transcript of interest (alphabetical index of all RNA transcripts targeted by siRNAs) and then adjust the level of threshold to identify siRNA duplexes that are of either low, intermediate or high in immunostimulatory activity (A). For example, using the threshold of 0.11 as described in example 17, a set of siRNA duplexes was identified for Homo sapiens vascular endothelial growth factor (VEGF) transcript variant 1 mRNA (NM_--001025366.1) with low immunostimulatory activity for both the sense and the antisense strand (B).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0038] As used herein, "a" and "an" refers to a group or species of entities, rather than one single individual.

oligonucleotide

[0039] As used herein, the term "oligonucleotide" refers to a polynucleotide formed from a plurality of linked nucleoside units. Such oligonucleotides can be obtained from existing nucleic acid sources, including genomic or cDNA, but are preferably produced by synthetic methods including chemical synthesis, in vitro and in vivo transcription. In preferred embodiments each nucleoside unit includes a heterocyclic base and a pentofuranosyl, trehalose, arabinose, 2'-deoxy-2'-substituted arabinose, 2'-O-substituted arabinose or hexose sugar group. The nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages. Such internucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, pyrophosphate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. The term "oligonucleotide" also encompasses polynucleosides having one or more stereospecific internucleoside linkage (e.g., (R_p)- or (S_p)-phosphorothioate, alkylphosphonate, or phosphotriester linkages).

[0040] The oligonucleotides of the invention can include naturally occurring nucleosides, modified nucleosides, or mixtures thereof. As used herein, the term "modified nucleoside" is a nucleoside that includes a modified heterocyclic base, a modified sugar moiety, or a combination thereof. In some embodiments, the modified nucleoside is a non-natural pyrimidine or purine nucleoside, as herein described. In some embodiments, the modified nucleoside is a 2'-substituted ribonucleoside an arabinonucleoside or a 2'-deoxy-2'-substituted-arabinoside.

[0041] As used herein, the term "2'-substituted ribonucleoside" or "2'-substituted arabinoside" includes ribonucleosides or arabinonucleoside in which the hydroxyl group at the 2' position of the pentose moiety is substituted to produce a 2'-substituted or 2'-O-substituted ribonucleoside. Preferably, such substitution is with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an aryl group having 6-10 carbon atoms, wherein such alkyl, or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups. Examples of 2'-O-substituted ribonucleosides or 2'-O-substituted-arabinosides include, without limitation 2'-O-methylribonucleosides or 2'-O-methylarabinosides and 2'-O-methoxyethylribonucleosides or 2'-O-methoxyethylarabinosides.

[0042] The term "2'-substituted ribonucleoside" or "2'-substituted arabinoside" also includes ribonucleosides or arabinonucleosides in which the 2'-hydroxyl group is replaced with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an amino or halo group. Examples of such 2'-substituted ribonucleosides or 2'-substituted arabinosides include, without limitation, 2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl ribonucleosides or arabinosides.

[0043] The term "oligonucleotide" includes hybrid and chimeric oligonucleotides. A "chimeric oligonucleotide" is an oligonucleotide having more than one type of internucleoside linkage. One preferred example of such a chimeric oligonucleotide is a chimeric oligonucleotide comprising a phosphorothioate, phosphodiester or phosphorodithioate region and non-ionic linkages such as alkylphosphonate or alkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Pat. Nos. 5,635,377 and 5,366,878).

[0044] A "hybrid oligonucleotide" is an oligonucleotide having more than one type of nucleoside. One preferred example of such a hybrid oligonucleotide comprises a ribonucleotide or 2'-substituted ribonucleotide region, and a deoxyribonucleotide region (see, e.g., Metelev and Agrawal, U.S. Pat. Nos. 5,652,355, 6,346,614 and 6,143,881).

[0045] RNA oligonucleotides discussed herein include otherwise unmodified RNA as well as RNA which have been modified (e.g., to improve efficacy), and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. 1994, Nucleic Acids Res 22: 2183-2196. Such rare or unusual RNAs, often termed modified RNAs (apparently because these are typically the result of a post-transcriptional modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occurs in nature, preferably different from that which occurs in the human body. While they are referred to as modified "RNAs," they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.

[0046] All nucleic acid sequences listed herein are in the 5' to 3' direction unless otherwise indicated.

[0047] The RNA oligonucleotide of the invention can be single-stranded, double stranded, or partially double-stranded.

[0048] A single-stranded RNA oligonucleotide may contain self-complementary sequences and forms a hairpin. For example, 5'-GACCUAGCCUAAAACUAGGUC-3'. The self-complementary sequence may be a palindromic sequence. For example, 5'AAAGAUCCGGAUCAAAA-3'.

[0049] A double stranded RNA oligonucleotide may have one- or two-nucleotide overhang at the 5' or 3' end of one or both strands.

[0050] A partially double-stranded RNA oligonucleotide may comprise two strands of the same or different length, wherein the at least one of the strands contains nucleotides outside the complementary sequence. For example,

TABLE-US-00001 Esample 1: 5'-AAAAGUUCAAAGCUCAAAA-3' 3'-CAAGUUUCGAG-5' Example 2: 5'-UCAAAGUCAAAAGCUCAAAGUUGAAAGUUUAAA-3' 3'-GACUUGAAAAUUUCAGUUUUCGAGUUUAAGUUGAAAACUCG-5' Example 3: 5'-UCAAAGUCAAAAGCUCAAAGUUGAAA-3' 3'-UUUCAGUUUUCGAGUUUAAGUUGAAAACUCG-5'

[0051] The length of a single-stranded RNA oligonucleotide is the number of nucleotides contained in the oligonucleotide.

[0052] In the case of a double-stranded or partially double-stranded oligonucleotide, the length of the oligonucleotide is the length of the individual strands. In other words, a partially double-stranded oligonucleotide can have two lengths.

Enhanced Nuclease Resistance

[0053] For increased nuclease resistance and/or binding affinity to the target, an oligonucleotide can include, for example, 2'-modified ribose units and/or phosphorothioate linkages and/or pyrophosphate linkages. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.

[0054] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_nCH₂CH₂OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; G-AMINE and aminoalkoxy, O(CH₂)_nAMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification. "Deoxy" modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH₂CH₂NH)_nCH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), --NHC(O)R(R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality.

[0055] Preferred substitutents are 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-- allyl, and 2'-fluoro. To maximize nuclease resistance, the 2' modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called "chimeric" oligonucleotides are those that contain two or more different modifications. The inclusion of furanose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage. An oligonucleotide agent can be further modified by including a 3' cationic group, or by inverting the nucleoside at the 3'-terminus with a 3'-3' linkage. In another alternative, the 3'-terminus can be blocked with an aminoalkyl group, e.g., a 3' C5-aminoalkyl dT. Other 3' conjugates can inhibit 3'-5' exonucleolytic cleavage. While not being bound by theory, a 3' conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3'-end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5'-exonucleases.

[0056] Similarly, 5' conjugates can inhibit 5'-3' exonucleolytic cleavage. While not being bound by theory, a 5' conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 5'-end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5'-exonucleases.

[0057] Single-stranded RNA oligonucleotides which contain self-complementary sequences and form a hairpin structure have enhanced nuclease resistance compared to single-stranded oligonucleotides which do not.

5'-Phosphate Modifications

[0058] The oligonucleotides of the present invention can be 5' phosphorylated or can include a phosphoryl analog at the 5' prime terminus. 5'-phosphate modifications of the antisense strand include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P--O-5'); 5'-diphosphate ((HO)2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate ((HO)2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure. Other suitable 5'-phosphate modifications will be known to the skilled person.

Tethered Ligands

[0059] The RNA oligonucleotides of the present invention also include those with tethered ligands. The properties of a RNA oligonucleotide, including its pharmacological properties, can be influenced and tailored by the introduction of ligands, e.g. tethered ligands.

[0060] The ligands may be coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RNA oligonucleotide. In preferred embodiments, the ligand is attached to the oligonucleotide via an intervening tether.

[0061] In preferred embodiments, a ligand alters the distribution, targeting or lifetime of a RNA oligonucleotide into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, a cellular or organ compartment, tissue, organ or region of the body.

[0062] Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

[0063] A wide variety of ligands may be used. Ligands may include agents that allow for the specific targeting of the oligonucleotide; diagnostic compounds or reporter groups which allow for the monitoring of oligonucletotide distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophilic moleculeses, lipids, lectins, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins, carbohydrates (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics.

[0064] The ligand may be a naturally occurring or recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic moieties, e.g., cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[0065] Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a thyrotropin, melanotropin, surfactant protein A, Mucin carbohydrate, a glycosylated polyaminoacid, transferrin, bisphosphonate, polyglutamate, polyaspartate, or an RGD peptide or RGD peptide mimetic.

[0066] Ligands can be proteins, e.g., glycoproteins, lipoproteins, e.g. low density lipoprotein (LDL), or albumins, e.g. human serum albumin (HSA), or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NE-κB. The ligand can be a substance, e.g., a drug, which can increase the uptake of the oligonucleotide agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0067] In one embodiment, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., liver tissue, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

[0068] A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

[0069] In another embodiment, the ligand is a moiety, e.g., a vitamin or nutrient, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include the B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells.

[0070] In another embodiment, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennapedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

[0071] In a preferred embodiment, the ligand is an antibody or a fragment thereof which is specific for a moiety present in a cell to be targeted. The moiety may be a protein, a carbohydrate structure, a polynucleotide, or a combination thereof. The moiety may be secreted, associated with the plasma membrane (e.g., on the extracellular or intracellular surface), cytosolic, associated with intracellular organelles (e.g., ER, Golgi complex, mitochondria, endosome, lysosome, secretory vesicle) or nuclear. The antibody may be monoclonal or polyclonal. The antibody may be chemeric or humanized. The antibody may be a single chain antibody. The antibody fragment may be a Fab fragment, a F(ab')₂ fragment, or any fragments that retain the antigen-binding specificity of the intact antibody.

Immunostimulatory Activity

[0072] As used herein, "immunostimulatory activity" refers to the capability of a molecule or a composition to induce an immune response. In one aspect, the immunostimulatory activity refers to the type I-IFN-inducing activity, in particular, the IFN-α-inducing activity.

[0073] As used herein, "inducing an immune response" means initiating or causing an increase in one or more of B-cell activation, T-cell activation, natural killer cell activation, activation of antigen presenting cells (e.g., B cells, dendritic cells, monocytes and macrophages), cytokine production, chemokine production, specific cell surface marker expression, in particular, expression of co-stimulatory molecules. In one aspect, such an immune response involves the production of type I IFN, in particular, IFN-α, in cells such as PDC.

[0074] As used herein, "IFN-α-inducing activity" refers to the capability of a molecule or composition to induce IFN-α production from a cell capable of producing IFN-α. Cells capable of producing IFN-α include, but are not limited to, peripheral blood mononuclear cells (PBMC) (e.g., B cells, dendritic cells (myeloid dendritic cells and plasmacytoid dendritic cells), macrophages, monocytes, natural killer cells, granulocytes), endothelial cells, and cell lines (e.g., THP1; cells transfected with expression vectors for TLR-7 and/or TLR-8 such as CHO cells, COS cells, HEK293 cells). Cells capable of producing IFN-α include those that express TLR7, TLR8, or both TLR7 and TLR8.

Gene Silencing Activity

[0075] As used herein, "gene silencing" refers to the downregulation or the abolition of the expression of a target gene. Gene silencing as used herein, occurs at the post-transcriptional level. Gene silencing may be directly or indirectly mediated by siRNA, shRNA and antisense RNA.

[0076] Both the antisense-strand of the siRNA and the antisense RNA have complementary to the target mRNA and are the effector strand of the gene silencing activity. The term complementary is well understood by those skilled in the art. For example, A is complementary to T, G is complementary to C, 5'-AG-3' is complementary to 5'-CT-3'.

[0077] The degree of complementarity between two oligonucleotides is the percentage of complementary bases in the overlapping region of the two oligonucleotides. The degree of complementarily can be determined manually or automatically by various engines such as BLAST. For example, ATCG has 100% complementarity to CGAT and CGATGG, and 75% complementarity to CGTT and CGTTGG. Furthermore, the degree of complementarity between a RNA oligonucleotide and any sequences present in the public databases (e.g., EMBL, GeneBank) can be determined by the BLAST program.

[0078] The degree of complementarity between the antisense strand of the siRNA or the antisense RNA and the target mRNA is at least 80% 81%, 82%, 83%, preferably at least 84%, 85%, 86%, 87%, 88%, more preferably at least 89%, 90%, 91%, 92%, 93%, even more preferably at least 94%, 95%, 96%, 97%, 98%, 99%, and most preferably 100%.

[0079] The gene silencing activity of a RNA oligonucleotide can be determined experimentally by methods well known in the art. For Example, the RNA oligonucleotide may be introduced into a cell by a method known in the art such as transfection and transduction; the mRNA level of the target gene can be determined by routine methods such as Northern blot analysis, quantitative PCR, RNase protection assay, and branching DNA; and the protein expression level can be determined by routine methods such as Western blotting, ELISA, and biological activity assays specific to the target protein. Furthermore, the mRNA level of all known and hypothetical genes can be determined at the global level using the microarray technology. Technologies in the field of proteonomics allow for the protein levels of a large number of genes to be determined at the global level as well.

[0080] Naked RNA oligonceotide may be transfected into a cell via electroporation. RNA oligonucleotide may be complexed with a complexation agent which facilitates the uptake of the oligonucletide into a cell. Such complexation agents include, but are not limited to cationic lipids (e.g., Lipofectamine, Oligofectamine, DOTAP), cationic peptides, and calcium phosphate.

[0081] The gene silencing activity of a RNA oligonucleotide can be predicted by algorithms such as the one disclosed in Reynolds et al. 2004, Nat Biotechnol 22:326-330.

siRNA

[0082] As used herein, "siRNA" stands for short interfering RNA, and has the same definition as that established in the art. siRNA is double-stranded and is usually between 19 and 27 nucleotide in length. In vivo, siRNA is the product of Dicer activity on long dsRNA. The antisense strand of siRNA is complementary to the target mRNA; it binds the target mRNA and induces RISC-mediated target mRNA degradation. siRNA can be chemically synthesized, produced in vitro by Dicer-mediated enzymatic degradation of long dsRNA, produced by in vitro transcription from linear (e.g. PCR products) or circular templates (e.g., viral or non-viral vectors), or produced by in vivo transcription from viral or non-viral vectors. Commercially available synthetic siRNA usually contain a core of 19 complemetary base pairs and a 2-nucleotide (UU or TT) 3' overhang on each strand. siRNA may be chemically modified to have enhanced stability in vitro (especially in serum-containing media) and in vivo. siRNA may also be chemically modified to have enhanced uptake by cells in vitro and in vivo. Furthermore, siRNA may be linked to tethered ligands to have enhanced target specificity and improved pharmacological properties (such as half-life, clearance, distribution).

shRNA

[0083] As used herein, "shRNA" stands for short hairpin RNA and has the same definition as that established in the art. shRNA is processed inside a cell into siRNA which mediates RNAi as described previously. The loop sequence in shRNA is not thought to be involved in RNAi, and it can be of various lengths and sequences. The preferred lengths and sequences of the loop are known to those skilled in the art.

[0084] Similar to siRNA, shRNA can be chemically synthesized, produced by in vitro transcription from linear (e.g. PCR products) or circular templates (e.g., viral or non-viral vectors), or produced by in vivo transcription from viral or non-viral vectors.

Antisense RNA

[0085] As used herein, "antisense RNA" has the same definition as that established in the art. Antisense RNA is complementary to target mRNA and it thought to interfere with the translation of the target mRNA. Antisense RNA molecules are usually 18-50 nucleotides in length. Antisense RNA may be modified to have enhanced stability, nuclease resistance, target specificity and improved pharmacological properties.

[0086] Similar to siRNA and shRNA, antisense RNA can be chemically synthesized, produced by in vitro transcription from linear (e.g. PCR products) or circular templates (e.g., viral or non-viral vectors), or produced by in vivo transcription from viral or non-viral vectors.

Disorder/Disease-Related Gene and Antigen

[0087] As used herein, "disorder/disease-related gene" refers to a gene that is expressed or overexpressed in a disease/disorder and that is not expressed or expressed in reduced amount under normal condition. For example, a mutant CF gene is expressed in cystic fibrosis patient but not in an individual without cystic fibrosis; ErbB2 (or Her2) is overexpressed in breast cancer cells compared to normal breast cells; a viral gene is expressed in infected cells but not in uninfected cells. The gene product of the disorder/disease-related gene is referred to herein as the "disorder/disease-related antigen".

Mammal

[0088] As used herein, the term "mammal" includes, without limitation, rats, mice, cats, dogs, horses, sheep, cattle, cows, pigs, rabbits, non-human primates, and humans.

Technology Platform

[0089] The vertebrate immune system established different ways to detect invading pathogens based on certain characteristics of their microbial nucleic acids. Detection of microbial nucleic acids alerts the immune system leading to the appropriate type of immune responses that is required for the defence against the respective type of pathogen detected. Detection of viral nucleic acids leads to the production of type I IFN, the key cytokine for anti-viral defence. While it is well established that the recognition of microbial DNA is sequence-specific, involving the so-called CpG motifs, the optimal motif for the recognition of microbial RNA has not been defined yet. The present application provides a technology platform for identifying the optimal motif for the recognition of microbial RNA and the induction of type I IFN.

[0090] The technology platform of the present invention comprises three key features: i) the transfection of peripheral blood mononuclear cells (PBMC) from healthy donors with RNA oligonucleotides; ii) the generation of a RNA oligonucleotide library containing 4mer motifs on a poly adenosine (polyA) backbone; iii) the development of algorithms based on the experimental data generated for the RNA oligonucleotide library to predict the immunostimulatory activity of any given RNA oligonucleotide.

[0091] The first key feature of the technology platform is the method of introducing RNA oligonucleotides into PBMC. Naked RNA oligonucleotides are not taken up by the cells to any significant degree. RNA oligonucleotides normally need to form complexes with complexation agent (or transfection agent) in order to be introduced into cells. In the literature, cationic lipids such as lipofectamine or DOTAP are routinely used as complexation agent for the transfection of RNA oligonucleotides. However, RNA-cationic lipid complexes lead to rapid cell death of myeloid cells. Although myeloid cells are not the cellular source of IFN-α within PBMC (the source is PDC), death of myeloid cells in the cell culture negatively affects the reproducibility of IFN-α induction in PBMC. Therefore, the use of cationic lipids is limited to isolated PDC. However, isolated PDC are not suitable for large scale screening assays because PDC make up 0.2-0.6% of the PBMC in a normal individual; it is difficult to obtain enough cells for the assays.

[0092] To identify a complexation agent that is suitable for use with PBMC, we compared different types of cationic peptides, poly-His, poly-L-Lys, and poly-L-arg. Poly-L-arg was found to provide the most potent support for the immunostimulatory activity of RNA oligonucleotides when compared to other cationic peptides and cationic lipids (FIG. 1). A protocol was then established that allows well-controlled and highly reproducible complex formation between the RNA oligonucleotide and the complexation agent and subsequent RNA transfection into cells. Complex formation could be controlled by salt concentration, phosphate content and incubation time. Complex formation was monitored by the size of complexes and the functional activity over a range of concentrations. The use of poly-L-arg did not affect the viability of myeloid cells and thus could be applied to PBMC without restrictions.

[0093] The second key issue of the technology platform was the generation of the RNA oligonucleotide library. An earlier study showed that a minimal length of 19 bases was required for the optimal immunostimulatory activity of an RNA oligonucleotide; furthermore, it showed that poly adenosine (poly A) was completely inactive (Hornung V et al. 2005, Nat Med 11:263-270). Therefore, the motif search was performed with a 19mer oligonucleotide on a poly A sequence background. By adding increasing numbers of uridine (U) in the center of such a poly A oligonucleotide, we found that a 4-nucleotide (4-mer) motif in the center was sufficient to confer marked immunostimulatory activity (FIG. 2). Importantly, after identifying the optimal 4mer sequence motifs for inducing IFN-α production, we found that changing the bases flanking the 4mer motifs did not further enhance the immunostimulatroy activity of the 4mer motifs (FIG. 7B). The library of 193 RNA oligonucleotides used covered all 256 possible 4mer motifs. The reduction from 258 to 193 was possible because of redundant motifs caused by the poly A flanking regions. In additional studies we found that the exact location of the 4mer motif within the poly A backbone is not critical for the immunostimulatory activity (FIG. 7A).

[0094] The third key feature of the technology platform was the generation of a data matrix and its mathematical analysis. Algorithms were developed that allowed an excellent prediction of the immunostimulatory activity of RNA oligonucleotides. The frequency of a given 4mer motif at a certain position within an oligonucleotide is only 1:256. Even though the most active 4mer motifs can be used as the core for constructing potent immunostimulatory RNA oligonucleotides, the IFN-α indices of the 4mer motifs are not particularly useful for predicting the activity of a given RNA oligonucleotide, or for designing RNA oligonucleotides with minimal immunostimulatory activity which is desired for an siRNA. Therefore, algorithms were established which based on parts of the 4mer motifs, namely 1, 2 or 3 bases either in a row (XXX) or with spacing (X*XX; XX*X). The highest predictive value was obtained with the algorithm using 3 bases (i.e., 3mer motifs). This 3mer-based algorithm allowed an impressively accurate prediction (correlation coefficient (r)=0.87) of the immunostimulatory activity of the 19mer RNA oligonucleoties carrying 4mer motifs in our library (FIG. 6E) and RNA oligonucleotides previously published in the literature by us and others (FIG. 8A-C).

[0095] There are a number of applications for the information generated by our technology platform: a) the 4mer motif data matrix can be used to design oligonucleotides with optimal IFN-α-inducing activity; b) 4mer motifs with minimal IFN-α-inducing activity can be used as the repertoire for selecting potential inhibitory sequence motifs; c) the 3mer-based algorithm (e.g., the IFN-α point score matrix) can be used to predict the immunostimulatory activity of a given RNA oligonucleotide; d) the 3mer-based algorithm (e.g., the IFN-α point score matrix) can be used to design RNA oligonucleotides with maximal immunostimulatory activity and additional sequence requirements for other functionalities such as gene silencing in the case of an siRNA (in this case the use of 4mer motif matrix is not useful since 4mer motifs are not frequent enough); e) the 3mer-based algorithm (e.g., the IFN-α point score matrix) can also be used to design RNA nucleotides with minimal immunostimulatory activity and additional sequence requirements for other functionalities such as gene silencing in the case of an siRNA (in this case the use of 4mer motif matrix is not useful since 4mer motifs are not frequent enough).

[0096] In the case of an immunostimulatory RNA, an oligonucleotide containing only one of the most potent 4mer motifs is 80% more active than the most active complex oligonucleotide containing a 9mer motif in the literature (Table 1). In a 19mer oligonucleotide, there is room for several potent 4mer motifs. A 19mer RNA olignucleotide containing more than one potent immunostimulatory 4mer motifs is expected to have even higher activity.

[0097] Furthermore, inhibitory motifs may exist that inhibit the immunostimulatory activity of a RNA oligonucleotide as in the case of CpG oligonucleotides. Such inhibitory motifs, by definition, are among the motifs with weak IFN-α-inducing activity. In the field of RNA interference, type I IFN induction usually is unwanted. The 3mer-based algorithm (e.g., the IFN-α point score matrix) described above can be used to select siRNA sequences with minimal immunostimulatory activity. A sequence analysis of cyclophylin B mRNA, one of the best studied targets for siRNA, identifies a number of siRNA sequences for which our algorithm (e.g., the IFN-α point score matrix) predicts minimal type I IFN induction and which still are known to be potent in gene silencing (Reynolds A et al. 2004, Nat Biotechnol 22: 326-330). This confirms our previous finding that RNA interference and IFN-α induction are two independent functional activities of a siRNA molecule.

[0098] Of note, the motif search performed in the present study focuses on the activity of RNA oligonucleotides to induce IFN-α. From previous studies, it is known that the cellular source of IFN-α within the PBMC is PDC. By analysing the level of IFN-α induction in PBMC, other activities of the RNA oligonucleotides on other cellular subsets of the PBMC, such as myeloid cells, are not addressed. Myeloid cells express TLR8 in addition to TLR7 and thus may show different nucleotide sequence specificities and may be induced to exhibit additional activities than IFN-α production. It therefore needs to be born in mind that ssRNA oligonucleotides are capable of inducing both PDC-dependent (i.e. IFN-α production) and PDC-independent activities (e.g., activation of myeloid cells). In contrast, we found that dsRNA oligonucleotides, such as siRNA, are only recognized by PDC but not myeloid cells. As a result, it is valid to predict the immunological activity of siRNA oligonculeotides based on their ability to induce IFN-α production.

Method for Determining the Immunostimulatory Activity of an RNA Oligonucleotide

[0099] The present invention provides a method for determining the immunostimulatory activity, in particular, the IFN-α-inducing activity, of a RNA oligonucleotide, comprising the steps of:

[0100] (a) complexing the RNA oligonucleotide with a complexation agent;

[0101] (b) contacting a cell with the complexed RNA oligonucleotide, wherein the cell expresses TLR7 or TLR8 or both TLR7 and TLR8; and

[0102] (c) determining the amount of IFN-α produced by the cell of step (b), an increase of IFN-α production indicating immunostimulatory activity of the RNA oligonucleotide.

[0103] In one embodiment of the invention, the complexation agent is a polycationic peptide, preferably poly-L-arginine (poly-L-arg). In one embodiment, the polycationic peptide, in particular, poly-L-arg, is at least 24 amino acids in length. The polycationic peptide, in particular, poly-L Arg, may be a heterogeneous mixture of peptides of different lengths.

[0104] The cells expressing TLR7 or TLR8 or both TLR7 or TLR8 include, but are not limited to, peripheral blood mononuclear cells (PBMC), plasmacytoid dendritric cells (PDC), myeloid dendritic cells (MDC), B cells, macrophages, monocytes, natural killer cells, granulocytes, endothelial cells, cell lines such as THP1, and cells containing exogenous DNA which directs the expression of TLR7 or TLR8 or both TLR7 or TLR8 such as transfected CHO, HEK293, and COS cells.

[0105] In one embodiment of the invention, the cell is a mammalian cell, preferably a human cell or a cell of human origin.

[0106] The RNA oligonucleotide can be single-stranded, double-stranded or partially double-stranded.

Method for Predicting the Immunostimulatory Activity of a RNA Oligonucleotide

[0107] The present invention provides a method for predicting the immunostimulatory activity, in particular the IFN-α-inducing activity, of a RNA oligonucleotide, comprising the steps of: [0108] (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; [0109] (b) assigning an IFN-α point score for each individual 3mer motif; [0110] (c) assigning the sum of the IFN-α point scores of individual 3mer motifs as the IFN-α score of the oligonucleotide; and [0111] (d) assigning to the oligonucleotide a high immunostimulatory activity if the IFN-α score is at least 23, an intermediate immunostimulatory activity if the IFN-α score is between -4 and 23, and a low immunostimulatory activity if the IFN-α score is at most -4, when n=6; [0112] assigning to the oligonucleotide a high immunostimulatory activity if the IFN-α score is at least 26, an intermediate immunostimulatory activity if the IFN-α score is between -4 and 26, and a low immunostimulatory activity if the IFN-α score is at most -4, when n=7; [0113] assigning to the oligonucleotide a high immunostimulatory activity if the IFN-α score is at least 28, an intermediate immunostimulatory activity if the IFN-α score is between -5 and 23, and a low immunostimulatory activity if the IFN-α score is at most -5, when n=8; [0114] assigning to the oligonucleotide a high immunostimulatory activity if the IFN-α score is at least 30, an intermediate immunostimulatory activity if the IFN-α score is between -5 and 30, and a low immunostimulatory activity if the IFN-α score is at most -9, when n=9; [0115] assigning to the oligonucleotide a high immunostimulatory activity if the IFN-α score is at least 1.4909×n+22.014, an intermediate immunostimulatory activity if the IFN-α score is between 0.005×n²-0.2671×n-3.5531 and 1.4909×n+22.014, and a low immunostimulatory activity if the IFN-α score is at most 0.005×n²-0.2671×n-3.5531, when n is greater than 9, [0116] wherein n is the length of the oligonucleotide.

[0117] The present invention also provides a method for assigning the IFN-α score of a RNA oligonucleotide comprising steps (a)-(c) described above.

[0118] A single-stranded RNA oligonucleotide of the length n (n≧6) is broken up into all possible 3mer motifs starting a the 5' end. This will result in a total number of n-2 possible 3mer motifs. For example the 20mer ssRNA oligonucleotide 5'-CAGAGCGGGAUGCGUUGGUC-3' can be broken up into the following 18 3mer motifs (5'-->3'): CAG, AGA, GAG, AGC, GCG, CGG, GGG, GGA, GAU, AUG, UGC, GCG, CGU, GUU, UUG, UGG, GGU, GUC.

[0119] Subsequently, all of the 3mer motifs are checked against the IFN-α point score matrix (Table 7).

TABLE-US-00002 TABLE 7 IFN-α point score matrix 3mer motif IFN-α point (5'→3') score ACA -2 ACC -2 AGA -2 AAC -1 AUA -1 UGG +1 GUA +3 GUG +3 GGU +4 UCA +4 UUC +4 UUU +5 AGU +6 UUG +6 GUC +8 UGU +8 GUU +9

[0120] Whenever a 3mer motif is present in the IFN-α point score matrix, the listed point score is added to the so-called predicted IFN-α score of the oligonucleotide analyzed. A 3mer motif which is absent from the IFN-α point score matrix has a point score of 0. Thus the predicted IFN-α score of an oligonucleotide is the sum of IFN-α scores of all 3mer motifs that are present in the IFN-α point score matrix.

[0121] For example, for the 20mer ssRNA oligonucleotide 5'-CAGAGCGGGAUGCGUUGGUC-3', a predicted IFN-α score can be calculated as follows:

TABLE-US-00003 3mer motifs in the Score in the IFN-α predicted 20mer ssRNA point score matrix IFN-α score CAG 0 0 AGA (-2) -2 GAG 0 0 AGC 0 0 GCG 0 0 CGG 0 0 GGG 0 0 GGA 0 0 GAU 0 0 AUG 0 0 UGC 0 0 GCG 0 0 CGU 0 0 GUU (+9) +9 UUG (+6) +6 UGG (+1) +1 GGU (+4) +4 GUC (+8) +8 Overall +26

[0122] This method is herein referred to as the "addition method".

[0123] The present application further provides an alternative method for predicting the immunostimulatory activity, in particular the IFN-α-inducing activity, of a RNA oligonucleotide, comprising the steps of: [0124] (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; [0125] (b) assigning an IFN-α point score for each individual 3mer motif according to Table 12A; [0126] (c) assigning the highest individual IFN-α point score as the IFN-α score of the oligonucleotide; and [0127] (d) assigning to the oligonucleotide a high immunostimulatory activity if the IFN-α score is at least 0.58, an intermediate immunostimulatory activity if the IFN-α score is between 0.11 and 0.58, and a low immunostimulatory activity if the IFN-α score is at most 0.11.

[0128] The present invention also provides a method for assigning the IFN-α score of a RNA oligonucleotide comprising steps (a)-(c) described above.

[0129] This method is herein referred to as the "simplified method".

[0130] The IFN-α score of a double-stranded RNA oligonucleotide is the higher of the two IFN-α scores for the two strands.

[0131] In the case of a double-stranded or partially double-stranded RNA oligonucleotide, the oligonucleotide is assigned high immunostimulatory activity if at least one of the strands meets the threshold for having high immunostimulatory activity as defined above; the oligonucleotide is assigned low immunostimulatory activity if both strands meet the threshold for having low immunostimulatory activity as defined above. The rest RNA oligonucleotides are assigned intermediate immunostimulatory activity.

Method for Designing and Preparing RNA Oligonucleotides

[0132] The present application provides a method for preparing a RNA oligonucleotide having immunostimulatory activity, in particular, high IFN-α-inducing activity, comprising the steps of: [0133] (a) providing candidate oligonucleotide sequence(s); [0134] (b) identifying oligonucleotide sequence(s) with high immunostimulatory activity predicted according to the method of prediction described in the previous section; [0135] (c) preparing the RNA oligonucleotide(s) identified for high immunostimulatory activity in step (b); and [0136] (d) optionally testing the immunostimulatory activity of the RNA oligonucleotide(s) prepared in step (c) according to the method of determination described previously; and [0137] (e) further optionally modifying the oligonucleotide(s) to optimize the immunostimulatory activity.

[0138] The present application also provides a method for preparing a RNA oligonucleotide having low immunostimulatory activity, in particular, low IFN-α-inducing activity, comprising the steps of: [0139] (a) providing candidate oligonucleotide sequence(s); [0140] (b) identifying oligonucleotide sequence(s) with low immunostimulatory activity predicted according to the method of prediction described in the previous section; [0141] (c) preparing the RNA oligonucleotide(s) identified for low immunostimulatory activity in step (b); and [0142] (d) optionally testing the RNA oligonucleotide(s) prepared in step (c) for the lack of immunostimulatory activity according to the method of determination described previously; and [0143] (e) further optionally modifying the oligonucleotide(s) to minimize the immunostimulatory activity.

[0144] The present invention further provides a method for preparing a RNA oligonucleotide having high immunostimulatory activity, in particular, high IFN-α-inducing activity, comprising the steps of: [0145] (a) providing an oligonucleotide sequence which comprises at least one, preferably at least two, more preferably at least three, even more preferably at least four, of the 4-nucleotide (4-mer) motifs selected from the group consisting of:

TABLE-US-00004 [0145] GUUC, (No. 1) GUCA, (No. 2) GCUC, (No. 3) GUUG, (No. 4) GUUU, (No. 5) GGUU, (No. 6) GUGU, (No. 7) GGUC, (No. 8) GUCU, (No. 9) GUCC, (No. 10) GCUU, (No. 11) UUGU, (No. 12) UGUC, (No. 13) CUGU, (No. 14) CGUC, (No. 15) UGUU, (No. 16) GUUA, (No. 17) UGUA, (No. 18) UUUC, (No. 19) UGUG, (No. 20) GGUA, (No. 21) GUCG, (No. 22) UUUG, (No. 23) UGGU, (No. 24) GUGG, (No. 25) GUGC, (No. 26) GUAC, (No. 27) GUAU, (No. 28) UAGU, (No. 29) GUAG, (No. 30) UUCA, (No. 31) UUGG, (No. 32) UCUC, (No. 33) CAGU, (No. 34) UUCG, (No. 35) CUUC, (No. 36) GAGU, (No. 37) GGUG, (No. 38) UUGC, (No. 39) UUUU, (No. 40) CUCA, (No. 41) UCGU, (No. 42) UUCU, (No. 43) UGGC, (No. 44) CGUU, (No. 45) CUUG, (No. 46) UUAC, (No. 47)

[0146] wherein the nucleotide sequences of the motifs are 5'→3', [0147] wherein the oligonucleotide is between 6 and 64, preferably between 12 and 50, more preferably between 14 and 40, even more preferably between 16 and 36, and most preferably between 18 and 25 nucleotides in length, [0148] wherein at least one strand of the RNA oligonucleotide has an IFN-α score of at least 23 when n=6; at least 26 when n=7; at least 28 when n=8; at least 30 when n=9; at least 1.4909×n+22.014 when n is greater than 9, wherein the IFN-α score is assigned according to the "addition method" as described above, and wherein n is the length of the oligonucleotide, [0149] or wherein at least one strand of the RNA oligonucleotide has an IFN-α score of at least 0.58, wherein the IFN-α score is assigned according to the "simplified method" as described above, [0150] (b) preparing the RNA oligonucleotide of step (a); and [0151] (c) optionally testing the immunostimulatory activity of the RNA oligonucleotide prepared in step (b) according to the method of determining the immunostimulatory activity as described above; and [0152] (d) further optionally modifying the oligonucleotide to optimize the immunostimulatory activity.

[0153] The RNA oligonucleotide can be single-stranded, double-stranded or partially double-stranded.

[0154] The RNA oligonucleotide can have other functionalities such as the gene silencing activity.

[0155] The methods provided by the present application can be used to prepare immunostimulatory RNA oligonucleotides, siRNA, shRNA or antisense RNA with high or low immunostimulatory activity.

[0156] Some of the RNA oligonucleotides which have low immunostimulatory activity, i.e., the non-immunostimulatory oligonucleotides, may in fact have inhibitory activity against immune activation. Such an immunoinhibitory oligonucleotide may be able to prevent immune activation induced by an immunostimulatory oligonucleotide when used in combination.

[0157] RNA oligonucleotides can be prepared by methods including, but are not limited to, chemical synthesis, in vitro and in vivo transcription from linear templates (e.g., PCR product) and circular templates (e.g., viral or non-viral vectors).

Method for Preparing siRNA Having High or Low Immunostimulatory Activity

[0158] The present invention provides a method for preparing an siRNA having gene silencing activity for a target gene and having immunostimulatory activity, in particular, IFN-α-inducing activity, comprising the steps of: [0159] (a) identifying all potential siRNA antisense sequences for a target mRNA; [0160] (b) identifying antisense sequences that have gene silencing activity; [0161] (c) predicting the immunostimulatory activity for the antisense sequences identified in step (b) and their complementary (i.e., sense) sequences; [0162] (d) identifying siRNA wherein at least one of the sense and antisense sequences has an IFN-α score of at least 1.4909×n+31,014, wherein the IFN-α score is assigned according to the "addition method" described above, wherein n is the length of the sequence and n is between 19 and 25; [0163] (e) decreasing the IFN-α score threshold by 1 if no siRNA is identified in step (d), until at least one siRNA is identified; [0164] (f) preparing the siRNA identified in step (d) or (e); [0165] (g) optionally testing the gene silencing and/or the immunostimulatory activity of the siRNA prepared in (f); [0166] (h) further optionally modify the siRNA prepared in (f) to optimize the gene silencing and/or immunostimulatory activity.

[0167] The present invention also provides an alternative method for preparing siRNA with gene silencing activity and immunostimulatory activity, comprising the steps of: [0168] (a) identifying all potential siRNA antisense sequences for a target mRNA; [0169] (b) predicting the immunostimulatory activity for all of the antisense sequences identified in (a) and their complementary (i.e., sense) sequences; [0170] (c) identifying 10 siRNA with the highest IFN-α scores, wherein the IFN-α score is assigned according to the "addition method" described above, wherein the IFN-α score of the siRNA is the higher of the two scores for the sense and the antisense strand; [0171] (d) identifying siRNA with gene silencing activity among the 10 siRNA identified in step (c); [0172] (e) identifying 10 siRNA with the next highest IFN-α scores if no siRNA can be identified in step (d); repeat steps (d) and (e) until at least one siRNA is identified; [0173] (f) preparing the siRNA identified in step (d) or (e); [0174] (g) optionally testing the gene silencing and/or the immunostimulatory activity of the siRNA prepared in (f); [0175] (h) further optionally modify the siRNA prepared in (f) to optimize the gene silencing and/or immunostimulatory activity.

[0176] The present invention further provides a method for preparing an siRNA having gene silencing activity for a target gene and having low (or minimal) immunostimulatory activity, in particular, IFN-α-inducing activity, comprising the steps of: [0177] (a) identifying all potential siRNA antisense sequences for a target mRNA; [0178] (b) identifying antisense sequences that have gene silencing activity; [0179] (c) predicting the immunostimulatory activity for the antisense sequences identified in step (b) and their complementary (i.e., sense) sequences; [0180] (d) identifying siRNA wherein both the sense and the antisense sequences have an IFN-α score of at most 0.6075×n-9.9484, wherein the IFN-α score is assigned according to the "addition method" described above, wherein n is the length of the sequence and n is between 19 and 25; [0181] (e) increasing the IFN-α score threshold by 1 if no siRNA is identified in step (b), until at least one siRNA is identified; [0182] (f) preparing the siRNA identified in step (d) or (e); [0183] (g) optionally testing the gene silencing and/or the immunostimulatory activity of the siRNA prepared in (f); [0184] (h) further optionally modify the siRNA prepared in (f) to optimize the gene silencing activity and/or to minimize the immunostimulatory activity.

[0185] The present invention also provide an alternative method for preparing siRNA with gene silencing activity and low (or minimal) immunostimulatory activity, comprising the steps of: [0186] (a) identifying all potential siRNA antisense sequences for a target mRNA; [0187] (b) predicting the immunostimulatory activity for all of the antisense sequences identified in (a) and their complementary (i.e., sense) sequences; [0188] (c) identifying 10 siRNA with the lowest IFN-α scores, wherein the IFN-α score is assigned according to the "addition method" described above, wherein the IFN-α score of the siRNA is the higher of the two scores for the sense and the antisense strand; [0189] (d) identifying siRNA with gene silencing activity among the 10 siRNA identified in step (c); [0190] (e) identifying 10 siRNA with the next lowest IFN-α scores if no siRNA can be identified in step (d); repeat steps (d) and (e) until at least one siRNA is identified; [0191] (f) preparing the siRNA identified in step (d) or (e); [0192] (g) optionally testing the gene silencing and/or the immunostimulatory activity of the siRNA prepared in (f); [0193] (h) further optionally modify the siRNA prepared in (f) to optimize the gene silencing activity and/or to minimize the immunostimulatory activity.

[0194] Candidate siRNA with gene silencing activity for a given gene can be identified using methods known to those skilled in the art, including, but not limited to, commercial engines such as those available from Dharmacon, Qiagen, Invitrogen. Furthermore, the gene silencing activity of an siRNA may be predicted using the algorithm of Reynolds et al. (2004, Nat Biotechnol 22:326-330) and may be determined experimentally.

[0195] The gene silencing activity of an siRNA can be determined experimentally by methods well known in the art. For Example, the RNA oligonucleotide may be introduced into a cell by a method known in the art such as transfection and transduction; the mRNA level of the target gene can be determined by routine methods such as Northern blot analysis, quantitative PCR, RNase protection assay, and branching DNA; and the protein expression level can be determined by routine methods such as Western blotting, ELISA, and biological activity assays specific to the target protein. Furthermore, the mRNA level of all known and hypothetical genes can be determined at the global level using the microarray technology. Technologies in the field of proteonomics allow for the protein levels of a large number of genes to be determined at the global level as well.

[0196] The same methods may be used for preparing an shRNA which comprises the sense and the antisense sequences of an siRNA identified by the above methods and a loop sequence. The preferred loop sequences for shRNA are known to those skilled in the art.

[0197] The same methods may also be used for preparing antisense RNA wherein only one strand, the antisense strand, needs to be identified and prepared.

[0198] The siRNA, shRNA and antisense RNA can be prepared by methods including, but are not limited to, chemical synthesis, in vitro and in vivo transcription from PCR products and viral or non-viral vectors.

Immunostimulatory RNA Oligonucleotides

[0199] The present invention provides an immunostimulatory RNA oligonucleotide having immunostimulatory activity, in particular, IFN-α-inducing activity, comprising at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, and most preferably at least six, of the motifs selected from the group consisting of GUY, GUNY, and GNUY, wherein Y is a pyrimidine (either a U or a C), wherein N is any one of nucleotides A, C, G, and U, and wherein the nucleotide sequences of the motifs are 5'→3'.

[0200] In one embodiment, the immunostimulatory RNA oligonucleotide is between 6 and 64, preferably between 12 and 50, more preferably between 14 and 40, even more preferably between 16 and 36, and most preferably between 18 and 25 nucleotides in length.

[0201] In a further embodiment, at least one strand of the immunostimulatory RNA oligonucleotide has an IFN-α score of at least 23 when n=6; at least 26 when n=7; at least 28 when n=8; at least 30 when n=9; at least 1.4909×n+22.014 when n is greater than 9, wherein the IFN-α score is assigned according to the "addition method" described above, wherein n is the length of the oligonucleotide, provided that the oligonucleotide of the present invention is not 5'-UUGAUGUGUUUAGUCGCUA-3' (Judge et al. 2005, Nat Biotechnol 23:457-462), 5'-GCACCACUAGUUGGUUGUC-3' (Sioud 2005, J Mol Biol 348:1079-1090), 5'-GUUGUAGUUGUACUCCAGC-3' (Sioud), 5'-GCCCGUCUGUUGUGUGACUC-3' (Heil et al. 2004 Science 303:1526-1529), 5'-GUCUGUUGUGUG-3' (Heil, et al.), 5'-GUUGUGGUUGUGGUUGUG-3' (WO 03/086280).

[0202] In an alternative embodiment, at least one strand of the immunostimulatory RNA oligonucleotide has an IFN-α score of at least 0.58, wherein the IFN-α score is assigned according to the "simplified method" described above, provided that the oligonucleotide of the present invention is not 5'-UUGAUGUGUUUAGUCGCUA-3' (Judge et al. 2005, Nat Biotechnol 23:457-462), 5'-GCACCACUAGUUGGUUGUC-3' (Sioud 2005, J Mol Biol 348:1079-1090), 5'-GUUGUAGUUGUACUCCAGC-3' (Sioud), 5'-GCCCGUCUGUUGUGUGACUC-3' (Heil et al. 2004 Science 303:1526-1529), 5'-GUCUGUUGUGUG-3' (Heil, et al.), 5'-GUUGUGGUUGUGGUUGUG-3' (WO 03/086280).

[0203] The present invention further provides an immunostimulatory RNA oligonucleotide having immunostimulatory activity, in particular, IFN-α-inducing activity, comprising at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, and most preferably at least six, of the 4-nucleotide (4-mer) motifs selected from the group consisting of:

TABLE-US-00005 GUUC, (No. 1) GUCA, (No. 2) GCUC, (No. 3) GUUG, (No. 4) GUUU, (No. 5) GGUU, (No. 6) GUGU, (No. 7) GGUC, (No. 8) GUCU, (No. 9) GUCC, (No. 10) GCUU, (No. 11) UUGU, (No. 12) UGUC, (No. 13) CUGU, (No. 14) CGUC, (No. 15) UGUU, (No. 16) GUUA, (No. 17) UGUA, (No. 18) UUUC, (No. 19) UGUG, (No. 20) GGUA, (No. 21) GUCG, (No. 22) UUUG, (No. 23) UGGU, (No. 24) GUGG, (No. 25) GUGC, (No. 26) GUAC, (No. 27) GUAU, (No. 28) UAGU, (No. 29) GUAG, (No. 30) UUCA, (No. 31) UUGG, (No. 32) UCUC, (No. 33) CAGU, (No. 34) UUCG, (No. 35) CUUC, (No. 36) GAGU, (No. 37) GGUG, (No. 38) UUGC, (No. 39) UUUU, (No. 40) CUCA, (No. 41) UCGU, (No. 42) UUCU, (No. 43) UGGC, (No. 44) CGUU, (No. 45) CUUG, (No. 46) UUAC, (No. 47)

wherein the nucleotide sequences of the motifs are 5'→3', wherein the oligonucleotide is between 6 and 64, preferably between 12 and 50, more preferably between 14 and 40, even more preferably between 16 and 36, and most preferably between 18 and 25 nucleotides in length, wherein at least one strand of the RNA oligonucleotide has an IFN-α score of at least 23 when n=6; at least 26 when n=7; at least 28 when n=8; at least 30 when n=9; at least 1.4909×n+22.014 when n is greater than 9, wherein the IFN-α score is assigned according to the "addition method" described above, or wherein at least one strand of the RNA oligonucleotide has an IFN-α score of at least 0.58, wherein the IFN-α score is assigned according to the "simplified method" described above, wherein n is the length of the oligonucleotide, and wherein the oligonucleotide is not 5'-UUGAUGUGUUUAGUCGCUA-3' (Judge et al. 2005, Nat Biotechnol 23:457-462), 5'-GCACCACUAGUUGGUUGUC-3' (Sioud 2005, J Mol Biol 348:1079-1090), 5'-GUUGUAGUUGUACUCCAGC-3' (Sioud), 5'-GCCCGUCUGUUGUGUGACUC-3' (Heil et al. 2004 Science 303:1526-1529), 5'-GUCUGUUGUGUG-3' (Heil, et al.), 5'-GUUGUGGUUGUGGUUGUG-3' (WO 03/086280).

[0204] In one embodiment, the 4mer motifs are selected from the group consisting of No. 1-19, No. 1-18, No. 1-17, No. 1-16, preferably, No. 1-15, No. 1-14, No. 1-13, No. 1-12, more preferably, No. 1-11, No. 1-10, No. 1-9, No. 1-8, No. 1-7, even more preferably, No. 1-6, No. 1-5, No. 1-4, No. 1-3, most preferably, No. 1-2 of the 4mer motifs.

[0205] The immunostimulatory RNA oligonucleotide of the invention may comprise one or more copies of the same 4mer motif, or one or more copies of different 4mer motifs.

[0206] The present invention also provide an immunostimulatory RNA oligonucleotide having immunostimulatory, in particular, IFN-α-inducing activity, comprising at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, most preferably at least six, of the 4mer motifs selected from the group consisting of No. 1-6, preferably No. 1-5, No. 1-4, No. 1-3, more preferably No. 1-2 of the 4mer motifs, wherein the spacer nucleotides which are not part of any of the 4mer motif(s) are identical, and wherein the spacer nucleotide is selected from the group consisting of A, T, C, G and variants and derivatives thereof.

[0207] In one embodiment, the spacer nucleotide is A or a derivative thereof.

[0208] In one embodiment, the immunostimulatory RNA oligonucleotide of the invention can comprise one or more copies of one type of 4mer motif (e.g., GUUC) on a poly A backbone. Examples of such an oligonucleotide includes, but are not limited to:

TABLE-US-00006 AAAAAAAGUUCAAAAAAAA AAAGUUCAAAAAAAAAAAA AAAAAAAAAAAAGUUCAAA AAAGUUCAAAAAGUUCAAA GUUCAAAGUUCAAAGUUCA

[0209] In another embodiment, the immunostimulatory RNA oligonucleotide of the invention can comprise one or more copies of more than one type of 4mer motif (e.g., GUUC GUCA, GCUC, GUUG, GUUU, GGUU) on a poly A backbone. Examples of such an oligonucleotide includes, but are not limited to:

TABLE-US-00007 AGUUCAAAGUCAAAAGCUC AGUUCAGUUCAAGUCAAAGCUC AAAGUUCAAAGUCAAAAGCUCAAAGUUGAAAGUUUAAAGGUUAAA

[0210] The more than one 4mer motifs in an immunostimulatory RNA oligonucleotide may overlap. For example, AAAAGUUGCUCAAAAAA.

[0211] Examples of immunostimulatory RNA oligonucleotides of the invention include, but are not limited to:

TABLE-US-00008 aaaguucaaaaaaguucaaa (SEQ ID NO: 391) aaagucaaaaaaaguucaaa (SEQ ID NO: 392) aaagcucaaaaaaguucaaa (SEQ ID NO: 393) aaaguugaaaaaaguucaaa (SEQ ID NO: 394) aaaguuuaaaaaaguucaaa (SEQ ID NO: 395) aaagguuaaaaaaguucaaa (SEQ ID NO: 396) aaaguguaaaaaaguucaaa (SEQ ID NO: 397) aaaggucaaaaaaguucaaa (SEQ ID NO: 398) aaagucuaaaaaaguucaaa (SEQ 1D NO: 399) aaaguccaaaaaaguucaaa (SEQ ID NO: 400) aaaguucaaaaaagucaaaa (SEQ ID NO: 401) aaagucaaaaaaagucaaaa (SEQ ID NO: 402) aaagcucaaaaaagucaaaa (SEQ ID NO: 403) aaaguugaaaaaagucaaaa (SEQ ID NO: 404) aaaguuuaaaaaagucaaaa (SEQ ID NO: 405) aaagguuaaaaaagucaaaa (SEQ ID NO: 406) aaaguguaaaaaagucaaaa (SEQ ID NO: 407) aaaggucaaaaaagucaaaa (SEQ ID NO: 408) aaagucuaaaaaagucaaaa (SEQ ID NO: 409) aaaguccaaaaaagucaaaa (SEQ ID NO: 410) aaaguucaaaaaagcucaaa (SEQ ID NO: 411) aaagucaaaaaaagcucaaa (SEQ ID NO: 412) aaagcucaaaaaagcucaaa (SEQ ID NO. 413) aaaguugaaaaaagcucaaa (SEQ ID NO: 414) aaaguuuaaaaaagcucaaa (SEQ ID NO: 415) aaagguuaaaaaagcucaaa (SEQ ID NO: 416) aaaguguaaaaaagcucaaa (SEQ ID NO: 417) aaaggucaaaaaagcucaaa (SEQ ID NO: 418) aaagucuaaaaaagcucaaa (SEQ ID NO: 419) aaaguccaaaaaagcucaaa (SEQ ID NO: 420) aaaguucaaaaaaguugaaa (SEQ ID NO: 421) aaagucaaaaaaaguugaaa (SEQ ID NO: 422) aaagcucaaaaaaguugaaa (SEQ ID NO: 423) aaaguugaaaaaaguugaaa (SEQ ID NO: 424) aaaguuuaaaaaaguugaaa (SEQ ID NO: 425) aaagguuaaaaaaguugaaa (SEQ ID NO: 426) aaaguguaaaaaaguugaaa (SEQ ID NO: 427) aaaggucaaaaaaguugaaa (SEQ ID NO: 428) aaagucuaaaaaaguugaaa (SEQ ID NO: 429) aaaguccaaaaaaguugaaa (SEQ ID NO: 430) aaaguucaaaaaaguuuaaa (SEQ ID NO: 431) aaagucaaaaaaaguuuaaa (SEQ ID NO: 432) aaagcucaaaaaaguuuaaa (SEQ ID NO: 433) aaaguugaaaaaaguuuaaa (SEQ ID NO: 434) aaaguuuaaaaaaguuuaaa (SEQ ID NO: 435) aaagguuaaaaaaguuuaaa (SEQ ID NO: 436) aaaguguaaaaaaguuuaaa (SEQ ID NO: 437) aaaggucaaaaaaguuuaaa (SEQ 10 NO: 438) aaagucuaaaaaaguuuaaa (SEQ ID NO: 439) aaaguccaaaaaaguuuaaa (SEQ ID NO: 440) aaaguucaaaaaagguuaaa (SEQ ID NO: 441) aaagucaaaaaaagguuaaa (SEQ ID NO: 442) aaagcucaaaaaagguuaaa (SEQ ID NO: 443) aaaguugaaaaaagguuaaa (SEQ ID NO: 444) aaaguuuaaaaaagguuaaa (SEQ ID NO: 445) aaagguuaaaaaagguuaaa (SEQ ID NO: 446) aaaguguaaaaaagguuaaa (SEQ ID NO: 447) aaaggucaaaaaagguuaaa (SEQ ID NO: 448) aaagucuaaaaaagguuaaa (SEQ ID NO: 449) aaaguccaaaaaagguuaaa (SEQ ID NO: 450) aaaguucaaaaaaguguaaa (SEQ ID NO: 451) aaagucaaaaaaaguguaaa (SEQ ID NO: 452) aaagcucaaaaaaguguaaa (SEQ ID NO: 453) aaaguugaaaaaaguguaaa (SEQ ID NO: 454) aaaguuuaaaaaaguguaaa (SEQ ID NO: 455) aaagguuaaaaaaguguaaa (SEQ ID NO: 456) aaaguguaaaaaaguguaaa (SEQ ID NO: 457) aaaggucaaaaaaguguaaa (SEQ ID NO: 458) aaagucuaaaaaaguguaaa (SEQ ID NO: 459) aaaguccaaaaaaguguaaa (SEQ ID NO: 460) aaaguucaaaaaaggucaaa (SEQ ID NO: 461) aaagucaaaaaaaggucaaa (SEQ ID NO: 462) aaagcucaaaaaaggucaaa (SEQ ID NO: 463) aaaguugaaaaaaggucaaa (SEQ ID NO: 464) aaaguuuaaaaaaggucaaa (SEQ ID NO: 465) aaagguuaaaaaaggucaaa (SEQ ID NO: 466) aaaguguaaaaaaggucaaa (SEQ ID NO: 467) aaaggucaaaaaaggucaaa (SEQ ID NO: 468) aaagucuaaaaaaggucaaa (SEQ ID NO: 469) aaaguccaaaaaaggucaaa (SEQ ID NO: 470) aaaguucaaaaaagucuaaa (SEQ ID NO: 471) aaagucaaaaaaagucuaaa (SEQ ID NO: 472) aaagcucaaaaaagucuaaa (SEQ ID NO: 473) aaaguugaaaaaagucuaaa (SEQ ID NO: 474) aaaguuuaaaaaagucuaaa (SEQ ID NO: 475) aaagguuaaaaaagucuaaa (SEQ ID NO: 476) aaaguguaaaaaagucuaaa (SEQ ID NO: 477) aaaggucaaaaaagucuaaa (SEQ ID NO: 478) aaagucuaaaaaagucuaaa (SEQ ID NO: 479) aaaguccaaaaaagucuaaa (SEQ ID NO: 480) aaaguucaaaaaaguccaaa (SEQ ID NO: 481) aaagucaaaaaaaguccaaa (SEQ ID NO: 482) aaagcucaaaaaaguccaaa (SEQ ID NO: 483) aaaguugaaaaaagucuaaa (SEQ ID NO: 484) aaaguuuaaaaaagucuaaa (SEQ ID NO: 485) aaagguuaaaaaagucuaaa (SEQ ID NO: 486) aaaguguaaaaaaguccaaa (SEQ ID NO: 487) aaaggucaaaaaaguccaaa (SEQ ID NO: 488) aaagucuaaaaaaguccaaa (SEQ ID NO: 489) aaaguccaaaaaaguccaaa (SEQ ID NO: 490)

[0212] In one embodiment, immunostimulatory RNA oligonucleotide of the invention does not have gene silencing activity for any known mammalian gene.

[0213] The immunostimulatory RNA oligonucleotide of the invention may be single-stranded, single-stranded containing a self-complementary sequence and can form a hairpin structure, double-stranded, or partially double-stranded.

[0214] Furthermore, the immunostimulatory RNA oligonucleotide of the invention may be covalently linked to one or more lipophilic groups which enhance the stability and the activity and facilitate the delivery of the RNA oligonucleotides.

[0215] As used herein, the term "lipophilic" or "lipophilic group" broadly refers to any compound or chemical moiety having an affinity for lipids. Lipophilic groups encompass compounds of many different types, including those having aromatic, aliphatic or alicyclic characteristics, and combinations thereof.

[0216] In specific embodiments, the lipophilic group is an aliphatic, alicyclic, or polyalicyclic substance, such as a steroid (e.g., sterol) or a branched aliphatic hydrocarbon. The lipophilic group generally comprises a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may comprise various substituents and/or at least one heteroatom, such as an oxygen atom. Such lipophilic aliphatic moieties include, without limitation, saturated or unsatarated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., the C₁₀ terpenes, C₁₅ sesquiterpenes, C₂₀ diterpenes, C₃₀ triterpenes, and C₄₀ tetraterpenes), and other polyalicyclic hydrocarbons.

[0217] The lipophilic group may be attached by any method known in the art, including via a functional grouping present in or introduced into the RNA oligonucleotide, such as a hydroxy group (e.g., --CO--CH₂--OH). Conjugation of the RNA oligonucleotide and the lipophilic group may occur, for example, through formation of an ether or a carboxylic or carbamoyl ester linkage between the hydroxy and an alkyl group R--, an alkanoyl group RCO--or a substituted carbamoyl group KNHCO--. The alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chained or branched; and saturated or unsaturated). Alkyl group R may be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group, or the like. Preferably, the lipophilic group is conjugated to the 5'-hydroxyl group of the terminal nucleotide. In a preferred embodiment, the liphophilic group is 12-hydroxydodeconoic acid bisdecylamide.

[0218] In another embodiment, the lipophilic group is a steroid, such as sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, without limitation, bile acids (e.g., cholic acid, deoxycholic acid and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol and cationic steroids, such as cortisone.

[0219] In a preferred embodiment, the lipophilic group is cholesterol or a derivative thereof. A "cholesterol derivative" refers to a compound derived from cholesterol, for example by substitution, addition or removal of substituents. The steroid may be attached to the RNA oligonucleotide by any method known in the art. In a preferred embodiment, the liphophilic group is cholesteryl (6-hydroxyhexyl) carbamate.

[0220] In another embodiment, the lipophilic group is an aromatic moiety. In this context, the term "aromatic" refers broadly to mono- and polyaromatic hydrocarbons. Aromatic groups include, without limitation, C₆-C₁₄ aryl moieties comprising one to three aromatic rings, which may be optionally substituted; "aralkyl" or "arylalkyl" groups comprising an aryl group covalently linked to an alkyl group, either of which may independently be optionally substituted or unsubstituted; and "heteroaryl" groups. As used herein, the term "heteroaryl" refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14π electrons shared in a cyclic array; and having, in addition to carbon atoms, between one and about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0221] As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is one having between one and about four, preferably between one and about three, more preferably one or two, non-hydrogen substituents. Suitable substituents include, without limitation, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.

[0222] The lipophilic group can be covalently linked directly or indirectly via a linker to the RNA oligonucleotide. The covalent linkage may or may not comprise a phosphodiester group. And the linker may be of various lengths. The preferred lengths of the linker are known to those skilled in the art and may be determined experimentally.

[0223] In one embodiment, the lipophilic group is covalently linked to the 5' end of at least one strand of the RNA oligonucleotide.

[0224] In addition, the immunostimulatory oligonucleotide of the invention may be coupled to a solid support. By "coupled" it is meant that the oligonucleotide is covalently or non-covalently, directly or indirectly, linked to the solid support. Suitable solid supports include, but are not limited to, silicon wafers, synthetic polymer support such as polystyrene, polypropylene, polyglycidylmethacrylate, substituted polystyrene (e.g., aminated or carboxylated polystyrene, polyacrlamides, polyamides, polyvinylchlorides, etc.), glass, agarose, nitrocellulose, nylon and gelatin nanoparticles. Solid support may enhance the stability and the activity of the oligonucleotide, especially short oligonucleotides less than 16 nucleotides in length.

Immunostimulatory RNA Oligonucleotide Conjugates

[0225] The present invention also provides immunomodulatory RNA oligonucleotide conjugates, comprising an immunomodulatory RNA oligonucleotide and an antigen conjugated to the oligonucleotide. In some embodiments, the antigen is conjugated to the oligonucleotide at a position other than its 3' end. In some embodiments, the antigen produces a vaccine effect.

[0226] The antigen is preferably selected from the group consisting of disease/disorder-related antigens. The disorder may be a cancer, a dermatological disorder, an immune disorder, a metabolic disorder, a neurological disorder, an ocular disease, an infection, or other hereditary and non-hereditary disorders. The antigen may be a protein, a polypeptide, a peptide, a carbohydrate, or a combination thereof.

[0227] The immunostimulatory RNA oligonucleotide may be covalently linked to the antigen, or it is otherwise operatively associated with the antigen. As used herein, the term "operatively associated with" refers to any association that maintains the activity of both the oligonucleotide and the antigen. Non-limiting examples of such operative associations include being part of the same liposome or other such delivery vehicle or reagent. In embodiments wherein the oligonucleotide agent is covalently linked to the antigen, such covalent linkage preferably is at any position on the oligonucleotide that does not interfere with the immunostimulatory activity of the oligonucleotide.

RNA Oligonucleotide with Gene Silencing Activity

[0228] The present invention provides a RNA oligonucleotide with gene silencing activity.

[0229] In one embodiment, the RNA oligonucleotide has both gene silencing activity and immunostimulatory activity, wherein at least one strand of the oligonucleotide has an IFN-α score of at least 1.4909×n+31.014, wherein the IFN-α score is assigned according to the "addition method" described above, wherein n is the length of the oligonucleotide.

[0230] In an alternative embodiment, the RNA oligonucleotide has both gene silencing activity and immunostimulatory activity, wherein at least one strand of the oligonucleotide has an IFN-α score of at least 0.58, wherein the IFN-α score is assigned according to the "simplified method" described above.

[0231] In another embodiment, the RNA oligonucleotide has gene silencing activity and low/minimal immunostimulatory activity, wherein all strand(s) of the oligonucleotide has(have) an IFN-α score of at most 0.6075×n-9.9484, wherein the IFN-α score is assigned according to the "addition method" described above, wherein n is the length of the oligonucleotide.

[0232] In an alternative embodiment, the RNA oligonucleotide has gene silencing activity and low/minimal immunostimulatory activity, wherein all strand(s) of the oligonucleotide has(have) an IFN-α score of at most 0.11, wherein the IFN-α score is assigned according to the "simplified method" described above.

[0233] The RNA oligonucleotide may be an siRNA, an shRNA or an antisense RNA. The siRNA is between 14 and 25 nucleotides in length; the shRNA is between 30 and 70 nucleotides in length; and the antisese RNA is between 14 and 50 nucleotides in length.

[0234] In the case of an immunostimulatory siRNA, the gene silencing activity resides on the antisense strands which is complementary to the target mRNA, whereas the immunostimulatory activity may reside on either the sense or the antisesen strand. In a preferred embodiment, the immunostimulatory activity resides on the sense strand; i.e., the sense strand has an IFN-α score of at least 1.4909×n+31.014 when the IFN-α score is assigned according to the "addition method" described above or the sense strand has and IFN-α score of at least 0.58 when the IFN-α score is assigned according to the "simplified method" described above.

[0235] In the case of an shRNA, the molecule is processed inside a cell to yield a siRNA molecule and the loop (linker) sequence. Therefore, the IFN-α score needs to be calculated not only for the intact molecule (a single-stranded RNA), but also for both strands of the resulting siRNA molecule and the single-stranded loop sequence. The shRNA is considered to have high immunostimulatory activity if at least one of the above-mentioned entities has an IFN-α score above the threshold of 1.4909×n+31.014 according to the "addition method" or 0.58 according to the "simplified method"; the molecule is considered to have low immunostimulatory activity if all of the entities mentioned above have an IFN-α score below the threshold of 0.6075×n-9.9484 according to the "addition method" or 0.11 according to the "simplified method". The gene silencing activity resides in the sequence that corresponds to the antisense strand of the siRNA; whereas the immunostimulatory activity may reside in any part of the molecule. In a preferred embodiment, the immunostimulatory activity resides in the portion of an shRNA that corresponds to the sense strand of the corresponding siRNA or in the loop sequence; i.e., said portion has an IFN-α score of at least 1.4909×n+31.014 according to the "addition method" or 0.58 according to the "simplified method".

[0236] In the case of an immunostimulatory antisense RNA, the immunostimulatory activity and the gene silencing activity have to reside on the same strand.

[0237] The gene silencing RNA oligonucleotide of the invention may be covalently linked to one or more lipophilic groups which enhance the stability and the activity and facilitate the delivery of the RNA oligonucleotides.

Pharmaceutical Compositions

[0238] The present invention provides pharmaceutical compositions comprising one or more of the RNA oligonucleotides of the invention and a pharmaceutically acceptable carrier. The more than one RNA oligonucleotides may have the same, similar, or different functionalities including, but are not limited to immunostimulatory activity and gene silencing activity.

[0239] For example, a RNA oligonucleotide having immunostimulatory activity but lacking gene silencing activity may be combined with a RNA oligonucleotide having gene silencing activity and low immunostimulatory activity in a pharmaceutical composition to achieve both immune activation and gene silencing. Such a combination composition may be useful for treating disorders such as cancers and viral infections. Such a combination composition may be necessary when the two activities cannot be optimized on a single RNA oligonucleotide.

[0240] In one embodiment, the pharmaceutical composition further comprises a RNA complexation agent. In a preferred embodiment, the complexation agent is a polycationic peptide, preferably poly-L-arginine (poly-L-arg). In a preferred embodiment, the polycationic peptide, in particular, poly-L-arg, is at least 24 amino acids in length. The polycationic peptide, in particular, poly-L Arg, may be a heterogeneous mixture of peptides of different lengths.

[0241] The pharmaceutical composition of the invention may further comprises another agent such as an agent that stabilizes the RNA oligonucleotide(s), e.g., a protein that complexes with the oligonucleotide agent to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

[0242] The pharmaceutical composition of the present invention can further comprise one or more additional pharmaceutically active (or therapeutic) agents which are selected from the group consisting of agents that are used for the treatment of cancer, dermatological disorders, immune disorders, metabolic disorders, neurological disorders, ocular diseases, infections, and other hereditary and non-hereditary disorders in a mammal.

[0243] In certain embodiments, the additional pharmaceutically active agent is selected from the group consisting of immunostimulatory RNA oligonucleotides, immunostimulatory DNA oligonucleotides, cytokines, chemokines, growth factors, antibiotics, anti-angiogenic factors, chemotherapeutic agents, anti-viral agents, anti-fungal agents, anti-parasitic agents, and antibodies. In one embodiment, the additional pharmaceutically active agent is natural or recombinant IFN-α polypeptide, or a CpG-containing or non-CpG-containing DNA oligonucleotide capable of inducing IFN-α (see e.g., WO 01/22990, WO 03/101375). In another embodiment, the additional pharmaceutically active agent is natural or recombinant IL-12, or an immunostimulatory RNA oligonucleotide capable inducing IL-12 (see e.g. our co-pending application; Sugiyama et al. 2005, J Immunol 174:2273-2279). In yet another embodiment, the additional pharmaceutically active agent is an anti-angiogenic factor such as vasostatin or an anti-VEGF antibody. In certain embodiments, the additional pharmaceutically active agent is a cancer-specific agent such as Herceptin®, Rituxan®, Gleevec®, Iressa®.

[0244] A formulated oligonucleotide composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the oligonucleotide agent is in an aqueous phase, e.g., in a solution that includes water, this form being the preferred form for administration via inhalation.

[0245] The aqueous phase or the crystalline compositions can be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase), or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the oligonucleotide composition is formulated in a manner that is compatible with the intended method of administration.

[0246] The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular, rectal, vaginal, and topical (including buccal and sublingual) administration. In preferred embodiments, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection. The pharmaceutical compositions can also be administered intraparenchymally, intrathecally, and/or by stereotactic injection.

[0247] For oral administration, the oligonucleotide agent useful in the invention will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.

[0248] Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

[0249] Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

[0250] For intramuscular, intraperitoneal, subcutaneous and intravenous use, the pharmaceutical compositions of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. In a preferred embodiment, the carrier consists exclusively of an aqueous buffer. In this context, "exclusively" means no auxiliary agents or encapsulating substances are present which might affect or mediate uptake of oligonucleotide agent in the cells that harbor the target gene or virus. Such substances include, for example, micellar structures, such as liposomes or capsids, as described below. Although microinjection, lipofection, viruses, viroids, capsids, capsoids, or other auxiliary agents are required to introduce oligonucleotide agent into cell cultures, surprisingly these methods and agents are not necessary for uptake of oligonucleotide agent in vivo. The oligonucleotide agent of the present invention are particularly advantageous in that they do not require the use of an auxiliary agent to mediate uptake of the oligonucleotide agent into the cell, many of which agents are toxic or associated with deleterious side effects. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0251] The pharmaceutical compositions can also include encapsulated formulations to protect the oligonucleotide agent against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodetradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811; PCT publication WO 91/06309; and European patent publication EP-A-43075.

[0252] In general, a suitable dose of a RNA oligonucleotide will be in the range of 0.001 to 500 milligrams per kilogram body weight of the recipient per day (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 100 milligrams per kilogram, about 1 milligrams per kilogram to about 75 milligrams per kilogram, about 10 micrograms per kilogram to about 50 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). The pharmaceutical composition may be administered once per day, or the oligonucleotide agent may be administered as two, three, four, five, six or more sub-doses at appropriate intervals throughout the day. In that case, the oligonucleotide agent contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the oligonucleotide agent over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0253] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the infection or disease/disorder, previous treatments, the general health and/or age of the subject, and other diseases/disorders present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual RNA oligonucleotide agent encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

[0254] Toxicity and therapeutic efficacy of the RNA oligonucleotide and the pharmaceutical composition of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. oligonucleotide agents that exhibit high therapeutic indices are preferred.

[0255] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosages of compositions of the invention are preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any oligonucleotide agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the oligonucleotide agent or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test oligonucleotide agent which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0256] The administering physician can adjust the amount and timing of the administration of the pharmaceutical composition of the invention on the basis of results observed using standard measures of efficacy known in the art or described herein.

Use of the RNA Oligonucleotide for Inducing an Immune Response

[0257] The present application provides the use of the immunostimulatory RNA oligonucleotide of the invention for the preparation of a pharmaceutical composition for inducing an immune response in a mammal.

[0258] Inducing an immune response means initiating or causing an increase in one or more of B-cell activation, T-cell activation, natural killer cell activation, activation of antigen presenting cells (e.g., B cells, dendritic cells, monocytes and macrophages), cytokine production, chemokine production, specific cell surface marker expression, in particular, expression of co-stimulatory molecules. In one aspect, such an immune response involves the production of type I IFN, in particular, IFN-α, in cells such as PDC.

Use of the RNA Oligonucleotide for Treating Diseases/Disorders

[0259] The present invention provides the use of the immunostimulatory RNA oligonucleotide of the invention for the preparation of a pharmaceutical composition for preventing and/or treating a disorder selected from immune disorders, infections, and cancers in a mammal, wherein the induction of an immune response is beneficial to the mammal.

[0260] The present invention also provides the use of the RNA oligonucleotide of the invention which has both immunostimulatory activity and gene silencing activity for the preparation of a pharmaceutical composition for preventing and/or treating a disorder selected from infections and cancers in a mammal, wherein the induction of an immune response together with the downregulation of a disorder-related gene are beneficial to the mammal.

[0261] The present invention further provides the use of the RNA oligonucleotide of the invention which has gene silencing activity and low/minimal immunostimulatory activity for the preparation of a pharmaceutical composition for preventing and/or treating a disorder in a mammal caused by the expression or overexpression of a disorder-related gene, wherein the induction of an immune disorder it to be avoided. The disorder may be selected from cancer, dermatological disorders, immune disorders, metabolic disorders, neurological disorders, ocular diseases, infections, and other hereditary and non-hereditary disorders.

[0262] The immune disorders include, but are not limited to, allergy, autoimmune disorders, inflammatory disorders, and immunodeficiency.

[0263] Allergies include, but are not limited to, food allergies and respiratory allergies.

[0264] Autoimmune diseases include, but are not limited to, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, automimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing, loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.

[0265] Inflammatory disorders include, without limitation, airway inflammation which includes, without limitation, asthma.

[0266] Immunodeficiencies include, but are not limited to, spontaneous immunodeficiency, acquired immunodeficiency (including AIDS), drug-induced immunodeficiency (such as that induced by immunosuppressants used in transplantation and chemotherapeutic agents used for treating cancer).

[0267] In one embodiment, the immune disorders include those caused by pathological Th2 responses.

[0268] The infections include, but are not limited to viral infections, bacterial infections, anthrax, parasitic infections, fungal infections and prion infection.

[0269] Viral infections include, but are not limited to, infection by hepatitis C, hepatitis B, herpes simplex virus (HSV), HIV-AIDS, poliovirus, and smallpox virus. Examples of (+) strand RNA viruses which can be targeted for inhibition include, without limitation, picornaviruses, caliciviruses, nodaviruses, coronaviruses, arteriviruses, flaviviruses, and togaviruses. Examples of picornaviruses include enterovirus (poliovirus 1), rhinovirus (human rhinovirus 1A), hepatovirus (hepatitis A virus), cardiovirus (encephalomyocarditis virus), aphthovirus (foot-and-mouth disease virus O), and parechovirus (human echovirus 22). Examples of caliciviruses include vesiculovirus (swine vesicular exanthema virus), lagovirus (rabbit hemorrhagic disease virus), "Norwalk-like viruses" (Norwalk virus), "Sapporo-like viruses" (Sapporo virus), and "hepatitis E-like viruses" (hepatitis E virus). Betanodavirus (striped jack nervous necrosis virus) is the representative nodavirus. Coronaviruses include coronavirus (avian infections bronchitis virus) and torovirus (Berne virus). Arterivirus (equine arteritis virus) is the representative arteriviridus. Togavirises include alphavirus (Sindbis virus) and rubivirus (Rubella virus).

[0270] Finally, the flaviviruses include flavivirus (Yellow fever virus), pestivirus (bovine diarrhea virus), and hepacivirus (hepatitis C virus).

[0271] In certain embodiments, the viral infections are selected from chornic hepatitis B, chornic hepatitis C, HIV infection, RSV infection, HSV infection, VSV infection, CMV infection, and influenza infection.

[0272] Cancers include, but are not limited to biliary tract cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasm, leukemia, lymphoma, liver cancer, lung cancer, melanoma, myelomas, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer and renal cancer.

[0273] In certain embodiments, cancers are selected from hairy cell leukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia, chronic myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma, breast carcinoma, ovarian carcinoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, basaliom, colon carcinoma, cervical dysplasia, and Kaposi's sarcoma (AIDS-related and non-AIDS related).

[0274] Dermatological disorders include, but are not limited to, psoriasis, acne, rosacea, eczema, molluscum contagious, seborrheic keratosis, actinic keratosis, verruca vulgaris.

[0275] Metabolic disorders include, but are not limited to, diabetes and obesity.

[0276] Ocular diseases include, but are not limited to, age-related macular degeneration.

[0277] Neurological disorders include, but are not limited to, Alzeimer` disease, Huntington's disease, Parkinson's disease, and spinal cord injury.

[0278] Hereditary diseases include, but are not limited to, cystic fibrosis.

[0279] In one embodiment, the pharmaceutical composition is for administration selected from the group consisting of airway, oral, ocular, parenteral (including intraveneous, intradermal, intramuscular, intraperitoneal, and subcutaneous), rectal, vaginal and topical (including buccal and sublingual) administration.

[0280] In another embodiment, the pharmaceutical composition is for use in combination with one or more treatments of disorders selected from treatments for cancer, dermatological disorders, immune disorders, metabolic disorders, neurological disorders, ocular diseases, infections, and other hereditary and non-hereditary disorders in a mammal. Such treatments include, but are not limited to, surgery, chemotherapy, radiation therapy, and the administration of pharmaceutically active (or therapeutic) agents such as immunostimulatory RNA oligonucleotides, immunostimulatory DNA oligonucleotides, cytokines, chemokines, growth factors, antibiotics, anti-angiogenic factors, chemotherapeutic agents, anti-viral agents, anti-fungal agents, anti-parasitic agents, and antibodies.

[0281] In one embodiment, pharmaceutically active agent is natural or recombinant IFN-α polypeptide, or a CpG-containing or non-CpG-containing DNA oligonucleotide capable of inducing IFN-α (see e.g., WO 01/22990, WO 03/101375). In another embodiment, the pharmaceutically active agent is natural or recombinant IL-12, or an immunostimulatory RNA oligonucleotide capable inducing IL-12 (see e.g. our co-pending application; Sugiyama et al. 2005, J Immunol 174:2273-2279). In yet another embodiment, the pharmaceutically active agent is an anti-angiogenic factor such as vasostatin or an anti-VEGF antibody. In certain embodiments, the pharmaceutically active agent is a cancer-specific agent such as Herceptin, Rituxan, Gleevec, Iressa.

[0282] Mammals include, but are not limited to, rats, mice, cats, dogs, horses, sheep, cattle, cows, pigs, rabbits, non-human primates, and humans. In a preferred embodiment, the mammal is human.

In Vitro Method for Inducing IFN-α Production

[0283] The present invention provides an in vitro method of inducing IFN-α production in a mammalian cell, comprising the steps of: [0284] (a) complexing an immunostimulatory RNA oligonucleotide of the invention with a complexation agent; and [0285] (b) contacting the cell with the complex prepared in step (a).

[0286] The mammalian cell is capable of producing IFN-α. In one embodiment, the mammalian cell expresses TLR7, TLR8, or both TLR7 and TLR8. The mammalian cells include, but are not limited to, peripheral blood mononuclear cells (PBMC), plasmacytoid dendritric cells (PDC), myeloid dendritic cells (MDC), B cells, macrophages, monocytes, natural killer cells, granulocytes, endothelial cells, cell lines such as THP1, and cells containing exogenous DNA which directs the expression of TLR7 or TLR8 or both TLR7 or TLR8 such as transfected CHO, HEK293 or COS cells.

[0287] In one embodiment of the invention, the complexation agent is a polycationic peptide, preferably poly-L-arginine (poly-L-arg). In one embodiment, the polycationic peptide, in particular, poly-L-arg, is at least 24 amino acids in length. The polycationic peptide, in particular, poly-L Arg, may be a heterogeneous mixture of peptides of different lengths.

[0288] In a preferred embodiment, the mammal is human.

In Vitro Method for Activating Mammalian Dendritic Cells

[0289] The present application provides an in vitro method of activating mammalian dendritic cells, comprising the steps of: [0290] (a) complexing an immunostimulatory RNA oligonucleotide of the invention with a complexation agent; [0291] (b) contacting dendritic cells isolated from a donor mammal with the complexed RNA oligonucleotide; and [0292] (c) contacting the dendritic cells with an antigen.

[0293] In one embodiment of the invention, the complexation agent is a polycationic peptide, preferably poly-L-arginine (poly-L-arg). In one embodiment, the polycationic peptide, in particular, poly-L-arg, is at least 24 amino acids in length. The polycationic peptide, in particular, poly-L Arg, may be a heterogeneous mixture of peptides of different lengths.

[0294] In one embodiment, the antigen is a disease/disorder-related antigen. The disorder may be a cancer, a dermatological disorder, an immune disorder, a metabolic disorder, a neurological disorder, an ocular disease, an infection, or other hereditary and non-hereditary disorders. The antigen may be a protein, a polypeptide, a peptide, a carbohydrate, or a combination thereof.

[0295] The present invention further provides the use of the in vitro activated dendritic cells for the preparation of a medicament for inducing an immune response in a mammal, wherein the in vitro activated dendritic cells are for transfer into a recipient that is the same or different from the donor.

[0296] In a preferred embodiment, the mammal is human.

[0297] The present invention is illustrated by the following examples.

EXAMPLES

Example 1

Stimulation of PBMC Using Poly-L-Arginine-Complexed Single-Stranded RNA Oligonucleotides

[0298] First we sought to develop an assay system for the comparison of the IFN-a inducing capacity of single-stranded RNA (ssRNA) oligonucleotides on a large scale. Peripheral blood mononuclear cells (PBMC) were selected as a biological system that is relevant for future clinical application. Within PBMC, the plasmacytoid dendritic cell (PDC) is responsible for IFN-α production upon stimulation with ssRNA oligonucleotides. In order to stimulate IFN-α production in PDC, ssRNA oligonucleotides require transfection. While cationic lipids such as lipofectamine are well-established to support IFN-α production in isolated PDC, the induction of IFN-α in PBMC was not satisfactory. In order to improve transfection of RNA in PBMC we tested a number of cationic polypeptides including poly-L-Lys, poly-His and poly-L-arg. Among these three, poly-L-arg (14 kD) was the most potent polycationic peptide and was even more potent than the cationic lipid lipofectamine to support the stimulatory activity of an established immunostimulatory RNA oligonucleotide (RNA9.2sense) (FIG. 1). Within PBMC, the source of IFN-α production were found to be PDC, since PDC depletion abrogated IFN-α production (data not shown). Importantly, poly-L-arg complexation maintained the same sequence dependency (sense versus antisense strand) of ssRNA oligonucleotide-mediated IFN-α induction that was previously seen for lipofectamine (Homung et al. 2005, Nat Med 11:263-270). This experimental system was found to be robust, since adding different concentrations of RNA oligonucleotide from 80% to 120% had little effect on the amount of IFN-α induced. This is in marked contrast to the use of cationic lipids that require exact adjustment of the net charges to avoid cytotoxicity. Together these data indicated that poly-L-arg complexation is an effective and reliable system to screen for ssRNA oligonucleotide-induced IFN-α production in PBMC.

[0299] The experimental procedures are described in more detail in the following:

PBMC Isolation

[0300] Human PBMC were prepared from whole blood donated by young healthy donors. PBMC were obtained from whole blood by Ficoll-Hypaque density gradient centrifugation (Biochrom, Berlin, Germany). PBMC were cultured in RPMI-Medium (Biochrom, Berlin, Germany) supplemented with human AB-Serum (2 vol %, Firma, Germany) at a density of 2×10⁶ PBMC/ml. Subsequently PBMC were plated into 96-well flat bottom wells at 200 μl/well. Cells were kept on ice until stimulation.

Resuspension and Annealing of ssRNA Oligonucleotides

[0301] Lyophilized ssRNA oligonucleotides (Eurogentec, Belgium) were resuspended in sterile, RNase free water at a concentration of 620 μg/ml. Subsequently 40 μl of the ssRNA oligonucleotides, 40 μl sterile, RNase free water and 20 μl 5× annealing buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA) were combined. This resulted in a final volume of 100 μl with a final concentration of the ssRNA oligonucleotide of 248 μg/ml. Subsequently the solution was incubated at 95° C. for 2 minutes and cooled down to 20° C. with a linear decrease in temperature in 60 minutes (-1.25° C./1 minute). The solution was then stored at -80° C. prior to usage (ssRNA oligonucleotide stock solution).

Stimulation

[0302] If not otherwise indicated, ssRNA oligonucleotides were complexed using the polycationic polymer Poly-L-arginine Hydrochloride with a molecular weight of 5.000-15.000 (Prod. Number: P4663, Sigma, Munich, Germany). Lyophilized Poly-L-arginine

[0303] Hydrochloride was dissolved in sterile, RNase free water at a concentration of 2000 μg/ml (Poly-L-arginine stock solution) and subsequently aliquoted and stored at a temperature of -20° C. ssRNA oligonucleotides were complexed using the following protocol:

1. The poly-L-arginine stock solution and the ssRNA oligonucleotide stock solution are thawed to 4° C. 2. Poly-L-arginine is diluted in Phosphate buffered saline to a concentration of 24 μg/ml (1:83.3 dilution from 2000 μg/ml stock solution). 3. Immediately the ssRNA oligonucleotide is added to a final concentration of 14.8 μg/ml (1:16.6 dilution from 248 μg/ml stock solution). 4. Subsequently, the solution is mixed thoroughly by vortexing for 10 seconds. 5. Next, 15 μl of this solution is added immediately to 200 μl PBMC in the 96-well flat bottom plate. The final concentration of poly-L-arginine is 1.67 μg/ml, whereas the final concentration of the ssRNA oligonucleotide is 1.03 μg/ml.

[0304] Throughout all procedures the temperature was kept between 20-25° C. On all 96-well plates PBMC from three individual donors were plated, whereas eleven different ssRNA oligonucleotides were tested. In addition, on each plate the ssRNA oligonucleotide 9.2 sense (5'-AGCUUAACCUGUCCUUCAA-3') was included as a positive control. All tested ssRNA oligonucleotides were run in duplicates. After stimulation cells were cultured at 37° C./5% CO2 for 44 hours.

ELISA

[0305] After 44 hours of cell culture supernatants were collected and stored in three individual aliquots at -80° C. Prior to ELISA-procedures frozen supernatants were thawed at 20-25° C. for two hours. To measure IFN-a the IFN-a module set Bender MedSystems (Prod. Number: BMS216MST, Graz, Austria) was used. This ELISA detects most of IFN-α isoforms at a detection range of 8-500 μg/ml. All ELISA procedures were performed according to manufacturer's recommendations.

Example 2

Rational Design of a 4mer-Motif Library to Screen for Potent Motifs within ssRNA

[0306] Previous experiments have shown that a minimal length of 19 bases is required for maximal IFN-α induction by ssRNA oligonucleotides. Since poly adenosine oligonucleotides proved to be inactive in terms of IFN-a induction, we decided to generate a ssRNA oligonucleotide library by placing putative motifs into the center of poly adenosine RNA oligonucleotides. In a first set of experiments we determined the minimal length of a motif for IFN-α induction in our system. We designed a panel of 19mer poly-adenosine ssRNA oligonucleotides with increasing numbers of uracil in the center: 5'-U-3',5'-UU-3',5'-UUU-3' and 5'-UUUU-3'. These oligonucleotides were compared to the previously published standard ssRNA oligonucleotide RNA9.2sense (5'-AGCUUAACCUGUCCUUCAA-3'): while the 1mer motif 5'-U-3' hardly induced IFN-α (0.8 of RNA9.2sense), considerable amounts of IFN-a were observed for the 2mer motif 5'-UU-3'(10.6% of RNA9.2sense), the 3mer motif 5'-UUU-3' (15.7% of RNA9.2sense) and the 4mer motif 5'-UUUU-3'(50.5% of RNA9.2sense) (FIG. 2). Since the largest increment of IFN-a induction was seen between the 3mer and 4mer motif, we generated a library of ssRNA oligonucleotides comprising all possible 4mer motifs in the centre of a poly adenosine oligonucleotide. In view of the fact that the flanking adenosine residues can be part of a 4mer RNA sequence motif, only 193 ssRNA oligonucleotides (table 1) were needed to cover all 256 possible 4mer motifs.

Example 3

Generation and Processing of Raw Data

[0307] All 193 ssRNA oligonucleotides were tested on PBMC of six individual healthy donors using poly-L-arg for complexation. At 44 hours after stimulation with RNA oligonucleotides, supernatants were collected and IFN-α production was measured by ELISA. Prior to statistical analysis the raw data were processed as follows: for each cell culture plate the mean IFN-α value of the experimental duplicates for each tested ssRNA oligonucleotide were normalized to the ssRNA oligonucleotide RNA9.2sense (5'-AGCUUAACCUGUCCUUCAA-3'). This standard RNA oligonucleotide was included as a positive control on all cell culture plates. Normalization was performed by calculating the ratio of IFN-α induced by the tested oligonucleotide and IFN-α induced by the standard oligonucleotide RNA9.2sense. Thus, for each tested oligonucleotide in an individual donor a mean ratio of IFN-a induction was obtained. In the following, this mean of the ratios is referred to as IFN-a index (one value of IFN-a index per donor). For example testing ssRNA oligonucleotide ANP144 (5'-AAAAAAAGUUGAAAAAAAA-3') in donor 1 gave the mean of the two raw values of the duplicates (IFN-α in supernatant) of 2024 pg/ml, whereas the control oligonucleotide RNA9.2sense (5'-AGCUUAACCUGUCCUUCAA-3') resulted in 1256 pg/ml. The corresponding IFN-α index of oligonucleotide ANP144 for donor 1 was calculated to be 1.61 (=2024 pg/ml divided by 1256 pg/ml).

[0308] Next, the means of all IFN-α indices for every individual donor were calculated. Then the adjusted IFN-α indices were calculated as IFN-α index minus the mean of all IFN-α indices of one individual donor. For example ssRNA oligonucleotide ANP144 of donor 1 (5'-AAAAAAAGUUGAAAAAAAA-3') had an IFN-α index of 1.61, whereas the mean of all IFN-a indices of donor 1 was 0.37. The adjusted IFN-a index of ANP144 was calculated: 1.61 minus 0.37=1.24 (=subtracting 0.37 from). The adjusted IFN-α indices from all six donors were summarized by calculating the means and the corresponding standard error of mean. The data are depicted in ascending order (FIG. 3A, B, C, D). The adjusted IFN-a indices of the top thirty ssRNA oligonucleotides of all six donors were compared using a two-tailed Student's t-test (FIG. 3E). For most combinations tested, a significant difference was observed when the interval between the analyzed pairs was at least six or seven places in the assortment.

[0309] The ssRNA oligonucleotides were split into two groups: group 1 (table 2) comprising all ssRNA oligonucleotides with an IFN-α index that lower than the mean IFN-α index off all ssRNA oligonucleotides (or in other words with a mean of the adjusted IFN-α index below 0) (table 1), and group 2 (table 3) comprising all ssRNA oligonucleotides with an IFN-a index higher than the mean IFN-α index off all tested ssRNA oligonucleotides (or in other words with a mean of the adjusted IFN-α index below 0) (table 1). Thus group 1 contained all ssRNA oligonucleotides with an adjusted IFN-α index below 0, whereas group 2 contained all ssRNA oligonucleotides with an adjusted IFN-a index above 0. Group 1 consisted of 148 ssRNA oligonucleotides (75%), whereas group 2 consisted of 45 ssRNA oligonucleotides (25%).

Example 4

Analyzing the Predictive Value of 1mer, 2mer and 3mer Motifs within the 4mer Motif Matrix

[0310] Next the frequency of 1 mer motifs (5'-X-3'), 2mer motifs (5'-XX-3',5'-X*X-3',5'-X**X-3') and 3mer motifs (5'-XXX-3',5'-XX*X-3',5'-X*XX-3') in ssRNA oligonucleotides with an IFN-a index below the mean IFN-a index (group 1) and above the mean IFN-α index (group 2) was analyzed. Multiple occurrences of a motif within one single ssRNA oligonucleotide was accounted for. For example, the motif 5'-GU-3' is present twice in the ssRNA oligonucleotide ANP142 (5'-AAAAAAAGUGUAAAAAAAA-3', group 2). Consequently the ssRNA oligonucleotide ANP142 contributed two counts for motif 5'-GU-3' within group 2. In order to compare the distribution of a specific motif between the two groups, the relative occurrence of a specific motif was calculated (ratio of the absolute number of occurrence of a specific motif over the total number of occurrence of all motifs). For example the motif 5'-GU-3' was found 7 times in group 1 (total number of 5'-XX-3'-motifs in group 1: 2482) and 33 times in group 2 (total number of 5'-XX-3'-motifs in group 2: 799). Thus the calculated relative occurrence for motif 5'-GU-3' in group 1 was 0.0028, whereas the respective relative occurrence for group 2 was 0.0413. In FIG. 4 the relative occurrence of 1 mer motifs (5'-X-3'), 2mer motifs (5'-XX-3', 5'-X*X-3',5'-X**X-3') and 3mer motifs (5'-XXX-3',5'-XX*X-3',5'-X*XX-3') within the two groups is shown. A significant overrepresentation or underrepresentation of a given motif was analyzed using a chi-square test. The null hypothesis of equal distribution within both groups was rejected when the calculated p-value was below 0.05 (significant differences in distribution are indicated by "*" in FIG. 4).

Example 5

Calculating an Individual IFN-α Score for 1mer-, 2mer- and 3mer-Motifs

[0311] A mean IFN-α index for all possible 1mer motifs (5'-X-3'), 2mer motifs (5'-XX-3',5'-X*X-3', 5'-X**X-3') or 3mer motifs (5'-XXX-3',5'-XX*X-3',5'-X*XX-3') was obtained by calculating a mean IFN-α index of all ssRNA oligonucleotides containing the corresponding motifs. This mean IFN-a index is referred to as the IFN-α score of a given motif. For example the 3mer motif 5'-GUC-3' was contained in ssRNA oligonucleotides ANP 35, 83, 131, 137, 138, 139 and 179 with respective adjusted IFN-α indices of 1.33, 0.68, 0.93, 0.79, 0.44, 0.84, 0.73. The IFN-α score of the 3mer motif 5'-GUC-3' was thus calculated to be 0.82 with a standard error of mean of 0.11. The calculation of the IFN-α score of a motif did not account for the position of the motif within the sequence of the corresponding ssRNA oligonucleotides. Multiple occurrences of one motif within the same ssRNA oligonucleotide was accounted for by adding the corresponding IFN-α index times the number of its occurrence within the oligonucleotide to the calculation of the corresponding IFN-α score of the motif. Consequently an IFN-α score could be assigned to all possible 1 mer motifs (5'-X-3'), 2mer motifs (5'-XX-3',5'-X*X-3',5'-X**X-3') or 3mer motifs (5'-XXX-3',5'-XX*X-3',5'-X*XX-3') (FIG. 5).

Example 6

Predicting the IFN-α Index of ssRNA Oligonucleotides Using the IFN-α Score of 1mer-, 2mer- and 3mer-Motifs

[0312] Next we tested the predictive value of the calculated 1mer-, 2mer- and 3mer-motif IFN-α scores to predict ssRNA oligonucleotides with a low or high IFN-α index. Thus for each ssRNA oligonucleotide the occurrence of a set of motifs was tested and the respective IFN-a scores were assigned to the ssRNA. For example for the panel of 3mer motifs with unspaced sequences (5'-XXX-3') the ssRNA oligonucleotide ANP 35 (5'-AAAAAAAAGUCAAAAAAAA-3') was analyzed the following way:

TABLE-US-00009 3mer motif IFN-α Occurrences within the ssRNA Assigned IFN-α (5'-XXX-3') score oligonucleotide ANP 35 score 5'-AAA-3' -0.0041 12 -0.0503 5'-AAG-3' +0.0990 1 +0.0990 5'-AGU-3' +0.5796 1 +0.5796 5'-GUC-3' +0.8115 1 +0.8115 5'-UCA-3' +0.3668 1 +0.3668 5'-CAA-3' +0.0033 1 +0.0033 overall +1.8099

[0313] All ssRNA oligonucleotides were assigned an individual IFN-a score for all possible motif-combinations (1mer-, 2mer- and 3mer-motifs). Next, the prediction that was obtained by using the assigned IFN-α scores was compared to the actual adjusted IFN-α indices for all ssRNA oligonucleotides and for each motif combination. Data were sorted in ascending order according to the adjusted IFN-α indices. For all predictions, the correlation coefficient was calculated: Using the IFN-α scores of 1 mer motifs (5'-X-3') to predict the actual adjusted IFN-α indices off all ssRNA oligonucleotides a correlation coefficient of 0.53 was obtained (FIG. 6A). When 2mer motifs were used to predict the adjusted IFN-α indices a correlation coefficient of 0.77 was obtained for 5'-XX-3'-motifs, a correlation coefficient of 0.58 for 5'-X*X-3'-motifs and a correlation coefficient of 0.60 for 5'-X**X-3'-motifs (FIG. 6B). Using 3mer motifs to predict the adjusted IFN-α indices, a correlation coefficient of 0.87 was calculated for 5'-XXX-3'-motifs, of 0.80 for 5'-XX*X-3'-motifs and of 0.80 for 5'-X*XX-3'-motifs.

[0314] Next, the most accurate prediction algorithm (using the individual IFN-α scores of the 5'-XXX-3'-motifs) was translated into a point score system. The matrix of the 64 different 3mer motifs (5'-XXX-3') was reduced to encompass only the motifs that were significantly over- or underrepresented in either group 1 or group 2 ssRNA oligonucleotides. Even though only 17 3mer motifs (5'-XXX-3') were left in this matrix, a correlation coefficient of 0.87 could still be calculated when using the respective IFN-α scores to predict the IFN-α indices of the complete ssRNA oligonucleotide library (data not shown). In addition, the respective IFN-α scores of the reduced 3mer motif matrix was translated in into a point score system by assigning a point score to each 3mer motif (FIG. 6D). Using this point score system, a prediction of the measured IFN-α indices of all 193 ssRNA oligonucleotides was calculated with a corresponding correlation coefficient of 0.87 (FIG. 6E).

Example 7

Influence of the Positioning and the Flanking Bases on the IFN-α-Inducing Activity of a Potent 4mer Motif within a 19mer ssRNA Oligonucleotide

[0315] Next we sought to address the impact of moving a potent 4mer motif to either the 5' or the 3' end within a 19mer ssRNA oligonucleotide. A panel of ssRNA oligonucleotides was designed that included the potent 4mer motif 5'-GUCA-3' within a 19mer poly-A ssRNA oligonucleotide (table 4). The positioning of the 4mer motif was chosen to be either 2, 6, 8, 10 or 14 bases from the 5'-end of the ssRNA oligonucleotide. PBMC were stimulated with the respective ssRNA oligonucleotides and IFN-α production was assessed 44 hours after stimulation. Positioning the 4mer motif two bases from the 5'-end of the ssRNA oligonucleotide slightly decreased IFN-α induction by 13%, when compared to the center positioning (8 bases from the 5'-end). A higher decrease in IFN-α production (29%) was seen, when the 4mer motif was positioned on the 3'-end of the ssRNA oligonucleotide (14 bases from the 5'-end). Nevertheless all ssRNA oligonucleotides tested were still more potent in terms of IFN-α induction than the positive control 9.2sense (FIG. 7A).

[0316] In addition, we addressed the influence of flanking bases on the stimulatory activity of the potent 4mer motif 5'-GUCA-3'. A panel of 16 ssRNA oligonucleotides, which included all possible oligonucleotides with permutated bases at the flanking positions to the 5'- and the 3'-end of the central 5'-GUCA-3'-motif (table 5), was tested (FIG. 7B). As soon as the 5'-GUCA-3'-motif was modified with a preceding 5'-C-3', a significant decrease to less than 50% of the original activity was obtained (FIG. 7C). In addition, when a 5'-U-3' preceded the 5'-GUCA-3'-motif, a significant decrease to about 77% of the original activity was observed (FIG. 7C). In contrast, changing the preceding base to a 5'-G-3' did not impact on the stimulatory activity of the 5'-GUCA-3'-motif. The modification of the flanking base to the 3'-end had little impact on the stimulatory activity of the 5'-GUCA-3'-motif. Nevertheless, a slight, yet significant decrease in activity was detected, as soon as the base to the 3'-end was changed into a 5'-C-3'.

Example 8

Predicting the Immunostimulatory Activity of Complex 19mer ssRNA Oligonucleotides Using the 3mer Motif 5'-XXX-3' Based IFN-α Point Score Matrix

[0317] Next, we employed the IFN-α point score matrix to predict the potency of complex (i.e., no longer on a poly A backbone) 19mer ssRNA oligonucleotides in terms of IFN-α induction. Modified versions of RNA9.2sense that have been previously described (Hornung et al. 2005, Nat Med 11:263-270) were used to stimulate PBMC; IFN-α production was measured 44 h after stimulation. In addition, respective sequences were analyzed using the above-described algorithm that is based on the IFN-α score of 3mer motifs 5'-XXX-3'. Both the measured and the predicted data were normalized to the ssRNA oligonucleotide RNA9.2sense (set to 100%). In FIG. 8A both measured data and predicted data are depicted. The correlation coefficient for this analysis was calculated to be 0.84.

Example 9

Validation of the Method of Prediction

[0318] Our method of predicting the immunostimulatory activity of an RNA oligonucleotide is further validated by data disclosed in various publications. To date, four publications describe IFN-α induction by RNA oligonucleotides in the human system: Heil F et al. 2004, Science 303: 1526-1529; Sioud M et al. 2005, J Mol Biol 348: 1079-1090; Homung V et al. 2005, Nat Med 11: 263-270; Judge A D et al. 2005, Nat Biotechnol 2005. 23: 457-462.

[0319] Heil and colleagues (Heil F et al. 2004, Science 303: 1526-1529) found that when added to human PBMC as a complex with cationic lipid DOTAP, RNA40 (5''-GCC CGU CUG UUG UGU GAO UC-3'', HIV-1 U5 region nt 108-127) but not RNA41 (U-A replacement of RNA40) or RNA42 (G-A replacement of RNA40) induced IFN-α, and that the source of IFN-α was PDC. In contrast, both RNA40 and RNA42, but not RNA41 induced TNF-α, IL-6 and IL-12p40; TNF-α was produced by CD11c+ cells. Similar results were found with isolated murine PDC and macrophages. RNA33 (5-GUAGUGUGUG-3'') and RNA34 (5''-GUCUGUUGUGUG-3''), both containing one phosphorothioate linkage at the 3''end, induced the same cytokine profile as RNA40 in the mouse system. Heil et al. stated that a sequence motif responsible for the IFN-α inducing activity of the RNA oligonucleotides tested could not be identified; subsequently, the activity was attributed to the high GU content of the sequence. Our analysis of the results of Heil et al. reveals that RNA40 contains a 4mer motif, GUUG, which is the fourth most potent motif in inducing IFN-α production in our matrix. Furthermore, our 3mer-based algorithm predicts high IFN-α-inducing activity for RNA40, RNA33 and RNA34, but not for RNA41 and RNA42, which is in agreement with the experimental data (FIG. 8D).

[0320] Another publication on ssRNA and IFN-α in the human system is from our own group (Hornung V et al. 2005, Nat Med 11: 263-270). In this publication, we identified a 9mer sequence motif which was responsible for the immunostimulatory activity of the ssRNA oligonucleotide RNA9.2 sense (5'-AGC UUA ACC UGU CCU UCA A-3', 9mer motif underlined). No motif shorter than the 9mer motif was characterized. The analysis of our previously published results reveals that the previously identified 9mer motif contains GUCC which is the tenth most potent immunostimulatory 4mer in our matrix. Furthermore, our 3mer-based IFN-α point score matrix predicts RNA9.2 sense to be a highly active IFN-α-inducing sequence. Moreover, our 3mer-based IFN-α point score matrix offers a prediction of the IFN-α-inducing activities of other sequences tested in Hornung et al.; our prediction correlates very well with the published experimental data.

[0321] Another study in the human system was published by Sioud and colleagues (Sioud M et al. 2005, J Mol Biol 348: 1079-1090). The authors examined a panel of 32 siRNAs for their ability to induce TNF-α and IL-6 in PBMC. The most active sequence was number 27 (sense: 5''-GUCCGGGCAGGUCUACUUUTT-3'') either as siRNA (double-stranded) or as the sense strand. As negative control, number 32 (sense: 5''-GCUGGAGAUCCUGAAGAACTT-3'') was used. Of note, the whole panel was not screened for IFN-α-inducing activity; only sequence 27 was assayed for IFN-α induction in PBMC. Both the number 27 siRNA and the corresponding sense strand were found to induce IFN-α. DOTAP was used for transfection. Our analysis of the panel of 32 siRNAs reveals that only oligonucleotide number 27, but none of the other oligonucleotides of the panel, contains the motif GUCC, which is ranked number 10 on our most potent immunostimulatory 4mer list and which is also contained in the 9mer motif of our earlier paper (Homung V et al. 2005, Nat Med 11: 263-270) discussed above. Furthermore, our 3mer-based IFN-α point score matrix predicts potent IFN-α-inducing activity for siRNA number 27. However, since siRNA number 27 was the only sequence examined for IFN-α-inducing activity in Sioud et al, a comprehensive analysis of the whole panel of siRNAs could not be carried out.

[0322] Besides our own previous publication (Hornung V et al. 2005, Nat Med 11: 263-270), Judge and colleagues are the only ones who proposed a sequence motif (UGUGU) for the IFN-α-inducing activity of RNA oligonucleotides (Judge A D et al. 2005, Nat Biotechnol 2005. 23: 457-462). Although most of their work was done with siRNA (double-stranded), for one of their potent immunostimulatory sequences, β-Gal control, both the sense and the antisense strand were tested. The sense strand, but not the antisense strand, was found to be active to inducing IFN-α in human PBMC. The sense strand (5''-UUGAUGUGUUUAGUCGCUA-3'') contained the proposed UGUGU motif, while the antisense strand (5''-UAGCGACUAAACACAUCAA-3'') did not. The introduction of one (UGCGU) or two (UGCGC) mismatches in the sense strand sequence of the β-Gal control siRNA led to the loss of IFN-α-inducing activity. On the other hand, the creation of the UGUGU motif, starting from UGGCU, in a primarily non-stimulatory siRNA, BP1, led to an enhanced IFN-α-inducing activity. Furthermore, Judge and colleagues showed that they could select non-stimulatory siRNA sequences by avoiding U-rich sequences and GUGU motifs. Indeed, in our 4mer matrix, GUGU is the 7th most active motif, and UGUG is the 20th most active motif. Furthermore, the relative IFN-α-inducing activities of βp-Gal control siRNA, BP1 siRNA and their derivatives predicated by our 3mer-based IFN-α point score matrix correlates extremely well with the experimental data of Judge et al. (FIG. 8B).

[0323] Additional publication reports the induction of IFN-α by RNA oligonucleotides in the mouse system.

[0324] Barchet W et al. (2005, Eur J Immunol 35: 236-242) reports IFN-α induction by RNA oliognucleotides in murine PDC. In this study, the RNA sequences examined were derived from the 5' and 3' untranslated regions (UTR) of Influenza virus. The following sequences were used:

TABLE-US-00010 5' UTR: 5'-AGUAGAAACAAGGUAGUUU-3' (19 mer) 3' UTR: 5'-UUAACUACCUGCUUUUGCU-3' (19 mer) 5'3' UTR: 5'-AGUAGAAACAAGGUAGUUUUUUGUUAACUACCUGCUUUUGCU-3' (42 mer), 5' UTR U-C replacement: 5'-AGCAGAAACAAGGCAGCCC-3' (19 mer) 5' UTR G-C replacement: 5'-ACUACAAACAACCUACUUU-3' (19 mer)

[0325] 5''UTR, 3'UTR and 5'3'UTR oligonucleotides all induced IFN-α production from murine PDC. The activity of 5'UTR was significantly reduced when the Gs were replaced by Cs, and abolished when the Us were replaced by Cs. No motif responsible for the IFN-α-inducing activity was defined in this study. According to our 4mer motif matrix, 5'UTR contains the 5th active motif GUUU, and 3'UTR contains the motif UUUU which is above average. The activity levels of the oligonucleotides used in Barchet et al. predicted by our 3mer-based IFN-α point score matrix correlates with the experimental data of Barchet et al.

[0326] It should be noted that in some studies double stranded RNA oligonucleotides were used. In such cases, a mean value for the individually analyzed single strands was calculated. Nevertheless, using the prediction algorithm, a good estimate of the actual IFN-α data could be obtained.

[0327] All of the publications discussed above validate the use of our 4mer matrix and algorithms for predicting the immunostimulatory activity of RNA oligonucleotides. The teaching in the prior art with regard to the prediction of IFN-α-inducing activity of RNA oligonucleotides has been limited. The only criteria available so far are the content of G and U (Heil et al. 2004, Science 303: 1526-1529), and the presence of the GUGU motif (Judge et al. 2005, Nat Biotechnol 2005. 23: 457-462). With our 3mer-based IFN-α point score matrix, we now can predict the immunostimulatory activity of any RNA oligonucleotide reliably.

Example 10

Determining the Threshold for High and Low Immunostimulatory Activity

[0328] The immunostimulatory activity of any given RNA oligonucleotide can be predicted using the 3mer-based IFN-α point score matrix as described previously (i.e., the "addition method". For research and drug discovery and development purposes, two groups of RNA oligonucleotide are of interest: Group A oligonucleotides which have high or maximal IFN-α-inducing activity, and Group B oligonucleotides which have low or minimal IFN-α-inducing activity. Among all possible ssRNA oligonucleotides of a certain length, 1% of the oligonucleotides with the highest IFN-α scores are assigned to Group A; where as 1% of the oligonucleotides with the lowest IFN-α scores are assigned to Group B. The cut-off IFN-α score for Group A oligonucleotide is the threshold for high or maximal immunostimulatory activity; the cut-off IFN-α score for Group B oligonucleotide is the threshold for low or minimal immunostimulatory activity.

[0329] The IFN-α score thresholds for high/maximal and low/minimal immunostimulatory activity for 19mer ssRNA oligonucleotides are determined as follows:

[0330] A pool of all possible sequences of 19mer RNA oligonucleotides consists of 4¹⁹=274,877,906,944 oligonucleotides. The IFN-α score for every single RNA oligonucleotide in the pool is calculated using the 3mer-based IFN-α point score matrix. All 4¹⁹ oligonucleotides are ranked based on their calculated predicted IFN-α scores. The threshold for group A is determined to be

1.4909×n+22.014 [0331] (n=length of the ssRNA oligonucleotide, and n>9).

[0332] All ssRNA oligonucleotides with a calculated IFN-α score above the threshold value are grouped into Group A. The Group A threshold for 19mer ssRNA oligonucleotides is 50.3411. Non-limiting examples of Group A 19mer ssRNA oligonucleotides include the following:

TABLE-US-00011 Sequence (5'-->3') Predicted IFN-α score GUUUGUUGCUUUGAUUGCC 60 UUGUAGUUCGUUGCUAGUG 60 AGUUCAUGGUGGGUUGUAC 62 UGUUUAAGUUGUUCUACCC 62 AAGUUUUGAUUUUUCAGUA 63 AGGCGUUUGUGUUCGGGUU 65 AGAUGUUGUAGGGUGUUUU 66 UAGUGUGUGUCAGUGUGAC 71 GGUUGCGUGUGGAGUUGUU 72 UGUAGUUUUGUUAGAGUCA 75 GUGUGGUUGCUGUUGUCAA 77

[0333] The threshold for Group B oligonucleotides is determined to be:

(0.005×n²)-(0.2671×n)-3.5531 [0334] (n=length of the ssRNA oligonucleotide, and X>9)

[0335] All ssRNA oligonucleotides with a calculated IFN-α score below the threshold value are grouped into Group B. The Group B threshold for 19mer ssRNA oligonucleotide is -6.823. Non-limiting examples of Group B 19mer oligonucleotide include the following:

TABLE-US-00012 Sequence (5'-->3') Predicted IFN-α score GGGACCGAAAGACCAGACC -10 UAAGACUAGAAGAGACAGA -10 AGAUCCGAACCACCGACCA -9 GAACCAGAAAAUAGAGCAG -8 CAUAUAAGAAGACCAGCCA -8 UAAGAACCAACUGCUAGAA -8 CCCCUACAGACAGAAUACC -7 CUGGCAGAUAGAUAGAAGC -7 CUAGACCAGAACAAUCUCG -7 UUAGAGACAUAACAACAUU -7 GGACCAAACCUCUCGACAU -7

[0336] For ssRNA oligonucleotides between 3 and 9 nucleotides in length, the Group A and Group B threshold values are given below in Table 8:

TABLE-US-00013 TABLE 8 Threshold IFN-α scores for Group A and Group B oligonucleotides 3-9 nucleotides in length. The predicted IFN-α score ssRNA (using the IFN-α point score matrix) oligonucleotide Threshold for GROUP B Threshold for GROUP A length ssRNA oligonucelotides ssRNA oligonucelotides 3 -2 9 4 -2 15 5 -3 20 6 -4 23 7 -4 26 8 -5 28 9 -5 30

Example 11

Designing siRNA with High or Low Immunostimulatory Activity

[0337] The threshold for Group A siRNA is

1.4909×n+31.014 [0338] (n=length of the ssRNA oligonucleotide and n>9) according to the "addition method".

[0339] An siRNA is considered a Group A siRNA, i.e., an siRNA with high or maximal immunostimulatory activity, if at least one of the strands, preferably the sense strand, has an IFN-α score above the threshold.

[0340] In order to maximize the chance of identifying at least one siRNA with optimized gene silencing as well as immunostimulatory activity, at least ten siRNA molecules need to be identified whose sense strands have an IFN-α score above the threshold for Group A siRNA. When fewer than ten siRNA can be identified that fit the above criteria, the threshold for Group A siRNA is decrease by 1 in a stepwise manner until ten siRNA can be identified.

[0341] Most commonly used siRNA are at least 19 nucleotides in length. The threshold for Group A siRNA for a 19mer is 59.3411.

[0342] The following example demonstrates the identification of Group A siRNA for mRNA of human cyclophilin B (hCyPB) (Accession No. M6087).

[0343] For hCyPB, 833 putative or potential siRNA duplexes (19mer) can be identified. The IFN-α score is calculated for both the sense and the antisense strand of all possible 833 19mer siRNA using the IFN-α point score matrix. All siRNA duplexes which contain at least one strand with an IFN-α score higher than 59.3411 are put into Group A. 11 siRNA duplexes are assigned to Group A because the IFN-α scores of their sense strands are above the threshold; 24 siRNA duplexes are assigned to Group A because the IFN-α scores of their antisense strands are above the threshold. The Group A hCyPB siRNA are listed in Table 9:

TABLE-US-00014 Sense strand IFN-α Antisene strand IFN-α (5'→3') score (5'→3') score UAACAAACUCCUACCAACA -9 UGUUGGUAGGAGUUUGUUA 74 AACAAACUCCUACCAACAC -9 GUGUUGGUAGGAGUUUGUU 77 UACCAACACUGACCAAUAA -8 UUAUUGGUCAGUGUUGGUA 63 CUACCAACACUGACCAAUA -8 UAUUGGUCAGUGUUGGUAG 63 ACCAACACUGACCAAUAAA -8 UUUAUUGGUCAGUGUUGGU 65 ACAAACUCCUACCAACACU -8 AGUGUUGGUAGGAGUUUGU 74 ACUCCUACCAACACUGACC -7 GGUCAGUGUUGGUAGGAGU 62 CCUACCAACACUGACCAAU -7 AUUGGUCAGUGUUGGUAGG 63 UCCUACCAACACUGACCAA -7 UUGGUCAGUGUUGGUAGGA 63 AAACUCCUACCAACACUGA -6 UCAGUGUUGGUAGGAGUUU 64 CCAACACUGACCAAUAAAA -6 UUUUAUUGGUCAGUGUUGG 66 CAAACUCCUACCAACACUG -6 CAGUGUUGGUAGGAGUUUG 66 AACUCCUACCAACACUGAC -6 GUCAGUGUUGGUAGGAGUU 67 AACACUGACCAAUAAAAAA -6 UUUUUUAUUGGUCAGUGUU 69 CAACACUGACCAAUAAAAA -6 UUUUUAUUGGUCAGUGUUG 70 GCUACAAAAACAGCAAAUU -5 AAUUUGCUGUUUUUGUAGC 60 GGCUACAAAAACAGCAAAU -5 AUUUGCUGUUUUUGUAGCC 60 ACACUGACCAAUAAAAAAA -5 UUUUUUUAUUGGUCAGUGU 65 UGGCUACAAAAACAGCAAA -4 UUUGCUGUUUUUGUAGCCA 60 GUAACAAACUCCUACCAAC -4 GUUGGUAGGAGUUUGUUAC 66 CACUGACCAAUAAAAAAAA -3 UUUUUUUUAUUGGUCAGUG 62 ACUGACCAAUAAAAAAAAA -3 UUUUUUUUUAUUGGUCAGU 64 UACAAAAACAGCAAAUUCC -1 GGAAUUUGCUGUUUUUGUA 60 CUACAAAAACAGCAAAUUC -1 GAAUUUGCUGUUUUUGUAG 60 AAAAUGUGGGUUUUUUUUU 60 AAAAAAAAACCCACAUUUU 5 UGUGGUGUUUGGCAAAGUU 63 AACUUUGCCAAACACCACA 3 AAAUGUGGGUUUUUUUUUU 65 AAAAAAAAAACCCACAUUU 0 GUUUUUUUUUUUUUUAAUA 68 UAUUAAAAAAAAAAAAAAC -1 AAUGUGGGUUUUUUUUUUU 70 AAAAAAAAAAACCCACAUU -5 GGUUUUUUUUUUUUUUAAU 73 AUUAAAAAAAAAAAAAACC -3 GGGUUUUUUUUUUUUUUAA 73 UUAAAAAAAAAAAAAACCC -3 UGGGUUUUUUUUUUUUUUA 74 UAAAAAAAAAAAAAACCCA -3 AUGUGGGUUUUUUUUUUUU 75 AAAAAAAAAAAACCCACAU -5 GUGGGUUUUUUUUUUUUUU 77 AAAAAAAAAAAAAACCCAC -3 UGUGGGUUUUUUUUUUUUU 80 AAAAAAAAAAAAACCCACA -5

[0344] The IFN-α score threshold for a Group B siRNA is

0.6075×n-9.9484 [0345] (n=length of the ssRNA oligonucleotide and n>13) according to the "addition method".

[0346] Both the sense and the antisense strands of an siRNA have to have an IFN-α score below the threshold for the siRNA to be assigned to Group B. In order to maximize the chance of identifying at least one siRNA with optimal gene silencing activity but minimal immunostimulatory activity, at least ten Group B siRNA duplexes should be identified for a given target mRNA. Should this condition not be met, the threshold IFN-α score is increased by 1 in a stepwise manner until ten Group B siRNA are identified.

[0347] The IFN-α score threshold for Group B 19mer siRNA duplexes is 1.5941.

[0348] None of the 833 potential hCyPB siRNA have an IFN-α score of lower than 1.5491 for both strands. When the threshold was increased to 2.5491, one siRNA is identified. In order to identify at least ten siRNA duplexes, the threshold IFN-α score has to be increased by 3 to 4.5491. The Group B siRNA thus identified are listed in Table 10:

TABLE-US-00015 Sense strand IFN-α Antisense strand IFN-α (5'→3') score (5'→3') score AAGAUCGAGGUGGAGAAGC 4 GCUUCUCCACCUCGAUCUU 2 AGAUCGAGGUGGAGAAGCC 4 GGCUUCUCCACCUCGAUCU 2 CCGCCGCCCUCAUCGCGGG 4 CCCGCGAUGAGGGCGGCGG 0 CCUUCUGCGGCCGAUGAGA 2 UCUCAUCGGCCGCAGAAGG 2 CGCCGCCCUCAUCGCGGGG 4 CCCCGCGAUGAGGGCGGCG 0 CUUCCUGCUGCUGCCGGGA 4 UCCCGGCAGCAGCAGGAAG 0 GAGCGCUUCCCCGAUGAGA 2 UCUCAUCGGGGAAGCGCUC 4 GCCGCCGCCCUCAUCGCGG 4 CCGCGAUGAGGGCGGCGGC 0 GGCAAGAUCGAGGUGGAGA 4 UCUCCACCUCGAUCUUGCC 4 UCUUCCUGCUGCUGCCGGG 4 CCCGGCAGCAGCAGGAAGA -2 UGCCGCCGCCCUCAUCGCG 4 CGCGAUGAGGGCGGCGGCA 0

Example 12

Rational Identification of a Potent 4mer ssRNA Motif with Maximal IFN-α Induction in PBMC

[0349] Previous experiments have shown that a minimal length of 19 bases is required for maximal IFN-α induction by ssRNA oligonucleotides. Since poly A oligonucleotides proved to be inactive in terms of IFN-α induction, we decided to analyze sequence requirements for IFN-α induction in PBMC by placing putative motifs into the center of poly A RNA oligonucleotides. RNA9.2sense (5'-AGCUUAACCUGUCCUUCAA-3') was used as an established positive control. We started by analyzing the impact of a single nucleotide exchange in the center of a poly A RNA oligonucleotide.

[0350] As expected, little IFN-α induction could be elicited by these oligonucleotides, yet a consistent induction was seen for the ssRNA oligonucleotide containing a Uracil (U) in the center of the poly-A chain (FIG. 9A).

[0351] Based on these data, a panel of ssRNA oligonucleotides was designed that contained a U in the center of a 19mer poly A oligonucleotide including all possible single base permutations to either the 5' or the 3' end (FIG. 9B). Compared to the 1mer motifs, a considerable increase in IFN-α induction by the tested 2mer motifs could be seen. A wide distribution with an approximately 100-fold difference between the weakest and the strongest motif was observed: 5'-CU-3', 0.24% of control vs. 5'-GU-3', 24.63% of control. Among all 2mer motifs tested, 5'-GU-3' turned out to be the most potent motif with a mean IFN-α induction of 24.63% of the control oligonucleotide 9.2 sense.

[0352] Following these results, oligonucleotides were designed that contained the 5'-GU-3' motif in the center of a 19mer poly A oligonucleotide while again all possible single base permutations to either the 5' or the 3' end of the central motif were tested (FIG. 90). Compared to 5'-GU-3', an almost 6-fold increase in IFN-α induction was seen for the most potent oligonucleotide that minimally contained the sequence 5'-GUC-3' as the central motif. This oligonucleotide exceeded the control oligonucleotide by 1.43-fold in terms of IFN-α induction. A considerable amount of IFN-α, yet significantly less, was induced by the second best motif combination 5'-GUU-3' (88% of control).

[0353] Based on these data, we again designed oligonucleotides that contained the identified minimal 3mer motif 5'-GUC-3' with single base permutations to either the 5' or the 3' end. While the transition from 1mer motifs to 2mer motifs and from 2mer motifs to 3mer motifs had resulted in a strong increase in IFN-α induction, no additional enhancement in IFN-α induction was seen with the elongation of the central motif to a 4mer motif (FIG. 9D). Nevertheless, these data indicated that A was required at the 3' end of the 5'-GUC-3' motif for maximal IFN-α induction. Placing either C or U at the 3' end of the 5'-GUC-3' motif resulted in a reduction of approximately one third in IFN-α induction, whereas the addition of G resulted in an almost two third decrease in IFN-α production. No further increase in IFN-α induction was seen when the position to the 5' end of the 5'-GUC-3' motif was permutated. While changing the A to G resulted in a slight, yet not significant decrease in IFN-α induction, a decrease of approximately one third was obtained when either U or C were positioned at the 5' end. Altogether these data identified the 4mer sequence 5'-GUCA-3' as a potent motif for the induction of IFN-α in PBMC. Additional modification of the 3' end by single base permutations did not result in an increase in IFN-α induction (FIG. 9E), thereby indicating the maximal requirement of a 4mer motif for potent IFN-α induction.

Example 13

The Effect of Position and Number of 4mer Motifs on Immunostimulatory Activity

[0354] The position of the 4mer motif within the poly A backbone had little impact on the immunostimulatory activity of the respective oligonucleotide (FIG. 7). Only a slight decrease (12.6%) in IFN-α induction was seen, when the 4mer motif was moved to 5' end of the oligonucleotide, while positioning of the 4mer motif at the 3' end resulted in a decrease of 28.8% in IFN-α induction. Moving the motif two nucleotides to the 5' end from the center position resulted in an almost identical IFN-α inducing activity.

[0355] Moreover, no inhibitory effect was seen, when non-stimulatory motifs were introduced into ssRNA oligonucleotides that contained the 4mer motif 5'-GUCA-3' (data not shown). More importantly, when additional 5'-GUCA-3' motifs were introduced into a ssRNA oligonucleotide, no further increase in IFN-α induction could be observed (FIG. 10).

Example 14

Identification of G-U-Pyrimidine as the Optimal IFN-α Inducing RNA Motif Using a ssRNA Oligonucleotide Library Encompassing all Possible 4mer Motifs

[0356] The approach of gradually refining a stimulatory motif by permutation of the adjoining bases is based on the assumption that the exact succession of specific bases is critical for the stimulatory capacity of the sequence. The toleration of per se "non-stimulatory inserts" into an active sequence cannot be accounted for by this experimental setup. Moreover, this approach would miss stimulatory motifs if the sequential combination of several by itself non-stimulatory bases could synergize to render a potent motif. To test for these possibilities a library of ssRNA oligonucleotides was designed that encompassed all possible 4mer motifs in the centre of a poly A oligonucleotide. In view of the fact that flanking A residues can be part of a 4mer RNA sequence motif, only 193 ssRNA oligonucleotides (Table 1) were needed to cover all 256 possible 4mer motifs.

[0357] All 193 ssRNA oligonucleotides were tested on PBMC from six individual healthy donors. RNA9.2sense (5'-AGCUUAACCUGUCCUUCAA-3') was included as a positive control on all cell culture plates and was used as a reference for the oligonucleotides tested (RNA9.2sense=1, all data are expressed as fold values). As typically performed for large data sets such as gene array data, a global normalization to the mean was performed for each individual donor by subtracting the mean of all data from a particular donor from the individual raw data. This allowed to control for the observed inter individual variability between the donors and made it possible to summarize all donors as mean values±SEM. Thus due to the normalization, a negative value was obtained for all oligonucleotides that were below the immunostimulatory activity of the mean of all oligonucleotides, whereas for all oligonucleotides that were above the immunostimulatory activity of the mean a positive value was obtained. A colored output was chosen to give an overview on all obtained data in one graph in ascending order (FIG. 11A), while the corresponding mean values are depicted next to it (FIG. 11B). A complete list of all data with the respective sequence information is depicted in FIG. 3. Confirming the validity of our prior approach, the 4mer motif 5'-GUCA-3' turned out to be the second highest hit in the obtained data set, whereas only the motif 5'-GUUC-3' turned out to be more active in terms of IFN-α induction.

[0358] To systematically identify motifs or patterns that were associated with high or low IFN-α induction, a statistical analysis was performed by analyzing the occurrence of all possible 3mer motifs within the tested oligonucleotide library. For all 3mer motifs the mean level of IFN-α induction was calculated by grouping all oligonucleotides that contained the respective 3mer motif.

[0359] For example the 3mer motif 5'-GUC-3' was contained in ssRNA oligonucleotides ANP 35, 83, 131, 137, 138, 139 and 179 with respective IFN-α induction levels of 1.33, 0.68, 0.93, 0.79, 0.44, 0.84 and 0.73. The mean IFN-α induction level of the 3mer motif 5'-GUC-3' was thus calculated to be 0.82 with a standard error of mean of 0.10.

[0360] 3mer motifs that were gapped by one nucleotide between either the first and the second nucleotide position or the second and third nucleotide position were also included in this analysis. A two-tailed T-Test was used to identify motifs that were either significantly higher or lower in IFN-α induction than the particular motif analyzed.

[0361] For ungapped 3mer motifs, the highest mean level of IFN-α induction was obtained for the motif 5'-GUU-3' (0.87) followed by the motif 5'-GUC-3' (0.82). Within 3mer motifs that contained a nucleotide gap between the first and second position 5'-GNUC-3' (0.87) and 5'-GNUU-3' (0.72) were the two highest hits, where N represents any one nucleotide A, G, U or C. Correspondingly 5'-GUNU-3' (0.83) and 5'-GUNC-3' (0.72) were the two most potent 3mer motifs within the group of 3mer motifs, which had a nucleotide gap between the second and the third base position (FIG. 12). A detailed list of all motifs and the respective mean levels of IFN-α induction is given in Table 12.

[0362] Among the top 5% of the tested ssRNA oligonucleotide library, all oligonucleotides contained at least one of the above-mentioned most potent motifs and this was also true for 79% of all top 10% oligonucleotides (FIG. 13). The mean level of IFN-α induction for all oligonucleotides that contained at least one of the above motifs was calculated to be 0.72±0.09 compared to -0.09±0.01 for the rest of the oligonucleotides. Altogether this analysis was able to identify the motif G-U-Pyrimidine with a one-gap tolerance between either the first and the second or the second and the third position as a potent motif for RNA-mediated IFN-α induction in the human system.

Example 15

Comparison of Immunostimulatory Activity

[0363] Comparing the motif 5'-GUUC-3' on a poly A backbone to previously published ssRNA oligonucleotides with high IFN-α inducing activity revealed an almost 1.4 fold higher level of IFN-α induction than 9.2sense 5'-AGCUUAACCUGUCCUUCAA-3' (Hornung et al. Nat Med 11:263:270), 1.5 fold higher level than RNA40 5'-GCCCGUCUGUUGUGUGACUC-3' (Heil et al. Science 303:1526-1529) and a more than 2.5 fold higher activity than p-Gal control sense 5'-UUGAUGUGUUUAGUCGCUA-3' (Judge et al. Nat Biotech 23:457-462) (FIG. 14).

Example 16

Using the Obtained Motif Information to Predict Low or High IFN-α Inducing ssRNA Oligonucleotides

[0364] An analysis of published ssRNA oligonucleotides that were described to induce IFN-α indicated a good correlation with our motif information. To systematically predict the IFN-α inducing activity of a ssRNA oligonucleotide, an algorithm was established based on the occurrence of 3mer motifs 5'-NNN-3'. Since above data had indicated that the major IFN-α inducing activity of a ssRNA oligonucleotide was independent of the position of the stimulatory motif and the presence of additional stimulatory or inhibitory motifs, an algorithm was developed that predicted the IFN-α inducting activity by accounting for the highest stimulatory motif within the oligonucleotide independent of its position. Only motifs that had been shown to significantly correlate with high or low IFN-α inducing activity were included in the algorithm: 18 ungapped 3mer motifs (5'-NNN-3'), indicated by "*" or "**" in Table 12A. Thus for a given oligonucleotide, a predicted IFN-α inducing activity was calculated by analyzing the occurrence of all 3mer motifs within this oligonucleotide and subsequently by assigning the highest obtained mean IFN-α induction level to this respective oligonucleotide.

[0365] Applying this algorithm (i.e., the "simplified method") to our own data set resulted in a correlation coefficient of 0.80 for the comparison of the predicted and the measured data (FIG. 15).

[0366] The predictive quality of this algorithm to define low IFN-α inducing ssRNA oligonucleotides was then tested on the 193 oligo data set. Low inducing oligonucleotides were predefined by an IFN-α induction level below the mean of all oligonucleotides. Various threshold levels for the prediction algorithm were tested for both for the positive predictive value and the sensitivity to identify ssRNA oligonucleotides below the mean IFN-α of all oligonucleotides (FIG. 16). A high positive predictive value and a yet high sensitivity was obtained when the threshold of the prediction algorithm was set at 0.11 (FIG. 16B, upper right panel). 118 oligonucleotides had a calculated predicted IFN-α induction level below the threshold of 0.11 and 117 of these oligonucleotides had the expected measured IFN-α level below 0 (positive predictive value of 99.15%). Only 28 oligonucleotides of a total of 145 oligonucleotides were not detected using the prediction (sensitivity of 80.69%). Using the prediction algorithm with the threshold of 0.11 therefore allowed to predict a large percentage of oligonucleotides that would be below the desired level of 0 with high accuracy.

[0367] Applying this algorithm to more complex oligonucleotides resulted in a considerable lower percentage of oligonucleotides that met the above criteria. Approximately 10% of a random pool of 19mer oligonucleotides fell below the threshold of 0.11 when analyzed using the IFN-α prediction algorithm (data not shown). When a random pool of 19mer duplexes was analyzed, approximately 1% of all duplexes were comprised of single stranded RNA oligonucleotides that were both below the threshold of 0.11.

Example 17

Preparing siRNA with High or Low Immunostimulatory Activity

[0368] To address the applicability of this algorithm for the identification of non-stimulatory RNA oligonucleotides, a panel of siRNA duplexes was designed to target the mRNA of human TLR9. From a total pool of 3850 siRNA duplexes that were considered, 116 could be identified (3.01%) that had a calculated predicted IFN-α induction below the threshold of 0.11 according to the "simplified method". These 116 predicted siRNA duplexes were distributed over 21 different target sites within in the mRNA, whereas seven major sites could be identified that could be targeted by more than five consecutive siRNA duplexes. Three different siRNA duplexes were chosen to target TLR9 using the above described algorithm with putatively low IFN-α induction (TLR9_--271, TLR9_--1122 and TLR9_--1949), whereas three different siRNA duplexes were picked that contained at least one strand with predicted high IFN-α induction (TLR9_--1019, TLR9_--1302 and TLR9_--1949). Altogether eight ssRNA oligonucleotides with presumably low IFN-α induction (TLR9_--271 sense, TLR9_--271 antisense, TLR9_--1019 sense, TLR9_--1122 sense, TLR9_--1122 antisense, TLR9_--1302 antisense, TLR9_--1949 sense, TLR9_--1949 antisense) and four ssRNA oligonucleotides with high IFN-α induction (TLR9_--1019 antisense, TLR9_--1302 sense, TLR9_--1500 sense anf TLR9_--1500 antisense) were contained in this selection (FIG. 17).

[0369] The functionality of theses siRNA duplexes in terms of posttranscriptional gene silencing was assessed by analyzing the knock down activity of TLR9 expression (FIG. 18). Two siRNA duplexes with good knock down activity could be identified within both groups of siRNA duplexes.

[0370] When analyzing the IFN-α induction of both the single stranded components and the respective siRNA duplexes, a high accuracy of the IFN-α prediction algorithm could be seen. Of all eight ssRNA oligonucleotides predicted to be low in IFN-α induction, seven oligonucleotides (TLR9_--271 antisense, TLR9_--1019 sense, TLR9_--1122 sense, TLR9_--1122 antisense, TLR9_--1302 antisense, TLR9_--1949 sense, TLR9_--1949 antisense) showed negligible to absent IFN-α induction, whereas one oligonucleotide (TLR9_--271 sense) showed minimal IFN-α induction. This was also true for the respective siRNA duplexes: TLR9_--1122 and TLR9_--1949 duplexes showed negligible to absent IFN-α induction, and TLR9_--271 duplex showed minimal IFN-α induciton. Likewise, all ssRNA oligonucleotides predicted to be high in IFN-α induction showed a strong IFN-α response (TLR9_--1019 antisense, TLR9_--1302 sense, TLR9_--1500 sense anf TLR9_--1500 antisense). As described previously, within a siRNA duplex, the strong induction of IFN-α by either one component dictated the immunostimulatory activity of the whole siRNA duplex. TLR9_--1019, TLR9_--1032 and TLR9_--1500 duplexes all showed high IFN-α induction.

[0371] Altogether these results indicated that using a motif-based algorithm we are able to rationally design both stimulatory and non-stimulatory functional siRNA duplexes.

Example 18

Systematic and Automated Identification of siRNA with Desired Immunolostimulatory Activity

[0372] Based on the algorithm described in example 17 (i.e., the "simplified method"), a computer program was written that applies the algorithm to all possible siRNA duplexes targeting all human RNA transcripts (50421 as of 09/2006) as published by the National Center for Biotechnology Information (NCBI). Each entry into the NCBI database (ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/human.rna.fna.gz) of all listed human RNA transcripts was analyzed the following way: A list of all possible 19mer siRNA duplexes targeting a given RNA transcript was generated. Of all siRNA duplexes the IFN-α induction of both the sense and the antisense strand was predicted using the method described in example 17. The obtained data is stored in a database (CD-ROM) and can be retrieved by a search engine. Using the search interface, the user can pick the transcript of interest (alphabetical index of all RNA transcripts targeted by siRNAs) and then adjust the level of threshold to identify siRNA duplexes that are of either low, intermediate or high in immunostimulatory activity (FIG. 19A). For example, using the threshold of 0.11 as described in example 17, a set of siRNA duplexes was identified for Homo sapiens vascular endothelial growth factor (VEGF) transcript variant 1 mRNA (NM_--001025366.1) with low immunostimulatory activity for both the sense and the antisense strand (FIG. 19B).

TABLE-US-00016 TABLE 1 ssRNA oligonucleotides containing all possible 4 mer motifs on the poly a backbone. Name Sequence ANP-Oligo 001 AAAAAAAAAAAAAAAAAAA ANP-Oligo 002 AAAAAAAACAAAAAAAAAA ANP-Oligo 003 AAAAAAAAGAAAAAAAAAA ANP-Oligo 004 AAAAAAAAUAAAAAAAAAA ANP-Oligo 005 AAAAAAAACCAAAAAAAAA ANP-Oligo 006 AAAAAAAACGAAAAAAAAA ANP-Oligo 007 AAAAAAAACUAAAAAAAAA ANP-Oligo 008 AAAAAAAAGCAAAAAAAAA ANP-Oligo 009 AAAAAAAAGGAAAAAAAAA ANP-Oligo 010 AAAAAAAAGUAAAAAAAAA ANP-Oligo 011 AAAAAAAAUCAAAAAAAAA ANP-Oligo 012 AAAAAAAAUGAAAAAAAAA ANP-Oligo 013 AAAAAAAAUUAAAAAAAAA ANP-Oligo 014 AAAAAAAACACAAAAAAAA ANP-Oligo 015 AAAAAAAACAGAAAAAAAA ANP-Oligo 016 AAAAAAAACAUAAAAAAAA ANP-Oligo 017 AAAAAAAACCCAAAAAAAA ANP-Oligo 018 AAAAAAAACCGAAAAAAAA ANP-Oligo 019 AAAAAAAACCUAAAAAAAA ANP-Oligo 020 AAAAAAAACGCAAAAAAAA ANP-Oligo 021 AAAAAAAACGGAAAAAAAA ANP-Oligo 022 AAAAAAAACGUAAAAAAAA ANP-Oligo 023 AAAAAAAACUCAAAAAAAA ANP-Oligo 024 AAAAAAAACUGAAAAAAAA ANP-Oligo 025 AAAAAAAACUUAAAAAAAA ANP-Oligo 026 AAAAAAAAGACAAAAAAAA ANP-Oligo 027 AAAAAAAAGAGAAAAAAAA ANP-Oligo 028 AAAAAAAAGAUAAAAAAAA ANP-Oligo 029 AAAAAAAAGCCAAAAAAAA ANP-Oligo 030 AAAAAAAAGCGAAAAAAAA ANP-Oligo 031 AAAAAAAAGCUAAAAAAAA ANP-Oligo 032 AAAAAAAAGGCAAAAAAAA ANP-Oligo 033 AAAAAAAAGGGAAAAAAAA ANP-Oligo 034 AAAAAAAAGGUAAAAAAAA ANP-Oligo 035 AAAAAAAAGUCAAAAAAAA ANP-Oligo 036 AAAAAAAAGUGAAAAAAAA ANP-Oligo 037 AAAAAAAAGUUAAAAAAAA ANP-Oligo 038 AAAAAAAAUACAAAAAAAA ANP-Oligo 039 AAAAAAAAUAGAAAAAAAA ANP-Oligo 040 AAAAAAAAUAUAAAAAAAA ANP-Oligo 041 AAAAAAAAUCCAAAAAAAA ANP-Oligo 042 AAAAAAAAUCGAAAAAAAA ANP-Oligo 043 AAAAAAAAUCUAAAAAAAA ANP-Oligo 044 AAAAAAAAUGCAAAAAAAA ANP-Oligo 045 AAAAAAAAUGGAAAAAAAA ANP-Oligo 046 AAAAAAAAUGUAAAAAAAA ANP-Oligo 047 AAAAAAAAUUCAAAAAAAA ANP-Oligo 048 AAAAAAAAUUGAAAAAAAA ANP-Oligo 049 AAAAAAAAUUUAAAAAAAA ANP-Oligo 050 AAAAAAACAACAAAAAAAA ANP-Oligo 051 AAAAAAACAAGAAAAAAAA ANP-Oligo 052 AAAAAAACAAUAAAAAAAA ANP-Oligo 053 AAAAAAACACCAAAAAAAA ANP-Oligo 054 AAAAAAACACGAAAAAAAA ANP-Oligo 055 AAAAAAACACUAAAAAAAA ANP-Oligo 056 AAAAAAACAGCAAAAAAAA ANP-Oligo 057 AAAAAAACAGGAAAAAAAA ANP-Oligo 058 AAAAAAACAGUAAAAAAAA ANP-Oligo 059 AAAAAAACAUCAAAAAAAA ANP-Oligo 060 AAAAAAACAUGAAAAAAAA ANP-Oligo 061 AAAAAAACAUUAAAAAAAA ANP-Oligo 062 AAAAAAACCACAAAAAAAA ANP-Oligo 063 AAAAAAACCAGAAAAAAAA ANP-Oligo 064 AAAAAAACCAUAAAAAAAA ANP-Oligo 065 AAAAAAACCCCAAAAAAAA ANP-Oligo 066 AAAAAAACCCGAAAAAAAA ANP-Oligo 067 AAAAAAACCCUAAAAAAAA ANP-Oligo 068 AAAAAAACCGCAAAAAAAA ANP-Oligo 069 AAAAAAACCGGAAAAAAAA ANP-Oligo 070 AAAAAAACCGUAAAAAAAA ANP-Oligo 071 AAAAAAACCUCAAAAAAAA ANP-Oligo 072 AAAAAAACCUGAAAAAAAA ANP-Oligo 073 AAAAAAACCUUAAAAAAAA ANP-Oligo 074 AAAAAAACGACAAAAAAAA ANP-Oligo 075 AAAAAAACGAGAAAAAAAA ANP-Oligo 076 AAAAAAACGAUAAAAAAAA ANP-Oligo 077 AAAAAAACGCCAAAAAAAA ANP-Oligo 078 AAAAAAACGCGAAAAAAAA ANP-Oligo 079 AAAAAAACGCUAAAAAAAA ANP-Oligo 080 AAAAAAACGGCAAAAAAAA ANP-Oligo 081 AAAAAAACGGGAAAAAAAA ANP-Oligo 082 AAAAAAACGGUAAAAAAAA ANP-Oligo 083 AAAAAAACGUCAAAAAAAA ANP-Oligo 084 AAAAAAACGUGAAAAAAAA ANP-Oligo 085 AAAAAAACGUUAAAAAAAA ANP-Oligo 086 AAAAAAACUACAAAAAAAA ANP-Oligo 087 AAAAAAACUAGAAAAAAAA ANP-Oligo 088 AAAAAAACUAUAAAAAAAA ANP-Oligo 089 AAAAAAACUCCAAAAAAAA ANP-Oligo 090 AAAAAAACUCGAAAAAAAA ANP-Oligo 091 AAAAAAACUCUAAAAAAAA ANP-Oligo 092 AAAAAAACUGCAAAAAAAA ANP-Oligo 093 AAAAAAACUGGAAAAAAAA ANP-Oligo 094 AAAAAAACUGUAAAAAAAA ANP-Oligo 095 AAAAAAACUUCAAAAAAAA ANP-Oligo 096 AAAAAAACUUGAAAAAAAA ANP-Oligo 097 AAAAAAACUUUAAAAAAAA ANP-Oligo 098 AAAAAAAGAACAAAAAAAA ANP-Oligo 099 AAAAAAAGAAGAAAAAAAA ANP-Oligo 100 AAAAAAAGAAUAAAAAAAA ANP-Oligo 101 AAAAAAAGACCAAAAAAAA ANP-Oligo 102 AAAAAAAGACGAAAAAAAA ANP-Oligo 103 AAAAAAAGACUAAAAAAAA ANP-Oligo 104 AAAAAAAGAGCAAAAAAAA ANP-Oligo 105 AAAAAAAGAGGAAAAAAAA ANP-Oligo 106 AAAAAAAGAGUAAAAAAAA ANP-Oligo 107 AAAAAAAGAUCAAAAAAAA ANP-Oligo 108 AAAAAAAGAUGAAAAAAAA ANP-Oligo 109 AAAAAAAGAUUAAAAAAAA ANP-Oligo 110 AAAAAAAGCACAAAAAAAA ANP-Oligo 111 AAAAAAAGCAGAAAAAAAA ANP-Oligo 112 AAAAAAAGCAUAAAAAAAA ANP-Oligo 113 AAAAAAAGCCCAAAAAAAA ANP-Oligo 114 AAAAAAAGCCGAAAAAAAA ANP-Oligo 115 AAAAAAAGCCUAAAAAAAA ANP-Oligo 116 AAAAAAAGCGCAAAAAAAA ANP-Oligo 117 AAAAAAAGCGGAAAAAAAA ANP-Oligo 118 AAAAAAAGCGUAAAAAAAA ANP-Oligo 119 AAAAAAAGCUCAAAAAAAA ANP-Oligo 120 AAAAAAAGCUGAAAAAAAA ANP-Oligo 121 AAAAAAAGCUUAAAAAAAA ANP-Oligo 122 AAAAAAAGGACAAAAAAAA

ANP-Oligo 123 AAAAAAAGGAGAAAAAAAA ANP-Oligo 124 AAAAAAAGGAUAAAAAAAA ANP-Oligo 125 AAAAAAAGGCCAAAAAAAA ANP-Oligo 126 AAAAAAAGGCGAAAAAAAA ANP-Oligo 127 AAAAAAAGGCUAAAAAAAA ANP-Oligo 128 AAAAAAAGGGCAAAAAAAA ANP-Oligo 129 AAAAAAAGGGGAAAAAAAA ANP-Oligo 130 AAAAAAAGGGUAAAAAAAA ANP-Oligo 131 AAAAAAAGGUCAAAAAAAA ANP-Oligo 132 AAAAAAAGGUGAAAAAAAA ANP-Oligo 133 AAAAAAAGGUUAAAAAAAA ANP-Oligo 134 AAAAAAAGUACAAAAAAAA ANP-Oligo 135 AAAAAAAGUAGAAAAAAAA ANP-Oligo 136 AAAAAAAGUAUAAAAAAAA ANP-Oligo 137 AAAAAAAGUCCAAAAAAAA ANP-Oligo 138 AAAAAAAGUCGAAAAAAAA ANP-Oligo 139 AAAAAAAGUCUAAAAAAAA ANP-Oligo 140 AAAAAAAGUGCAAAAAAAA ANP-Oligo 141 AAAAAAAGUGGAAAAAAAA ANP-Oligo 142 AAAAAAAGUGUAAAAAAAA ANP-Oligo 143 AAAAAAAGUUCAAAAAAAA ANP-Oligo 144 AAAAAAAGUUGAAAAAAAA ANP-Oligo 145 AAAAAAAGUUUAAAAAAAA ANP-Oligo 146 AAAAAAAUAACAAAAAAAA ANP-Oligo 147 AAAAAAAUAAGAAAAAAAA ANP-Oligo 148 AAAAAAAUAAUAAAAAAAA ANP-Oligo 149 AAAAAAAUACCAAAAAAAA ANP-Oligo 150 AAAAAAAUACGAAAAAAAA ANP-Oligo 151 AAAAAAAUACUAAAAAAAA ANP-Oligo 152 AAAAAAAUAGCAAAAAAAA ANP-Oligo 153 AAAAAAAUAGGAAAAAAAA ANP-Oligo 154 AAAAAAAUAGUAAAAAAAA ANP-Oligo 155 AAAAAAAUAUCAAAAAAAA ANP-Oligo 156 AAAAAAAUAUGAAAAAAAA ANP-Oligo 157 AAAAAAAUAUUAAAAAAAA ANP-Oligo 158 AAAAAAAUCACAAAAAAAA ANP-Oligo 159 AAAAAAAUCAGAAAAAAAA ANP-Oligo 160 AAAAAAAUCAUAAAAAAAA ANP-Oligo 161 AAAAAAAUCCCAAAAAAAA ANP-Oligo 162 AAAAAAAUCCGAAAAAAAA ANP-Oligo 163 AAAAAAAUCCUAAAAAAAA ANP-Oligo 164 AAAAAAAUCGCAAAAAAAA ANP-Oligo 165 AAAAAAAUCGGAAAAAAAA ANP-Oligo 166 AAAAAAAUCGUAAAAAAAA ANP-Oligo 167 AAAAAAAUCUCAAAAAAAA ANP-Oligo 168 AAAAAAAUCUGAAAAAAAA ANP-Oligo 169 AAAAAAAUCUUAAAAAAAA ANP-Oligo 170 AAAAAAAUGACAAAAAAAA ANP-Oligo 171 AAAAAAAUGAGAAAAAAAA ANP-Oligo 172 AAAAAAAUGAUAAAAAAAA ANP-Oligo 173 AAAAAAAUGCCAAAAAAAA ANP-Oligo 174 AAAAAAAUGCGAAAAAAAA ANP-Oligo 175 AAAAAAAUGCUAAAAAAAA ANP-Oligo 176 AAAAAAAUGGCAAAAAAAA ANP-Oligo 177 AAAAAAAUGGGAAAAAAAA ANP-Oligo 178 AAAAAAAUGGUAAAAAAAA ANP-Oligo 179 AAAAAAAUGUCAAAAAAAA ANP-Oligo 180 AAAAAAAUGUGAAAAAAAA ANP-Oligo 181 AAAAAAAUGUUAAAAAAAA ANP-Oligo 182 AAAAAAAUUACAAAAAAAA ANP-Oligo 183 AAAAAAAUUAGAAAAAAAA ANP-Oligo 184 AAAAAAAUUAUAAAAAAAA ANP-Oligo 185 AAAAAAAUUCCAAAAAAAA ANP-Oligo 186 AAAAAAAUUCGAAAAAAAA ANP-Oligo 187 AAAAAAAUUCUAAAAAAAA ANP-Oligo 188 AAAAAAAUUGCAAAAAAAA ANP-Oligo 189 AAAAAAAUUGGAAAAAAAA ANP-Oligo 190 AAAAAAAUUGUAAAAAAAA ANP-Oligo 191 AAAAAAAUUUCAAAAAAAA ANP-Oligo 192 AAAAAAAUUUGAAAAAAAA ANP-Oligo 193 AAAAAAAUUUUAAAAAAAA

TABLE-US-00017 TABLE 2 Group 1 ssRNA-Oligonucleotides adjusted Name Sequence 5' → 3' IFN-α index SEM ANP-Oligo 018 AAAAAAAACCGAAAAAAAA -0.23 0.03 ANP-Oligo 020 AAAAAAAACGCAAAAAAAA -0.23 0.03 ANP-Oligo 029 AAAAAAAAGCCAAAAAAAA -0.23 0.03 ANP-Oligo 051 AAAAAAACAAGAAAAAAAA -0.23 0.03 ANP-Oligo 053 AAAAAAACACCAAAAAAAA -0.23 0.03 ANP-Oligo 075 AAAAAAACGAGAAAAAAAA -0.23 0.03 ANP-Oligo 080 AAAAAAACGGCAAAAAAAA -0.23 0.03 ANP-Oligo 128 AAAAAAAGGGCAAAAAAAA -0.23 0.03 ANP-Oligo 130 AAAAAAAGGGUAAAAAAAA -0.23 0.03 ANP-Oligo 009 AAAAAAAAGGAAAAAAAAA -0.23 0.03 ANP-Oligo 017 AAAAAAAACCCAAAAAAAA -0.23 0.03 ANP-Oligo 003 AAAAAAAAGAAAAAAAAAA -0.23 0.03 ANP-Oligo 081 AAAAAAACGGGAAAAAAAA -0.23 0.03 ANP-Oligo 113 AAAAAAAGCCCAAAAAAAA -0.23 0.03 ANP-Oligo 014 AAAAAAAACACAAAAAAAA -0.23 0.03 ANP-Oligo 077 AAAAAAACGCCAAAAAAAA -0.23 0.03 ANP-Oligo 126 AAAAAAAGGCGAAAAAAAA -0.23 0.03 ANP-Oligo 125 AAAAAAAGGCCAAAAAAAA -0.23 0.03 ANP-Oligo 050 AAAAAAACAACAAAAAAAA . -0.23 0.03 ANP-Oligo 015 AAAAAAAACAGAAAAAAAA -0.22 0.03 ANP-Oligo 114 AAAAAAAGCCGAAAAAAAA -0.22 0.03 ANP-Oligo 027 AAAAAAAAGAGAAAAAAAA -0.22 0.03 ANP-Oligo 117 AAAAAAAGCGGAAAAAAAA -0.22 0.03 ANP-Oligo 006 AAAAAAAACGAAAAAAAAA -0.22 0.03 ANP-Oligo 005 AAAAAAAACCAAAAAAAAA -0.22 0.03 ANP-Oligo 069 AAAAAAACCGGAAAAAAAA -0.22 0.03 ANP-Oligo 066 AAAAAAACCCGAAAAAAAA -0.22 0.03 ANP-Oligo 007 AAAAAAAACUAAAAAAAAA -0.22 0.03 ANP-Oligo 060 AAAAAAACAUGAAAAAAAA -0.22 0.03 ANP-Oligo 021 AAAAAAAACGGAAAAAAAA -0.22 0.03 ANP-Oligo 042 AAAAAAAAUCGAAAAAAAA -0.22 0.03 ANP-Oligo 086 AAAAAAACUACAAAAAAAA -0.22 0.03 ANP-Oligo 064 AAAAAAACCAUAAAAAAAA -0.22 0.03 ANP-Oligo 102 AAAAAAAGACGAAAAAAAA -0.22 0.03 ANP-Oligo 068 AAAAAAACCGCAAAAAAAA -0.22 0.03 ANP-Oligo 129 AAAAAAAGGGGAAAAAAAA -0.22 0.03 ANP-Oligo 008 AAAAAAAAGCAAAAAAAAA -0.22 0.03 ANP-Oligo 002 AAAAAAAACAAAAAAAAAA -0.22 0.03 ANP-Oligo 038 AAAAAAAAUACAAAAAAAA -0.22 0.03 ANP-Oligo 026 AAAAAAAAGACAAAAAAAA -0.22 0.03 ANP-Oligo 116 AAAAAAAGCGCAAAAAAAA -0.22 0.03 ANP-Oligo 054 AAAAAAACACGAAAAAAAA -0.22 0.03 ANP-Oligo 057 AAAAAAACAGGAAAAAAAA -0.22 0.03 ANP-Oligo 001 AAAAAAAAAAAAAAAAAAA -0.21 0.03 ANP-Oligo 074 AAAAAAACGACAAAAAAAA -0.21 0.03 ANP-Oligo 033 AAAAAAAAGGGAAAAAAAA -0.21 0.03 ANP-Oligo 123 AAAAAAAGGAGAAAAAAAA -0.21 0.03 ANP-Oligo 059 AAAAAAACAUCAAAAAAAA -0.21 0.03 ANP-Oligo 110 AAAAAAAGCACAAAAAAAA -0.21 0.03 ANP-Oligo 065 AAAAAAACCCCAAAAAAAA -0.21 0.03 ANP-Oligo 055 AAAAAAACACUAAAAAAAA -0.21 0.03 ANP-Oligo 122 AAAAAAAGGACAAAAAAAA -0.21 0.03 ANP-Oligo 104 AAAAAAAGAGCAAAAAAAA -0.21 0.03 ANP-Oligo 078 AAAAAAACGCGAAAAAAAA -0.21 0.03 ANP-Oligo 032 AAAAAAAAGGCAAAAAAAA -0.21 0.04 ANP-Oligo 030 AAAAAAAAGCGAAAAAAAA -0.21 0.03 ANP-Oligo 004 AAAAAAAAUAAAAAAAAAA -0.21 0.03 ANP-Oligo 105 AAAAAAAGAGGAAAAAAAA -0.21 0.03 ANP-Oligo 161 AAAAAAAUCCCAAAAAAAA -0.21 0.02 ANP-Oligo 061 AAAAAAACAUUAAAAAAAA -0.2 0.02 ANP-Oligo 041 AAAAAAAAUCCAAAAAAAA -0.2 0.03 ANP-Oligo 063 AAAAAAACCAGAAAAAAAA -0.2 0.04 ANP-Oligo 072 AAAAAAACCUGAAAAAAAA -0.2 0.02 ANP-Oligo 101 AAAAAAAGACCAAAAAAAA -0.2 0.04 ANP-Oligo 056 AAAAAAACAGCAAAAAAAA -0.2 0.04 ANP-Oligo 162 AAAAAAAUCCGAAAAAAAA -0.2 0.02 ANP-Oligo 099 AAAAAAAGAAGAAAAAAAA -0.2 0.05 ANP-Oligo 111 AAAAAAAGCAGAAAAAAAA -0.2 0.04 ANP-Oligo 011 AAAAAAAAUCAAAAAAAAA -0.2 0.04 ANP-Oligo 062 AAAAAAACCACAAAAAAAA -0.19 0.04 ANP-Oligo 016 AAAAAAAACAUAAAAAAAA -0.19 0.03 ANP-Oligo 177 AAAAAAAUGGGAAAAAAAA -0.19 0.03 ANP-Oligo 019 AAAAAAAACCUAAAAAAAA -0.19 0.03 ANP-Oligo 098 AAAAAAAGAACAAAAAAAA -0.19 0.05 ANP-Oligo 052 AAAAAAACAAUAAAAAAAA -0.19 0.02 ANP-Oligo 089 AAAAAAACUCCAAAAAAAA -0.18 0.05 ANP-Oligo 067 AAAAAAACCCUAAAAAAAA -0.18 0.02 ANP-Oligo 040 AAAAAAAAUAUAAAAAAAA -0.18 0.02 ANP-Oligo 039 AAAAAAAAUAGAAAAAAAA -0.18 0.02 ANP-Oligo 043 AAAAAAAAUCUAAAAAAAA -0.18 0.02 ANP-Oligo 073 AAAAAAACCUUAAAAAAAA -0.18 0.01 ANP-Oligo 024 AAAAAAAACUGAAAAAAAA -0.17 0.02 ANP-Oligo 028 AAAAAAAAGAUAAAAAAAA -0.17 0.01 ANP-Oligo 103 AAAAAAAGACUAAAAAAAA -0.17 0.01 ANP-Oligo 091 AAAAAAACUCUAAAAAAAA -0.17 0.01 ANP-Oligo 158 AAAAAAAUCACAAAAAAAA -0.17 0.02 ANP-Oligo 012 AAAAAAAAUGAAAAAAAAA -0.16 0.02 ANP-Oligo 163 AAAAAAAUCCUAAAAAAAA -0.15 0.03 ANP-Oligo 025 AAAAAAAACUUAAAAAAAA -0.15 0.02 ANP-Oligo 112 AAAAAAAGCAUAAAAAAAA -0.15 0.01 ANP-Oligo 070 AAAAAAACCGUAAAAAAAA -0.14 0.02 ANP-Oligo 172 AAAAAAAUGAUAAAAAAAA -0.14 0.03 ANP-Oligo 150 AAAAAAAUACGAAAAAAAA -0.14 0.04 ANP-Oligo 146 AAAAAAAUAACAAAAAAAA -0.14 0.03 ANP-Oligo 115 AAAAAAAGCCUAAAAAAAA -0.14 0.01 ANP-Oligo 160 AAAAAAAUCAUAAAAAAAA -0.13 0.02 ANP-Oligo 151 AAAAAAAUACUAAAAAAAA -0.13 0.03 ANP-Oligo 013 AAAAAAAAUUAAAAAAAAA -0.13 0.03 ANP-Oligo 090 AAAAAAACUCGAAAAAAAA -0.13 0.03 ANP-Oligo 048 AAAAAAAAUUGAAAAAAAA -0.13 0.02 ANP-Oligo 092 AAAAAAACUGCAAAAAAAA -0.13 0.02 ANP-Oligo 093 AAAAAAACUGGAAAAAAAA -0.13 0.03 ANP-Oligo 031 AAAAAAAAGCUAAAAAAAA -0.13 0.02 ANP-Oligo 157 AAAAAAAUAUUAAAAAAAA -0.12 0.02 ANP-Oligo 108 AAAAAAAGAUGAAAAAAAA -0.12 0.02 ANP-Oligo 076 AAAAAAACGAUAAAAAAAA -0.12 0.03 ANP-Oligo 165 AAAAAAAUCGGAAAAAAAA -0.11 0.03 ANP-Oligo 087 AAAAAAACUAGAAAAAAAA -0.11 0.01 ANP-Oligo 109 AAAAAAAGAUUAAAAAAAA -0.11 0.01 ANP-Oligo 183 AAAAAAAUUAGAAAAAAAA -0.11 0.05 ANP-Oligo 127 AAAAAAAGGCUAAAAAAAA -0.11 0.03 ANP-Oligo 079 AAAAAAACGCUAAAAAAAA -0.1 0.02 ANP-Oligo 088 AAAAAAACUAUAAAAAAAA -0.1 0.03 ANP-Oligo 159 AAAAAAAUCAGAAAAAAAA -0.1 0.05 ANP-Oligo 152 AAAAAAAUAGCAAAAAAAA -0.1 0.04 ANP-Oligo 084 AAAAAAACGUGAAAAAAAA -0.1 0.03 ANP-Oligo 184 AAAAAAAUUAUAAAAAAAA -0.1 0.03 ANP-Oligo 175 AAAAAAAUGCUAAAAAAAA -0.1 0.03 ANP-Oligo 164 AAAAAAAUCGCAAAAAAAA -0.09 0.04 ANP-Oligo 149 AAAAAAAUACCAAAAAAAA -0.09 0.06 ANP-Oligo 185 AAAAAAAUUCCAAAAAAAA -0.08 0.05 ANP-Oligo 044 AAAAAAAAUGCAAAAAAAA -0.08 0.03

ANP-Oligo 022 AAAAAAAACGUAAAAAAAA -0.08 0.02 ANP-Oligo 049 AAAAAAAAUUUAAAAAAAA -0.08 0.02 ANP-Oligo 147 AAAAAAAUAAGAAAAAAAA -0.07 0.06 ANP-Oligo 120 AAAAAAAGCUGAAAAAAAA -0.07 0.03 ANP-Oligo 107 AAAAAAAGAUCAAAAAAAA -0.07 0.05 ANP-Oligo 118 AAAAAAAGCGUAAAAAAAA -0.07 0.03 ANP-Oligo 170 AAAAAAAUGACAAAAAAAA -0.07 0.06 ANP-Oligo 124 AAAAAAAGGAUAAAAAAAA -0.07 0.05 ANP-Oligo 148 AAAAAAAUAAUAAAAAAAA -0.07 0.05 ANP-Oligo 097 AAAAAAACUUUAAAAAAAA -0.07 0.02 ANP-Oligo 156 AAAAAAAUAUGAAAAAAAA -0.06 0.04 ANP-Oligo 045 AAAAAAAAUGGAAAAAAAA -0.06 0.06 ANP-Oligo 171 AAAAAAAUGAGAAAAAAAA -0.05 0.08 ANP-Oligo 173 AAAAAAAUGCCAAAAAAAA -0.05 0.05 ANP-Oligo 174 AAAAAAAUGCGAAAAAAAA -0.05 0.07 ANP-Oligo 153 AAAAAAAUAGGAAAAAAAA -0.05 0.05 ANP-Oligo 100 AAAAAAAGAAUAAAAAAAA -0.04 0.02 ANP-Oligo 169 AAAAAAAUCUUAAAAAAAA -0.04 0.05 ANP-Oligo 155 AAAAAAAUAUCAAAAAAAA -0.03 0.04 ANP-Oligo 010 AAAAAAAAGUAAAAAAAAA -0.03 0.04 ANP-Oligo 036 AAAAAAAAGUGAAAAAAAA -0.02 0.04 ANP-Oligo 082 AAAAAAACGGUAAAAAAAA -0.01 0.03 ANP-Oligo 168 AAAAAAAUCUGAAAAAAAA -0.01 0.06

TABLE-US-00018 TABLE 3 Group 2 ssRNA-Oligonucleotides adjusted Name Sequence 5' → 3' IFN-α index SEM ANP-Oligo 071 AAAAAAACCUCAAAAAAAA 0 0.05 ANP-Oligo 182 AAAAAAAUUACAAAAAAAA 0.01 0.1 ANP-Oligo 096 AAAAAAACUUGAAAAAAAA 0.02 0.06 ANP-Oligo 085 AAAAAAACGUUAAAAAAAA 0.02 0.04 ANP-Oligo 176 AAAAAAAUGGCAAAAAAAA 0.04 0.04 ANP-Oligo 187 AAAAAAAUUCUAAAAAAAA 0.05 0.04 ANP-Oligo 166 AAAAAAAUCGUAAAAAAAA 0.07 0.04 ANP-Oligo 023 AAAAAAAACUCAAAAAAAA 0.08 0.06 ANP-Oligo 193 AAAAAAAUUUUAAAAAAAA 0.13 0.08 ANP-Oligo 188 AAAAAAAUUGCAAAAAAAA 0.14 0.04 ANP-Oligo 132 AAAAAAAGGUGAAAAAAAA 0.18 0.04 ANP-Oligo 106 AAAAAAAGAGUAAAAAAAA 0.21 0.07 ANP-Oligo 095 AAAAAAACUUCAAAAAAAA 0.22 0.07 ANP-Oligo 186 AAAAAAAUUCGAAAAAAAA 0.22 0.09 ANP-Oligo 058 AAAAAAACAGUAAAAAAAA 0.22 0.09 ANP-Oligo 167 AAAAAAAUCUCAAAAAAAA 0.23 0.07 ANP-Oligo 189 AAAAAAAUUGGAAAAAAAA 0.24 0.12 ANP-Oligo 047 AAAAAAAAUUCAAAAAAAA 0.27 0.09 ANP-Oligo 135 AAAAAAAGUAGAAAAAAAA 0.3 0.04 ANP-Oligo 154 AAAAAAAUAGUAAAAAAAA 0.32 0.03 ANP-Oligo 136 AAAAAAAGUAUAAAAAAAA 0.36 0.07 ANP-Oligo 134 AAAAAAAGUACAAAAAAAA 0.37 0.08 ANP-Oligo 140 AAAAAAAGUGCAAAAAAAA 0.38 0.04 ANP-Oligo 141 AAAAAAAGUGGAAAAAAAA 0.39 0.06 ANP-Oligo 178 AAAAAAAUGGUAAAAAAAA 0.41 0.03 ANP-Oligo 192 AAAAAAAUUUGAAAAAAAA 0.42 0.11 ANP-Oligo 138 AAAAAAAGUCGAAAAAAAA 0.44 0.05 ANP-Oligo 034 AAAAAAAAGGUAAAAAAAA 0.46 0.05 ANP-Oligo 180 AAAAAAAUGUGAAAAAAAA 0.46 0.07 ANP-Oligo 191 AAAAAAAUUUCAAAAAAAA 0.54 0.13 ANP-Oligo 046 AAAAAAAAUGUAAAAAAAA 0.59 0.04 ANP-Oligo 037 AAAAAAAAGUUAAAAAAAA 0.62 0.13 ANP-Oligo 181 AAAAAAAUGUUAAAAAAAA 0.64 0.07 ANP-Oligo 083 AAAAAAACGUCAAAAAAAA 0.68 0.15 ANP-Oligo 094 AAAAAAACUGUAAAAAAAA 0.73 0.09 ANP-Oligo 179 AAAAAAAUGUCAAAAAAAA 0.73 0.04 ANP-Oligo 190 AAAAAAAUUGUAAAAAAAA 0.74 0.07 ANP-Oligo 121 AAAAAAAGCUUAAAAAAAA 0.77 0.07 ANP-Oligo 137 AAAAAAAGUCCAAAAAAAA 0.79 0.06 ANP-Oligo 139 AAAAAAAGUCUAAAAAAAA 0.84 0.08 ANP-Oligo 131 AAAAAAAGGUCAAAAAAAA 0.93 0.2 ANP-Oligo 142 AAAAAAAGUGUAAAAAAAA 0.96 0.08 ANP-Oligo 133 AAAAAAAGGUUAAAAAAAA 1.04 0.13 ANP-Oligo 145 AAAAAAAGUUUAAAAAAAA 1.17 0.08 ANP-Oligo 144 AAAAAAAGUUGAAAAAAAA 1.22 0.08 ANP-Oligo 119 AAAAAAAGCUCAAAAAAAA 1.26 0.15 ANP-Oligo 035 AAAAAAAAGUCAAAAAAAA 1.33 0.28 ANP-Oligo 143 AAAAAAAGUUCAAAAAAAA 1.35 0.11

TABLE-US-00019 TABLE 4 ssRNA oligonucleotides - FIG. 7A Name Sequence 5' → 3' ANP-Oligo 194 AAGUCAAAAAAAAAAAAAA ANP-Oligo 195 AAAAAAGUCAAAAAAAAAA ANP-Oligo 035 AAAAAAAAGUCAAAAAAAA ANP-Oligo 196 AAAAAAAAAAGUCAAAAAA ANP-Oligo 197 AAAAAAAAAAAAAAGUCAA

TABLE-US-00020 TABLE 5 ssRNA oligonucleotides - FIG 7B Name Sequence 5' → 3' ANP-Oligo 035 AAAAAAAAGUCAAAAAAAA ANP-Oligo 198 AAAAAAAAGUCACAAAAAA ANP-Oligo 199 AAAAAAAAGUCAGAAAAAA ANP-Oligo 200 AAAAAAAAGUCAUAAAAAA ANP-Oligo 083 AAAAAAACGUCAAAAAAAA ANP-Oligo 201 AAAAAAACGUCACAAAAAA ANP-Oligo 202 AAAAAAACGUCAGAAAAAA ANP-Oligo 203 AAAAAAACGUCAUAAAAAA ANP-Oligo 131 AAAAAAAGGUCAAAAAAAA ANP-Oligo 204 AAAAAAAGGUCACAAAAAA ANP-Oligo 205 AAAAAAAGGUCAGAAAAAA ANP-Oligo 206 AAAAAAAGGUCAUAAAAAA ANP-Oligo 179 AAAAAAAUGUCAAAAAAAA ANP-Oligo 207 AAAAAAAUGUCACAAAAAA ANP-Oligo 208 AAAAAAAUGUCAGAAAAAA ANP-Oligo 209 AAAAAAAUGUCAUAAAAAA

TABLE-US-00021 TABLE 6 ssRNA oligonucleotides -- FIG. 8A Oligo-name Sequence 5' → 3' 9.2 sense AGCUUAACCUGUCCUUCAA L7A AAAAAAACCUGUCCUUCAA L8A AAAAAAAACUGUCCUUCAA L9A AAAAAAAAAUGUCCUUCAA L10A AAAAAAAAAAGUCCUUCAA L11A AAAAAAAAAAAUCCUUCAA L12A AAAAAAAAAAAACCUUCAA R9A AGCUUAACCUAAAAAAAAA R8A AGCUUAACCUGAAAAAAAA R7A AGCUUAACCUGUAAAAAAA R6A AGCUUAACCUGUCAAAAAA R5A AGCUUAACCUGUCCAAAAA R4A AGCUUAACCUGUCCUAAAA R3A AGCUUAACCUGUCCUUAAA

TABLE-US-00022 TABLE 12 3mer motifs and their mean levels of IFN-a induction A. 3mer motifs 5'-NNN-3' Mean (IFN-α point Motif Occurrences score) Sem p-value AAA 2192 0.00 0.01 0.389 AAC 67 -0.14 0.02 ** 0.001 AAG 67 0.11 0.06 ** 0.009 AAU 67 0.01 0.03 0.841 ACA 31 -0.17 0.02 ** 0.007 ACC 19 -0.19 0.01 * 0.017 ACG 19 -0.12 0.05 0.128 ACU 19 -0.07 0.05 0.407 AGA 31 -0.14 0.02 * 0.021 AGC 19 -0.05 0.09 0.508 AGG 19 -0.01 0.09 0.877 AGU 19 0.59 0.10 ** <0.001 AUA 31 -0.10 0.02 0.118 AUC 19 -0.11 0.03 0.176 AUG 19 0.08 0.07 0.318 AUU 19 0.09 0.06 0.253 CAA 67 0.00 0.05 0.913 CAC 7 -0.21 0.01 0.11 CAG 7 -0.13 0.06 0.313 CAU 7 -0.19 0.01 0.143 CCA 19 -0.14 0.05 0.077 CCC 7 -0.21 0.01 0.102 CCG 7 -0.21 0.01 0.11 CCU 7 -0.15 0.03 0.252 CGA 19 -0.14 0.04 0.082 CGC 7 -0.19 0.02 0.155 CGG 7 -0.18 0.03 0.172 CGU 7 0.05 0.11 0.678 CUA 19 -0.09 0.05 0.251 CUC 7 0.16 0.19 0.229 CUG 7 0.00 0.12 0.979 CUU 7 0.08 0.12 0.529 GAA 67 -0.08 0.03 * 0.044 GAC 7 -0.19 0.02 0.153 GAG 7 -0.13 0.06 0.306 GAU 7 -0.11 0.01 0.385 GCA 19 -0.12 0.04 0.114 GCC 7 -0.19 0.03 0.149 GCG 7 -0.17 0.03 0.184 GCU 7 0.22 0.21 0.094 GGA 19 -0.13 0.04 0.111 GGC 7 -0.17 0.04 0.194 GGG 7 -0.22 0.00 0.095 GGU 7 0.40 0.18 ** 0.002 GUA 19 0.27 0.08 ** <0.001 GUC 7 0.82 0.10 ** <0.001 GUG 7 0.32 0.13 * 0.014 GUU 7 0.87 0.18 ** <0.001 UAA 67 0.06 0.04 0.135 UAC 7 -0.06 0.08 0.647 UAG 7 0.01 0.08 0.94 UAU 7 -0.03 0.07 0.793 UCA 19 0.35 0.12 ** <0.001 UCC 7 -0.03 0.14 0.794 UCG 7 0.02 0.09 0.848 UCU 7 0.10 0.13 0.421 UGA 19 0.04 0.08 0.611 UGC 7 0.01 0.07 0.91 UGG 7 0.10 0.09 0.448 UGU 7 0.69 0.06 ** <0.001 UUA 19 0.17 0.10 * 0.036 UUC 7 0.37 0.18 ** 0.005 UUG 7 0.38 0.18 ** 0.004 UUU 7 0.32 0.17 * 0.013 B. 3mer motifs 5'-NN-N-3' Motif Occurences Mean Sem p-value AANA 1864 -0.01 0.01 0.096 AANC 112 -0.12 0.02 ** <0.001 AANG 112 0.07 0.04 * 0.021 AANU 112 0.11 0.03 ** <0.001 ACNA 40 -0.17 0.02 ** 0.002 ACNC 16 -0.18 0.02 * 0.035 ACNG 16 -0.10 0.06 0.244 ACNU 16 -0.05 0.06 0.533 AG NA 40 -0.09 0.03 0.111 AGNC 16 0.06 0.12 0.46 AGNG 16 -0.02 0.09 0.805 AGNU 16 0.52 0.15 ** <0.001 AUNA 40 -0.11 0.02 * 0.038 AUNC 16 -0.07 0.04 0.398 AU NG 16 0.05 0.07 0.556 AUNU 16 0.19 0.08 * 0.027 CANA 76 -0.02 0.04 0.668 CANC 4 -0.22 0.01 0.209 CANG 4 -0.22 0.00 0.201 CANU 4 -0.10 0.11 0.577 CCNA 28 -0.16 0.04 * 0.015 CCNC 4 -0.16 0.05 0.365 CCNG 4 -0.21 0.01 0.216 CCNU 4 -0.18 0.02 0.295 CGNA 28 -0.14 0.03 * 0.036 CGNC 4 0.00 0.23 0.981 CGNG 4 -0.19 0.03 0.268 CGNU 4 -0.05 0.03 0.765 CUNA 28 0.01 0.07 0.896 CUNC 4 -0.08 0.10 0.649 CUNG 4 -0.09 0.04 0.616 CUNU 4 0.10 0.21 0.567 GANA 76 -0.09 0.03 * 0.017 GANC 4 -0.17 0.03 0.324 GANG 4 -0.19 0.02 0.281 GANU 4 -0.03 0.08 0.87 GCNA 28 -0.13 0.03 * 0.044 GCNC 4 0.15 0.37 0.38 GONG 4 -0.18 0.04 0.298 GCNU 4 0.10 0.22 0.548 GGNA 28 -0.10 0.04 0.131 GGNC 4 0.07 0.29 0.697 GGNG 4 -0.12 0.10 0.492 GGNU 4 0.16 0.30 0.353 GUNA 28 0.38 0.08 ** <0.001 GUNC 4 0.72 0.23 ** <0.001 GUNG 4 0.59 0.21 ** <0.001 GUNU 4 0.83 0.17 ** <0.001 UANA 76 0.06 0.04 0.15 UANC 4 -0.09 0.02 0.599 UANG 4 -0.08 0.02 0.641 UANU 4 0.00 0.11 0.999 UCNA 28 0.30 0.10 ** <0.001 UCNC 4 -0.06 0.10 0.738 UCNG 4 -0.11 0.04 0.541

UCNU 4 -0.07 0.05 0.706 UGNA 28 0.17 0.07 ** 0.008 UGNC 4 0.16 0.19 0.351 UGNG 4 0.04 0.14 0.817 UGNU 4 0.20 0.19 0.239 UUNA 28 0.30 0.09 ** <0.001 UUNC 4 0.15 0.14 0.377 UUNG 4 0.19 0.11 0.267 UUNU 4 0.21 0.18 0.228 C. 3mer motifs 5'-N-NN-3' Motif Occurences Mean Sem p-value ANAA 1864 -0.01 0.01 0.107 ANAC 76 -0.14 0.02 ** <0.001 ANAG 76 0.09 0.05 * 0.019 ANAU 76 -0.01 0.03 0.78 ANCA 40 -0.18 0.01 ** 0.001 ANCC 28 -0.20 0.01 ** 0.003 ANCG 28 -0.13 0.03 * 0.048 ANCU 28 0.02 0.07 0.767 ANGA 40 -0.16 0.01 ** 0.004 ANGC 28 -0.08 0.06 0.207 ANGG 28 -0.04 0.07 0.533 ANGU 28 0.57 0.08 ** <0.001 ANUA 40 -0.05 0.03 0.366 ANUC 28 0.06 0.07 0.334 ANUG 28 0.18 0.07 ** 0.007 ANUU 28 0.27 0.08 ** <0.001 CNAA 112 -0.04 0.03 0.218 CNAC 4 -0.21 0.01 0.215 CNAG 4 -0.19 0.03 0.265 CNAU 4 -0.16 0.03 0.361 CNCA 16 -0.05 0.09 0.537 CNCC 4 -0.21 0.01 0.218 CNCG 4 -0.20 0.02 0.258 CNCU 4 -0.17 0.02 0.334 CNGA 16 -0.18 0.02 * 0.039 CNGC 4 -0.19 0.02 0.262 CNGG 4 -0.20 0.02 0.251 CNGU 4 0.20 0.19 0.244 CNUA 16 -0.07 0.06 0.389 CNUC 4 0.17 0.19 0.318 CNUG 4 -0.13 0.06 0.464 CNUU 4 -0.11 0.05 0.538 GNAA 112 -0.04 0.03 0.186 GNAC 4 -0.06 0.14 0.717 GNAG 4 -0.08 0.12 0.649 GNAU 4 0.02 0.11 0.89 GNCA 16 0.10 0.13 0.243 GNCC 4 0.03 0.25 0.842 GNCG 4 -0.06 0.17 0.745 GNCU 4 0.11 0.24 0.534 GNGA 16 -0.11 0.05 0.203 GNGC 4 -0.07 0.15 0.688 GNGG 4 -0.07 0.15 0.706 GNGU 4 0.22 0.26 0.207 GNUA 16 0.13 0.10 0.14 GNUC 4 0.87 0.33 ** <0.001 GNUG 4 0.30 0.31 0.08 GNUU 4 0.72 0.29 ** <0.001 UNAA 112 0.14 0.04 ** <0.001 UNAC 4 -0.09 0.04 0.603 UNAG 4 -0.08 0.01 0.626 UNAU 4 -0.11 0.02 0.522 UNCA 16 0.18 0.11 * 0.032 UNCC 4 -0.11 0.03 0.528 UNCG 4 -0.04 0.09 0.807 UNCU 4 -0.08 0.05 0.634 UNGA 16 0.14 0.09 0.113 UNGC 4 -0.01 0.06 0.975 UNGG 4 -0.03 0.09 0.86 UNGU 4 0.39 0.14 * 0.025 UNUA 16 0.29 0.12 ** 0.001 UNUC 4 0.37 0.17 * 0.032 UNUG 4 0.20 0.14 0.24 UNUU 4 0.15 0.17 0.375

TABLE-US-00023 TABLE 13 RNA oligonucleotide containing multiple copies of GUCA Oligo-name Sequence 5' → 3' 9.2 sense AGCUUAACCUGUCCUUCAA Poly GUCA GUCAAGUCAAGUCAAGUCAA ANP35 AAAAAAAAGUCAAAAAAAA

Sequence CWU 1

390119RNAArtificial SequenceSynthetic 1aaaaaaaaaa aaaaaaaaa 19219RNAArtificial SequenceSynthetic 2aaaaaaaaca aaaaaaaaa 19319RNAArtificial SequenceSynthetic 3aaaaaaaaga aaaaaaaaa 19419RNAArtificial SequenceSynthetic 4aaaaaaaaua aaaaaaaaa 19519RNAArtificial SequenceSynthetic 5aaaaaaaacc aaaaaaaaa 19619RNAArtificial SequenceSynthetic 6aaaaaaaacg aaaaaaaaa 19719RNAArtificial SequenceSynthetic 7aaaaaaaacu aaaaaaaaa 19819RNAArtificial SequenceSynthetic 8aaaaaaaagc aaaaaaaaa 19919RNAArtificial SequenceSynthetic 9aaaaaaaagg aaaaaaaaa 191019RNAArtificial SequenceSynthetic 10aaaaaaaagu aaaaaaaaa 191119RNAArtificial SequenceSynthetic 11aaaaaaaauc aaaaaaaaa 191219RNAArtificial SequenceSynthetic 12aaaaaaaaug aaaaaaaaa 191319RNAArtificial SequenceSynthetic 13aaaaaaaauu aaaaaaaaa 191419RNAArtificial SequenceSynthetic 14aaaaaaaaca caaaaaaaa 191519RNAArtificial SequenceSynthetic 15aaaaaaaaca gaaaaaaaa 191619RNAArtificial SequenceSynthetic 16aaaaaaaaca uaaaaaaaa 191719RNAArtificial SequenceSynthetic 17aaaaaaaacc caaaaaaaa 191819RNAArtificial SequenceSynthetic 18aaaaaaaacc gaaaaaaaa 191919RNAArtificial SequenceSynthetic 19aaaaaaaacc uaaaaaaaa 192019RNAArtificial SequenceSynthetic 20aaaaaaaacg caaaaaaaa 192119RNAArtificial SequenceSynthetic 21aaaaaaaacg gaaaaaaaa 192219RNAArtificial SequenceSynthetic 22aaaaaaaacg uaaaaaaaa 192319RNAArtificial SequenceSynthetic 23aaaaaaaacu caaaaaaaa 192419RNAArtificial SequenceSynthetic 24aaaaaaaacu gaaaaaaaa 192519RNAArtificial SequenceSynthetic 25aaaaaaaacu uaaaaaaaa 192619RNAArtificial SequenceSynthetic 26aaaaaaaaga caaaaaaaa 192719RNAArtificial SequenceSynthetic 27aaaaaaaaga gaaaaaaaa 192819RNAArtificial SequenceSynthetic 28aaaaaaaaga uaaaaaaaa 192919RNAArtificial SequenceSynthetic 29aaaaaaaagc caaaaaaaa 193019RNAArtificial SequenceSynthetic 30aaaaaaaagc gaaaaaaaa 193119RNAArtificial SequenceSynthetic 31aaaaaaaagc uaaaaaaaa 193219RNAArtificial SequenceSynthetic 32aaaaaaaagg caaaaaaaa 193319RNAArtificial SequenceSynthetic 33aaaaaaaagg gaaaaaaaa 193419RNAArtificial SequenceSynthetic 34aaaaaaaagg uaaaaaaaa 193519RNAArtificial SequenceSynthetic 35aaaaaaaagu caaaaaaaa 193619RNAArtificial SequenceSynthetic 36aaaaaaaagu gaaaaaaaa 193719RNAArtificial SequenceSynthetic 37aaaaaaaagu uaaaaaaaa 193819RNAArtificial SequenceSynthetic 38aaaaaaaaua caaaaaaaa 193919RNAArtificial SequenceSynthetic 39aaaaaaaaua gaaaaaaaa 194019RNAArtificial SequenceSynthetic 40aaaaaaaaua uaaaaaaaa 194119RNAArtificial SequenceSynthetic 41aaaaaaaauc caaaaaaaa 194219RNAArtificial SequenceSynthetic 42aaaaaaaauc gaaaaaaaa 194319RNAArtificial SequenceSynthetic 43aaaaaaaauc uaaaaaaaa 194419RNAArtificial SequenceSynthetic 44aaaaaaaaug caaaaaaaa 194519RNAArtificial SequenceSynthetic 45aaaaaaaaug gaaaaaaaa 194619RNAArtificial SequenceSynthetic 46aaaaaaaaug uaaaaaaaa 194719RNAArtificial SequenceSynthetic 47aaaaaaaauu caaaaaaaa 194819RNAArtificial SequenceSynthetic 48aaaaaaaauu gaaaaaaaa 194919RNAArtificial SequenceSynthetic 49aaaaaaaauu uaaaaaaaa 195019RNAArtificial SequenceSynthetic 50aaaaaaacaa caaaaaaaa 195119RNAArtificial SequenceSynthetic 51aaaaaaacaa gaaaaaaaa 195219RNAArtificial SequenceSynthetic 52aaaaaaacaa uaaaaaaaa 195319RNAArtificial SequenceSynthetic 53aaaaaaacac caaaaaaaa 195419RNAArtificial SequenceSynthetic 54aaaaaaacac gaaaaaaaa 195519RNAArtificial SequenceSynthetic 55aaaaaaacac uaaaaaaaa 195619RNAArtificial SequenceSynthetic 56aaaaaaacag caaaaaaaa 195719RNAArtificial SequenceSynthetic 57aaaaaaacag gaaaaaaaa 195819RNAArtificial SequenceSynthetic 58aaaaaaacag uaaaaaaaa 195919RNAArtificial SequenceSynthetic 59aaaaaaacau caaaaaaaa 196019RNAArtificial SequenceSynthetic 60aaaaaaacau gaaaaaaaa 196119RNAArtificial SequenceSynthetic 61aaaaaaacau uaaaaaaaa 196219RNAArtificial SequenceSynthetic 62aaaaaaacca caaaaaaaa 196319RNAArtificial SequenceSynthetic 63aaaaaaacca gaaaaaaaa 196419RNAArtificial SequenceSynthetic 64aaaaaaacca uaaaaaaaa 196519RNAArtificial SequenceSynthetic 65aaaaaaaccc caaaaaaaa 196619RNAArtificial SequenceSynthetic 66aaaaaaaccc gaaaaaaaa 196719RNAArtificial SequenceSynthetic 67aaaaaaaccc uaaaaaaaa 196819RNAArtificial SequenceSynthetic 68aaaaaaaccg caaaaaaaa 196919RNAArtificial SequenceSynthetic 69aaaaaaaccg gaaaaaaaa 197019RNAArtificial SequenceSynthetic 70aaaaaaaccg uaaaaaaaa 197119RNAArtificial SequenceSynthetic 71aaaaaaaccu caaaaaaaa 197219RNAArtificial SequenceSynthetic 72aaaaaaaccu gaaaaaaaa 197319RNAArtificial SequenceSynthetic 73aaaaaaaccu uaaaaaaaa 197419RNAArtificial SequenceSynthetic 74aaaaaaacga caaaaaaaa 197519RNAArtificial SequenceSynthetic 75aaaaaaacga gaaaaaaaa 197619RNAArtificial SequenceSynthetic 76aaaaaaacga uaaaaaaaa 197719RNAArtificial SequenceSynthetic 77aaaaaaacgc caaaaaaaa 197819RNAArtificial SequenceSynthetic 78aaaaaaacgc gaaaaaaaa 197919RNAArtificial SequenceSynthetic 79aaaaaaacgc uaaaaaaaa 198019RNAArtificial SequenceSynthetic 80aaaaaaacgg caaaaaaaa 198119RNAArtificial SequenceSynthetic 81aaaaaaacgg gaaaaaaaa 198219RNAArtificial SequenceSynthetic 82aaaaaaacgg uaaaaaaaa 198319RNAArtificial SequenceSynthetic 83aaaaaaacgu caaaaaaaa 198419RNAArtificial SequenceSynthetic 84aaaaaaacgu gaaaaaaaa 198519RNAArtificial SequenceSynthetic 85aaaaaaacgu uaaaaaaaa 198619RNAArtificial SequenceSynthetic 86aaaaaaacua caaaaaaaa 198719RNAArtificial SequenceSynthetic 87aaaaaaacua gaaaaaaaa 198819RNAArtificial SequenceSynthetic 88aaaaaaacua uaaaaaaaa 198919RNAArtificial SequenceSynthetic 89aaaaaaacuc caaaaaaaa 199019RNAArtificial SequenceSynthetic 90aaaaaaacuc gaaaaaaaa 199119RNAArtificial SequenceSynthetic 91aaaaaaacuc uaaaaaaaa 199219RNAArtificial SequenceSynthetic 92aaaaaaacug caaaaaaaa 199319RNAArtificial SequenceSynthetic 93aaaaaaacug gaaaaaaaa 199419RNAArtificial SequenceSynthetic 94aaaaaaacug uaaaaaaaa 199519RNAArtificial SequenceSynthetic 95aaaaaaacuu caaaaaaaa 199619RNAArtificial SequenceSynthetic 96aaaaaaacuu gaaaaaaaa 199719RNAArtificial SequenceSynthetic 97aaaaaaacuu uaaaaaaaa 199819RNAArtificial SequenceSynthetic 98aaaaaaagaa caaaaaaaa 199919RNAArtificial SequenceSynthetic 99aaaaaaagaa gaaaaaaaa 1910019RNAArtificial SequenceSynthetic 100aaaaaaagaa uaaaaaaaa 1910119RNAArtificial SequenceSynthetic 101aaaaaaagac caaaaaaaa 1910219RNAArtificial SequenceSynthetic 102aaaaaaagac gaaaaaaaa 1910319RNAArtificial SequenceSynthetic 103aaaaaaagac uaaaaaaaa 1910419RNAArtificial SequenceSynthetic 104aaaaaaagag caaaaaaaa 1910519RNAArtificial SequenceSynthetic 105aaaaaaagag gaaaaaaaa 1910619RNAArtificial SequenceSynthetic 106aaaaaaagag uaaaaaaaa 1910719RNAArtificial SequenceSynthetic 107aaaaaaagau caaaaaaaa 1910819RNAArtificial SequenceSynthetic 108aaaaaaagau gaaaaaaaa 1910919RNAArtificial SequenceSynthetic 109aaaaaaagau uaaaaaaaa 1911019RNAArtificial SequenceSynthetic 110aaaaaaagca caaaaaaaa 1911119RNAArtificial SequenceSynthetic 111aaaaaaagca gaaaaaaaa 1911219RNAArtificial SequenceSynthetic 112aaaaaaagca uaaaaaaaa 1911319RNAArtificial SequenceSynthetic 113aaaaaaagcc caaaaaaaa 1911419RNAArtificial SequenceSynthetic 114aaaaaaagcc gaaaaaaaa 1911519RNAArtificial SequenceSynthetic 115aaaaaaagcc uaaaaaaaa 1911619RNAArtificial SequenceSynthetic 116aaaaaaagcg caaaaaaaa 1911719RNAArtificial SequenceSynthetic 117aaaaaaagcg gaaaaaaaa 1911819RNAArtificial SequenceSynthetic 118aaaaaaagcg uaaaaaaaa 1911919RNAArtificial SequenceSynthetic 119aaaaaaagcu caaaaaaaa 1912019RNAArtificial SequenceSynthetic 120aaaaaaagcu gaaaaaaaa 1912119RNAArtificial SequenceSynthetic 121aaaaaaagcu uaaaaaaaa 1912219RNAArtificial SequenceSynthetic 122aaaaaaagga caaaaaaaa 1912319RNAArtificial SequenceSynthetic 123aaaaaaagga gaaaaaaaa 1912419RNAArtificial SequenceSynthetic 124aaaaaaagga uaaaaaaaa 1912519RNAArtificial SequenceSynthetic 125aaaaaaaggc caaaaaaaa 1912619RNAArtificial SequenceSynthetic 126aaaaaaaggc gaaaaaaaa 1912719RNAArtificial SequenceSynthetic 127aaaaaaaggc uaaaaaaaa 1912819RNAArtificial SequenceSynthetic 128aaaaaaaggg caaaaaaaa 1912919RNAArtificial SequenceSynthetic 129aaaaaaaggg gaaaaaaaa 1913019RNAArtificial SequenceSynthetic 130aaaaaaaggg uaaaaaaaa 1913119RNAArtificial SequenceSynthetic 131aaaaaaaggu caaaaaaaa 1913219RNAArtificial SequenceSynthetic 132aaaaaaaggu gaaaaaaaa 1913319RNAArtificial SequenceSynthetic 133aaaaaaaggu uaaaaaaaa 1913419RNAArtificial SequenceSynthetic 134aaaaaaagua caaaaaaaa 1913519RNAArtificial SequenceSynthetic 135aaaaaaagua gaaaaaaaa 1913619RNAArtificial SequenceSynthetic 136aaaaaaagua uaaaaaaaa 1913719RNAArtificial SequenceSynthetic 137aaaaaaaguc caaaaaaaa 1913819RNAArtificial SequenceSynthetic 138aaaaaaaguc gaaaaaaaa 1913919RNAArtificial SequenceSynthetic 139aaaaaaaguc uaaaaaaaa 1914019RNAArtificial SequenceSynthetic 140aaaaaaagug caaaaaaaa 1914119RNAArtificial SequenceSynthetic 141aaaaaaagug gaaaaaaaa 1914219RNAArtificial SequenceSynthetic 142aaaaaaagug uaaaaaaaa 1914319RNAArtificial SequenceSynthetic 143aaaaaaaguu caaaaaaaa 1914419RNAArtificial SequenceSynthetic 144aaaaaaaguu gaaaaaaaa 1914519RNAArtificial SequenceSynthetic 145aaaaaaaguu uaaaaaaaa 1914619RNAArtificial SequenceSynthetic 146aaaaaaauaa caaaaaaaa 1914719RNAArtificial SequenceSynthetic 147aaaaaaauaa gaaaaaaaa 1914819RNAArtificial SequenceSynthetic 148aaaaaaauaa uaaaaaaaa 1914919RNAArtificial SequenceSynthetic 149aaaaaaauac caaaaaaaa 1915019RNAArtificial SequenceSynthetic 150aaaaaaauac gaaaaaaaa 1915119RNAArtificial SequenceSynthetic 151aaaaaaauac uaaaaaaaa 1915219RNAArtificial SequenceSynthetic 152aaaaaaauag caaaaaaaa 1915319RNAArtificial SequenceSynthetic 153aaaaaaauag gaaaaaaaa 1915419RNAArtificial SequenceSynthetic 154aaaaaaauag uaaaaaaaa 1915519RNAArtificial SequenceSynthetic 155aaaaaaauau caaaaaaaa 1915619RNAArtificial SequenceSynthetic 156aaaaaaauau gaaaaaaaa 1915719RNAArtificial SequenceSynthetic 157aaaaaaauau uaaaaaaaa 1915819RNAArtificial SequenceSynthetic 158aaaaaaauca caaaaaaaa 1915919RNAArtificial SequenceSynthetic 159aaaaaaauca gaaaaaaaa 1916019RNAArtificial SequenceSynthetic 160aaaaaaauca uaaaaaaaa 1916119RNAArtificial SequenceSynthetic 161aaaaaaaucc

caaaaaaaa 1916219RNAArtificial SequenceSynthetic 162aaaaaaaucc gaaaaaaaa 1916319RNAArtificial SequenceSynthetic 163aaaaaaaucc uaaaaaaaa 1916419RNAArtificial SequenceSynthetic 164aaaaaaaucg caaaaaaaa 1916519RNAArtificial SequenceSynthetic 165aaaaaaaucg gaaaaaaaa 1916619RNAArtificial SequenceSynthetic 166aaaaaaaucg uaaaaaaaa 1916719RNAArtificial SequenceSynthetic 167aaaaaaaucu caaaaaaaa 1916819RNAArtificial SequenceSynthetic 168aaaaaaaucu gaaaaaaaa 1916919RNAArtificial SequenceSynthetic 169aaaaaaaucu uaaaaaaaa 1917019RNAArtificial SequenceSynthetic 170aaaaaaauga caaaaaaaa 1917119RNAArtificial SequenceSynthetic 171aaaaaaauga gaaaaaaaa 1917219RNAArtificial SequenceSynthetic 172aaaaaaauga uaaaaaaaa 1917319RNAArtificial SequenceSynthetic 173aaaaaaaugc caaaaaaaa 1917419RNAArtificial SequenceSynthetic 174aaaaaaaugc gaaaaaaaa 1917519RNAArtificial SequenceSynthetic 175aaaaaaaugc uaaaaaaaa 1917619RNAArtificial SequenceSynthetic 176aaaaaaaugg caaaaaaaa 1917719RNAArtificial SequenceSynthetic 177aaaaaaaugg gaaaaaaaa 1917819RNAArtificial SequenceSynthetic 178aaaaaaaugg uaaaaaaaa 1917919RNAArtificial SequenceSynthetic 179aaaaaaaugu caaaaaaaa 1918019RNAArtificial SequenceSynthetic 180aaaaaaaugu gaaaaaaaa 1918119RNAArtificial SequenceSynthetic 181aaaaaaaugu uaaaaaaaa 1918219RNAArtificial SequenceSynthetic 182aaaaaaauua caaaaaaaa 1918319RNAArtificial SequenceSynthetic 183aaaaaaauua gaaaaaaaa 1918419RNAArtificial SequenceSynthetic 184aaaaaaauua uaaaaaaaa 1918519RNAArtificial SequenceSynthetic 185aaaaaaauuc caaaaaaaa 1918619RNAArtificial SequenceSynthetic 186aaaaaaauuc gaaaaaaaa 1918719RNAArtificial SequenceSynthetic 187aaaaaaauuc uaaaaaaaa 1918819RNAArtificial SequenceSynthetic 188aaaaaaauug caaaaaaaa 1918919RNAArtificial SequenceSynthetic 189aaaaaaauug gaaaaaaaa 1919019RNAArtificial SequenceSynthetic 190aaaaaaauug uaaaaaaaa 1919119RNAArtificial SequenceSynthetic 191aaaaaaauuu caaaaaaaa 1919219RNAArtificial SequenceSynthetic 192aaaaaaauuu gaaaaaaaa 1919319RNAArtificial SequenceSynthetic 193aaaaaaauuu uaaaaaaaa 1919419RNAArtificial SequenceSynthetic 194aagucaaaaa aaaaaaaaa 1919519RNAArtificial SequenceSynthetic 195aaaaaaguca aaaaaaaaa 1919619RNAArtificial SequenceSynthetic 196aaaaaaaagu caaaaaaaa 1919719RNAArtificial SequenceSynthetic 197aaaaaaaaaa gucaaaaaa 1919819RNAArtificial SequenceSynthetic 198aaaaaaaaaa aaaagucaa 1919919RNAArtificial SequenceSynthetic 199aaaaaaaagu caaaaaaaa 1920019RNAArtificial SequenceSynthetic 200aaaaaaaagu cacaaaaaa 1920119RNAArtificial SequenceSynthetic 201aaaaaaaagu cagaaaaaa 1920219RNAArtificial SequenceSynthetic 202aaaaaaaagu cauaaaaaa 1920319RNAArtificial SequenceSynthetic 203aaaaaaacgu caaaaaaaa 1920419RNAArtificial SequenceSynthetic 204aaaaaaacgu cacaaaaaa 1920519RNAArtificial SequenceSynthetic 205aaaaaaacgu cagaaaaaa 1920619RNAArtificial SequenceSynthetic 206aaaaaaacgu cauaaaaaa 1920719RNAArtificial SequenceSynthetic 207aaaaaaaggu caaaaaaaa 1920819RNAArtificial SequenceSynthetic 208aaaaaaaggu cacaaaaaa 1920919RNAArtificial SequenceSynthetic 209aaaaaaaggu cagaaaaaa 1921019RNAArtificial SequenceSynthetic 210aaaaaaaggu cauaaaaaa 1921119RNAArtificial SequenceSynthetic 211aaaaaaaugu caaaaaaaa 1921219RNAArtificial SequenceSynthetic 212aaaaaaaugu cacaaaaaa 1921319RNAArtificial SequenceSynthetic 213aaaaaaaugu cagaaaaaa 1921419RNAArtificial SequenceSynthetic 214aaaaaaaugu cauaaaaaa 1921519RNAArtificial SequenceSynthetic 215agcuuaaccu guccuucaa 1921619RNAArtificial SequenceSynthetic 216aaaaaaaccu guccuucaa 1921719RNAArtificial SequenceSynthetic 217aaaaaaaacu guccuucaa 1921819RNAArtificial SequenceSynthetic 218aaaaaaaaau guccuucaa 1921919RNAArtificial SequenceSynthetic 219aaaaaaaaaa guccuucaa 1922019RNAArtificial SequenceSynthetic 220aaaaaaaaaa auccuucaa 1922119RNAArtificial SequenceSynthetic 221aaaaaaaaaa aaccuucaa 1922219RNAArtificial SequenceSynthetic 222agcuuaaccu aaaaaaaaa 1922319RNAArtificial SequenceSynthetic 223agcuuaaccu gaaaaaaaa 1922419RNAArtificial SequenceSynthetic 224agcuuaaccu guaaaaaaa 1922519RNAArtificial SequenceSynthetic 225agcuuaaccu gucaaaaaa 1922619RNAArtificial SequenceSynthetic 226agcuuaaccu guccaaaaa 1922719RNAArtificial SequenceSynthetic 227agcuuaaccu guccuaaaa 1922819RNAArtificial SequenceSynthetic 228agcuuaaccu guccuuaaa 1922920RNAArtificial SequenceSynthetic 229gcccgucugu ugugugacuc 2023019RNAArtificial SequenceSynthetic 230uugauguguu uagucgcua 1923119RNAArtificial SequenceSynthetic 231caucucuccc ugcucucug 1923219RNAArtificial SequenceSynthetic 232cagagagcag ggagagaug 1923319RNAArtificial SequenceSynthetic 233ucgccggccu gcaugcccu 1923419RNAArtificial SequenceSynthetic 234agggcaugca ggccggcga 1923519RNAArtificial SequenceSynthetic 235gaggcagaug gaggggaga 1923619RNAArtificial SequenceSynthetic 236ucuccccucc aucugccuc 1923719RNAArtificial SequenceSynthetic 237aguaucugcu guuguccua 1923819RNAArtificial SequenceSynthetic 238uaggacaaca gcagauacu 1923919RNAArtificial SequenceSynthetic 239ugguguugaa ggacaguuc 1924019RNAArtificial SequenceSynthetic 240gaacuguccu ucaacacca 1924119RNAArtificial SequenceSynthetic 241aaccugagcu acaacaaca 1924219RNAArtificial SequenceSynthetic 242uguuguugua gcucagguu 1924321RNAArtificial SequenceSynthetic 243gaccuagccu aaaacuaggu c 2124417RNAArtificial SequenceSynthetic 244aaagauccgg aucaaaa 1724519RNAArtificial SequenceSynthetic 245aaaaguucaa agcucaaaa 1924611RNAArtificial SequenceSynthetic 246caaguuucga g 1124733RNAArtificial SequenceSynthetic 247ucaaagucaa aagcucaaag uugaaaguuu aaa 3324841RNAArtificial SequenceSynthetic 248gacuugaaaa uuucaguuuu cgaguuuaag uugaaaacuc g 4124926RNAArtificial SequenceSynthetic 249ucaaagucaa aagcucaaag uugaaa 2625031RNAArtificial SequenceSynthetic 250uuucaguuuu cgaguuuaag uugaaaacuc g 3125120RNAArtificial SequenceSynthetic 251cagagcggga ugcguugguc 2025219RNAArtificial SequenceSynthetic 252uugauguguu uagucgcua 1925319RNAArtificial SequenceSynthetic 253gcaccacuag uugguuguc 1925419RNAArtificial SequenceSynthetic 254guuguaguug uacuccagc 1925520RNAArtificial SequenceSynthetic 255gcccgucugu ugugugacuc 2025612RNAArtificial SequenceSynthetic 256gucuguugug ug 1225718RNAArtificial SequenceSynthetic 257guugugguug ugguugug 1825819RNAArtificial SequenceSynthetic 258aaaaaaaguu caaaaaaaa 1925919RNAArtificial SequenceSynthetic 259aaaguucaaa aaaaaaaaa 1926019RNAArtificial SequenceSynthetic 260aaaaaaaaaa aaguucaaa 1926119RNAArtificial SequenceSynthetic 261aaaguucaaa aaguucaaa 1926219RNAArtificial SequenceSynthetic 262guucaaaguu caaaguuca 1926319RNAArtificial SequenceSynthetic 263aguucaaagu caaaagcuc 1926422RNAArtificial SequenceSynthetic 264aguucaguuc aagucaaagc uc 2226545RNAArtificial SequenceSynthetic 265aaaguucaaa gucaaaagcu caaaguugaa aguuuaaagg uuaaa 4526617RNAArtificial SequenceSynthetic 266aaaaguugcu caaaaaa 1726710RNAArtificial SequenceSynthetic 267guagugugug 1026821RNAArtificial SequenceSynthetic 268guccgggcag gucuacuuuu u 2126921RNAArtificial SequenceSynthetic 269gcuggagauc cugaagaacu u 2127019RNAArtificial SequenceSynthetic 270uugauguguu uagucgcua 1927119RNAArtificial SequenceSynthetic 271uagcgacuaa acacaucaa 1927219RNAArtificial SequenceSynthetic 272aguagaaaca agguaguuu 1927319RNAArtificial SequenceSynthetic 273uuaacuaccu gcuuuugcu 1927442RNAArtificial SequenceSynthetic 274aguagaaaca agguaguuuu uuguuaacua ccugcuuuug cu 4227519RNAArtificial SequenceSynthetic 275agcagaaaca aggcagccc 1927619RNAArtificial SequenceSynthetic 276acuacaaaca accuacuuu 1927719RNAArtificial SequenceSynthetic 277guuuguugcu uugauugcc 1927819RNAArtificial SequenceSynthetic 278uuguaguucg uugcuagug 1927919RNAArtificial SequenceSynthetic 279aguucauggu ggguuguac 1928019RNAArtificial SequenceSynthetic 280uguuuaaguu guucuaccc 1928119RNAArtificial SequenceSynthetic 281aaguuuugau uuuucagua 1928219RNAArtificial SequenceSynthetic 282aggcguuugu guucggguu 1928319RNAArtificial SequenceSynthetic 283agauguugua ggguguuuu 1928419RNAArtificial SequenceSynthetic 284uagugugugu cagugugac 1928519RNAArtificial SequenceSynthetic 285gguugcgugu ggaguuguu 1928619RNAArtificial SequenceSynthetic 286uguaguuuug uuagaguca 1928719RNAArtificial SequenceSynthetic 287gugugguugc uguugucaa 1928819RNAArtificial SequenceSynthetic 288gggaccgaaa gaccagacc 1928919RNAArtificial SequenceSynthetic 289uaagacuaga agagacaga 1929019RNAArtificial SequenceSynthetic 290agauccgaac caccgacca 1929119RNAArtificial SequenceSynthetic 291gaaccagaaa auagagcag 1929219RNAArtificial SequenceSynthetic 292cauauaagaa gaccagcca 1929319RNAArtificial SequenceSynthetic 293uaagaaccaa cugcuagaa 1929419RNAArtificial SequenceSynthetic 294ccccuacaga cagaauacc 1929519RNAArtificial SequenceSynthetic 295cuggcagaua gauagaagc 1929619RNAArtificial SequenceSynthetic 296cuagaccaga acaaucucg 1929719RNAArtificial SequenceSynthetic 297uuagagacau aacaacauu 1929819RNAArtificial SequenceSynthetic 298ggaccaaacc ucucgacau 1929919RNAArtificial SequenceSynthetic 299uaacaaacuc cuaccaaca 1930019RNAArtificial SequenceSynthetic 300uguugguagg aguuuguua 1930119RNAArtificial SequenceSynthetic 301aacaaacucc uaccaacac 1930219RNAArtificial SequenceSynthetic 302guguugguag gaguuuguu 1930319RNAArtificial SequenceSynthetic 303uaccaacacu gaccaauaa 1930419RNAArtificial SequenceSynthetic 304uuauugguca guguuggua 1930519RNAArtificial SequenceSynthetic 305cuaccaacac ugaccaaua 1930619RNAArtificial SequenceSynthetic 306uauuggucag uguugguag 1930719RNAArtificial SequenceSynthetic 307accaacacug accaauaaa 1930819RNAArtificial SequenceSynthetic 308uuuauugguc aguguuggu 1930919RNAArtificial SequenceSynthetic 309acaaacuccu accaacacu 1931019RNAArtificial SequenceSynthetic 310aguguuggua ggaguuugu 1931119RNAArtificial SequenceSynthetic 311acuccuacca acacugacc

1931219RNAArtificial SequenceSynthetic 312ggucaguguu gguaggagu 1931319RNAArtificial SequenceSynthetic 313ccuaccaaca cugaccaau 1931419RNAArtificial SequenceSynthetic 314auuggucagu guugguagg 1931519RNAArtificial SequenceSynthetic 315uccuaccaac acugaccaa 1931619RNAArtificial SequenceSynthetic 316uuggucagug uugguagga 1931719RNAArtificial SequenceSynthetic 317aaacuccuac caacacuga 1931819RNAArtificial SequenceSynthetic 318ucaguguugg uaggaguuu 1931919RNAArtificial SequenceSynthetic 319ccaacacuga ccaauaaaa 1932019RNAArtificial SequenceSynthetic 320uuuuauuggu caguguugg 1932119RNAArtificial SequenceSynthetic 321caaacuccua ccaacacug 1932219RNAArtificial SequenceSynthetic 322caguguuggu aggaguuug 1932319RNAArtificial SequenceSynthetic 323aacuccuacc aacacugac 1932419RNAArtificial SequenceSynthetic 324gucaguguug guaggaguu 1932519RNAArtificial SequenceSynthetic 325aacacugacc aauaaaaaa 1932619RNAArtificial SequenceSynthetic 326uuuuuuauug gucaguguu 1932719RNAArtificial SequenceSynthetic 327caacacugac caauaaaaa 1932819RNAArtificial SequenceSynthetic 328uuuuuauugg ucaguguug 1932919RNAArtificial SequenceSynthetic 329gcuacaaaaa cagcaaauu 1933019RNAArtificial SequenceSynthetic 330aauuugcugu uuuuguagc 1933119RNAArtificial SequenceSynthetic 331ggcuacaaaa acagcaaau 1933219RNAArtificial SequenceSynthetic 332auuugcuguu uuuguagcc 1933319RNAArtificial SequenceSynthetic 333acacugacca auaaaaaaa 1933419RNAArtificial SequenceSynthetic 334uuuuuuuauu ggucagugu 1933519RNAArtificial SequenceSynthetic 335uggcuacaaa aacagcaaa 1933619RNAArtificial SequenceSynthetic 336uuugcuguuu uuguagcca 1933719RNAArtificial SequenceSynthetic 337guaacaaacu ccuaccaac 1933819RNAArtificial SequenceSynthetic 338guugguagga guuuguuac 1933919RNAArtificial SequenceSynthetic 339cacugaccaa uaaaaaaaa 1934019RNAArtificial SequenceSynthetic 340uuuuuuuuau uggucagug 1934119RNAArtificial SequenceSynthetic 341acugaccaau aaaaaaaaa 1934219RNAArtificial SequenceSynthetic 342uuuuuuuuua uuggucagu 1934319RNAArtificial SequenceSynthetic 343uacaaaaaca gcaaauucc 1934419RNAArtificial SequenceSynthetic 344ggaauuugcu guuuuugua 1934519RNAArtificial SequenceSynthetic 345cuacaaaaac agcaaauuc 1934619RNAArtificial SequenceSynthetic 346gaauuugcug uuuuuguag 1934719RNAArtificial SequenceSynthetic 347aaaauguggg uuuuuuuuu 1934819RNAArtificial SequenceSynthetic 348aaaaaaaaac ccacauuuu 1934919RNAArtificial SequenceSynthetic 349ugugguguuu ggcaaaguu 1935019RNAArtificial SequenceSynthetic 350aacuuugcca aacaccaca 1935119RNAArtificial SequenceSynthetic 351aaaugugggu uuuuuuuuu 1935219RNAArtificial SequenceSynthetic 352aaaaaaaaaa cccacauuu 1935319RNAArtificial SequenceSynthetic 353guuuuuuuuu uuuuuaaua 1935419RNAArtificial SequenceSynthetic 354uauuaaaaaa aaaaaaaac 1935519RNAArtificial SequenceSynthetic 355aauguggguu uuuuuuuuu 1935619RNAArtificial SequenceSynthetic 356aaaaaaaaaa acccacauu 1935719RNAArtificial SequenceSynthetic 357gguuuuuuuu uuuuuuaau 1935819RNAArtificial SequenceSynthetic 358auuaaaaaaa aaaaaaacc 1935919RNAArtificial SequenceSynthetic 359ggguuuuuuu uuuuuuuaa 1936019RNAArtificial SequenceSynthetic 360uuaaaaaaaa aaaaaaccc 1936119RNAArtificial SequenceSynthetic 361uggguuuuuu uuuuuuuua 1936219RNAArtificial SequenceSynthetic 362uaaaaaaaaa aaaaaccca 1936319RNAArtificial SequenceSynthetic 363auguggguuu uuuuuuuuu 1936419RNAArtificial SequenceSynthetic 364aaaaaaaaaa aacccacau 1936519RNAArtificial SequenceSynthetic 365guggguuuuu uuuuuuuuu 1936619RNAArtificial SequenceSynthetic 366aaaaaaaaaa aaaacccac 1936719RNAArtificial SequenceSynthetic 367uguggguuuu uuuuuuuuu 1936819RNAArtificial SequenceSynthetic 368aaaaaaaaaa aaacccaca 1936919RNAArtificial SequenceSynthetic 369aagaucgagg uggagaagc 1937019RNAArtificial SequenceSynthetic 370gcuucuccac cucgaucuu 1937119RNAArtificial SequenceSynthetic 371agaucgaggu ggagaagcc 1937219RNAArtificial SequenceSynthetic 372ggcuucucca ccucgaucu 1937319RNAArtificial SequenceSynthetic 373ccgccgcccu caucgcggg 1937419RNAArtificial SequenceSynthetic 374cccgcgauga gggcggcgg 1937519RNAArtificial SequenceSynthetic 375ccuucugcgg ccgaugaga 1937619RNAArtificial SequenceSynthetic 376ucucaucggc cgcagaagg 1937719RNAArtificial SequenceSynthetic 377cgccgcccuc aucgcgggg 1937819RNAArtificial SequenceSynthetic 378ccccgcgaug agggcggcg 1937919RNAArtificial SequenceSynthetic 379cuuccugcug cugccggga 1938019RNAArtificial SequenceSynthetic 380ucccggcagc agcaggaag 1938119RNAArtificial SequenceSynthetic 381gagcgcuucc ccgaugaga 1938219RNAArtificial SequenceSynthetic 382ucucaucggg gaagcgcuc 1938319RNAArtificial SequenceSynthetic 383gccgccgccc ucaucgcgg 1938419RNAArtificial SequenceSynthetic 384ccgcgaugag ggcggcggc 1938519RNAArtificial SequenceSynthetic 385ggcaagaucg agguggaga 1938619RNAArtificial SequenceSynthetic 386ucuccaccuc gaucuugcc 1938719RNAArtificial SequenceSynthetic 387ucuuccugcu gcugccggg 1938819RNAArtificial SequenceSynthetic 388cccggcagca gcaggaaga 1938919RNAArtificial SequenceSynthetic 389ugccgccgcc cucaucgcg 1939019RNAArtificial SequenceSynthetic 390cgcgaugagg gcggcggca 19

Claims

1-84. (canceled)

85. A pharmaceutical composition comprising an oligonucleotide and a pharmaceutically acceptable carrier, wherein the oligonucleotide comprises at least one of the 4-nucleotide (4-mer) motifs selected from the group consisting of: GUUC, GUCA, GCUC, GUUG, GUUU, GGUU, GUGU, GGUC, GUCU, GUCC, GCUU, UUGU, UGUC, CUGU, CGUC, UGUU, GUUA, UGUA, UUUC, UGUG, GGUA, GUCG, UUUG, UGGU, GUGG, GUGC, GUAC, GUAU, UAGU, GUAG, UUCA, UUGG, UCUC, CAGU, UUCG, CUUC, GAGU, GGUG, UUGC, UUUU, CUCA, UCGU, UUCU, UGGC, CGUU, CUUG, and UUAC, wherein the nucleotide sequences of the motifs are 5'→3', wherein the oligonucleotide is between 6 and 64 nucleotides in length, wherein: (i) at least one strand of the RNA oligonucleotide has an IFN-.alpha. score of at least 23 when n=6; at least 26 when n=7; at least 28 when n=8; at least 30 when n=9; at least 1.4909.times.n+22.014 when n is greater than 9, wherein the IFN-.alpha. score is assigned according to a method comprising: (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; (b) assigning an IFN-.alpha. point score for each individual 3mer motif: (i) for a 3mer motif which appears in Table 7, assigning an IFN-.alpha. point score according to Table 7; (ii) for a 3mer motif which does not appear in Table 7, assigning an IFN-.alpha. point score of 0; and (c) assigning the sum of the IFN-.alpha. point scores of individual 3mer motifs as the IFN-.alpha. score of the oligonucleotide; or (ii) at least one strand of the RNA oligonucleotide has an IFN-.alpha. score of at least 0.58, wherein the IFN-.alpha. score is assigned according to a method comprising the steps of: (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; (b) assigning an IFN-.alpha. point score for each individual 3mer motif according to Table 12A; and (c) assigning the highest individual IFN-.alpha. point score as the IFN-.alpha. score of the oligonucleotide; wherein n is length of the oligonucleotide; and provided that the oligonucleotide is not 5'-UUGAUGUGUUUAGUCGCUA-3',5'-GCACCACUAGUUGGUUGUC-3',5'-GUUGUAGUUGUACUCCAG- C-3',5'-GCCCGUCUGUUGUGUGACUC-3',5'-GUCUGUUGUGUG-3', or 5'-GUUGUGGUUGUGGUUGUG-3'.

86. The pharmaceutical composition of claim 85, wherein the 4-nucleotide motifs are selected from the group consisting of GUUC, GUCA, GCUC, GUUG, GUUU, and GGUU.

87. The pharmaceutical composition claims 86, wherein spacer nucleotides between the 4-nucleotide motifs are identical, and the spacer nucleotide are selected from the group consisting of A, T, C, G, and variants and derivatives thereof.

88. The pharmaceutical composition of claim 85, wherein the oligonucleotides does not have gene silencing activity for any known mammalian gene.

89. A pharmaceutical composition comprising an oligonucleotide and a pharmaceutically acceptable carrier, wherein: at least one strand of the oligonucleotide has an IFN-.alpha. score of at least 1.4909.times.n+31.014, wherein the IFN-.alpha. score is assigned according to a method comprising: (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; (b) assigning an IFN-.alpha. point score for each individual 3mer motif: (i) for a 3mer motif which appears in Table 7, assigning an IFN-.alpha. point score according to Table 7; (ii) for a 3mer motif which does not appear in Table 7, assigning an IFN-.alpha. point score of 0; and (c) assigning the sum of the IFN-.alpha. point scores of individual 3mer motifs as the IFN-.alpha. score of the oligonucleotide; or (ii) at least one strand of the RNA oligonucleotide has an IFN-.alpha. score of at least 0.58, wherein the IFN-.alpha. score is assigned according to a method comprising the steps of: (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; (b) assigning an IFN-.alpha. point score for each individual 3mer motif according to Table 12A; and (c) assigning the highest individual IFN-.alpha. point score as the IFN-.alpha. score of the oligonucleotide; and wherein the oligonucleotide is an siRNA and n is between 14 and 25, or wherein the oligonucleotide is an antisense RNA and n is between 14 and 50.

90. A pharmaceutical composition comprising an oligonucleotide and a pharmaceutically acceptable carrier, wherein: (i) all strand(s) of the oligonucleotide has(have) an IFN-.alpha. score of at most 0.6075.times.-9.9484, wherein the IFN-.alpha. score is assigned according to a method comprising: (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; (b) assigning an IFN-.alpha. point score for each individual 3mer motif: (i) for a 3mer motif which appears in Table 7, assigning an IFN-.alpha. point score according to Table 7; (ii) for a 3mer motif which does not appear in Table 7, assigning an IFN-.alpha. point score of 0; and (c) assigning the sum of the IFN-.alpha. point scores of individual 3mer motifs as the IFN-.alpha. score of the oligonucleotide; or (ii) wherein all strand(s) of the oligonucleotide has(have) an IFN-.alpha. score of at most 0.11, wherein the IFN-.alpha. score is assigned according to a method comprising the steps of: (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; (b) assigning an IFN-.alpha. point score for each individual 3mer motif according to Table 12A; and (c) assigning the highest individual IFN-.alpha. point score as the IFN-.alpha. score of the oligonucleotide; and wherein the oligonucleotide is an siRNA and n is between 19 and 25, or wherein the oligonucleotide is an antisense RNA and n is between 18 and 50.

91. A pharmaceutical composition comprising an in vitro activated dendritic cell and a pharmaceutically acceptable carrier, wherein the in vitro activated dendritic cell is produced by a method comprising: (a) complexing an RNA oligonucleotide with a complexation agent; (b) contacting dendritic cells isolated from a donor mammal with the complexed RNA oligonucleotide; and (c) contacting the dendritic cells with an antigen, wherein:wherein the oligonucleotide comprises at least one of the 4-nucleotide (4-mer) motifs selected from the group consisting of: GUUC, GUCA, GCUC, GUUG, GUUU, GGUU, GUGU, GGUC, GUCU, GUCC, GCUU, UUGU, UGUC, CUGU, CGUC, UGUU, GUUA, UGUA, UUUC, UGUG, GGUA, GUCG, UUUG, UGGU, GUGG, GUGC, GUAC, GUAU, UAGU, GUAG, UUCA, UUGG, UCUC, CAGU, UUCG, CUUC, GAGU, GGUG, UUGC, UUUU, CUCA, UCGU, UUCU, UGGC, CGUU, CUUG, and UUAC, wherein the nucleotide sequences of the motifs are 5'→3', wherein the oligonucleotide is between 6 and 64 nucleotides in length, wherein: (i) at least one strand of the RNA oligonucleotide has an IFN-.alpha. score of at least 23 when n=6; at least 26 when n=7; at least 28 when n=8; at least 30 when n=9; at least 1.4909.times.n+22.014 when n is greater than 9, wherein the IFN-.alpha. score is assigned according to a method comprising: (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; (b) assigning an IFN-.alpha. point score for each individual 3mer motif: (i) for a 3mer motif which appears in Table 7, assigning an IFN-.alpha. point score according to Table 7; (ii) for a 3mer motif which does not appear in Table 7, assigning an IFN-.alpha. point score of 0; and (c) assigning the sum of the IFN-.alpha. point scores of individual 3mer motifs as the IFN-.alpha. score of the oligonucleotide; or (ii) at least one strand of the RNA oligonucleotide has an IFN-.alpha. score of at least 0.58, wherein the IFN-.alpha. score is assigned according to a method comprising the steps of: (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; (b) assigning an IFN-.alpha. point score for each individual 3mer motif according to Table 12A; and (c) assigning the highest individual IFN-.alpha. point score as the IFN-.alpha. score of the oligonucleotide; wherein n is length of the oligonucleotide; and provided that the oligonucleotide is not 5'-UUGAUGUGUUUAGUCGCUA-3',5'-GCACCACUAGUUGGUUGUC-3',5'-GUUGUAGUUGUACUCCAG- C-3',5'-GCCCGUCUGUUGUGUGACUC-3',5'-GUCUGUUGUGUG-3', or 5'-GUUGUGGUUGUGGUUGUG-3'.

92. The pharmaceutical composition of claim 91, wherein the 4-nucleotide motifs are selected from the group consisting of GUUC, GUCA, GCUC, GUUG, GUUU, and GGUU.

93. The pharmaceutical composition claims 91, wherein spacer nucleotides between the 4-nucleotide motifs are identical, and the spacer nucleotides are selected from the group consisting of A, T, C, G, and variants and derivatives thereof.

94. The pharmaceutical composition of claim 93, wherein the oligonucleotide does not have gene silencing activity for any known mammalian gene.

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