US 2008O132691A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0132691 A1 KhVOrOVa et al. (43) Pub. Date: Jun. 5, 2008
(54) SIRNA TARGETING KINASE INSERT Related U.S. Application Data DOMAIN RECEPTOR (KDR) (63) Continuation-in-part of application No. 10/940,892, filed on Sep. 14, 2004, which is a continuation of (75) Inventors: Anastasia Khvorova, Boulder, CO application No. PCT/US04/14885, filed on May 12, (US); Angela Reynolds, Conifer, 2004, Continuation-in-part of application No. 10/714, CO (US); Devin Leake, Denver, 333, filed on Nov. 14, 2003. CO (US); William Marshall, (60) Provisional application No. 60/426,137, filed on Nov. Boulder, CO (US); Steven Read, 14, 2002, provisional application No. 60/502,050, Denver, CO (US); Stephen filed on Sep. 10, 2003. Scaringe, Lafayette, CO (US) Publication Classification Correspondence Address: (51) Int. C. KALOW & SPRINGUT LLP C7H 2L/02 (2006.01) 488 MADISONAVENUE, 19TH FLOOR (52) U.S. Cl...... 536/24.5 NEW YORK, NY 10022 (57) ABSTRACT Efficient sequence specific gene silencing is possible through (73) Assignee: DHARMACON, INC., Lafayette, the use of siRNA technology. By selecting particular siRNAs CO (US) by rational design, one can maximize the generation of an effective gene silencing reagent, as well as methods for (21) Appl. No.: 11/978,107 silencing genes. Methods, compositions, and kits generated through rational design of siRNAS are disclosed including (22) Filed: Oct. 26, 2007 those directed to nucleotide sequences for KDR.
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(2002) Dicer APPLICATIONS Functions in RNA Interference and in Synthesis of Small 0001. This application is a continuation-in-part of U.S. RNA Involved in Developmental Timing in C. elegans; Mar Ser. No. 10/714,333, filed Nov. 14, 2003, which claims the tinez et al., Single-Stranded Antisense siRNAs Guide Target benefit of U.S. Provisional Application No. 60/426,137, filed RNA Cleavage in RNAi, Cell 110(5):563; Hutvagner & Nov. 14, 2002, and also claims the benefit of U.S. Provisional Zamore (2002) A microRNA in a multiple-turnover RNAi Application No. 60/502,050, filed Sep. 10, 2003; this appli enzyme complex, Science 297:2056. cation is also a continuation-in-part of U.S. Ser. No. 10/940, 0008. From a mechanistic perspective, introduction of 892, filed Sep. 14, 2004, which is a continuation of PCT long double stranded RNA into plants and invertebrate cells is Application No. PCT/US04/14885, international filing date May 12, 2004. The disclosures of the priority applications, broken down into siRNA by a Type III endonuclease known including the sequence listings and tables Submitted in elec as Dicer. Sharp, RNA interference 2001, Genes Dev. 2001, tronic form in lieu of paper, are incorporated by reference into 15:485. Dicer, a ribonuclease-III-like enzyme, processes the the instant specification. dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs. Bernstein, Caudy, Ham SEQUENCE LISTING mond, & Hannon (2001) Role for abidentate ribonuclease in the initiation step of RNA interference, Nature 409:363. The 0002 The sequence listing for this application has been siRNAs are then incorporated into an RNA-induced silencing submitted in accordance with 37 CFRS 1.52(e) and 37 CFR complex (RISC) where one or more helicases unwind the S 1.821 on CD-ROM in lieu of paper on a disk containing the siRNA duplex, enabling the complementary antisense Strand sequence listing file entitled “DHARMA 2100-US80 CRF. to guide target recognition. Nykanen, Haley, & Zamore txt created Oct. 18, 2007, 108 kb. Applicants hereby incor (2001) ATP requirements and small interfering RNA struc porate by reference the sequence listing provided on CD ture in the RNA interference pathway, Cell 107:309. Upon ROM in lieu of paper into the instant specification. binding to the appropriate target mRNA, one or more endo nucleases within the RISC cleaves the target to induce silenc FIELD OF INVENTION ing. Elbashir, Lendeckel, & Tuschl (2001) RNA interference 0003. The present invention relates to RNA interference is mediated by 21- and 22-nucleotide RNAs, Genes Dev. (“RNAi). 15:188, Figure 1. 0009. The interference effect can be long lasting and may BACKGROUND OF THE INVENTION be detectable after many cell divisions. Moreover, RNAi 0004 Relatively recently, researchers observed that exhibits sequence specificity. Kisielow, M. et al. (2002) Iso double stranded RNA (dsRNA) could be used to inhibit form-specific knockdown and expression of adaptor protein protein expression. This ability to silence a gene has broad ShcA using small interfering RNA. J. Biochem. 363: 1-5. potential for treating human diseases, and many researchers Thus, the RNAi machinery can specifically knock down one and commercial entities are currently investing considerable type of transcript, while not affecting closely related mRNA. resources in developing therapies based on this technology. These properties make siRNA a potentially valuable tool for 0005 Double stranded RNA induced gene silencing can inhibiting gene expression and studying gene function and occur on at least three different levels: (i) transcription inac drug target validation. Moreover, siRNAs are potentially use tivation, which refers to RNA guided DNA or histone methy ful as therapeutic agents against: (1) diseases that are caused lation; (ii) siRNA induced mRNA degradation; and (iii) by over-expression or misexpression of genes; and (2) dis mRNA induced transcriptional attenuation. eases brought about by expression of genes that contain muta 0006. It is generally considered that the major mechanism tions. of RNA induced silencing (RNA interference, or RNAi) in 0010. Successful siRNA-dependent gene silencing mammalian cells is mRNA degradation. Initial attempts to depends on a number of factors. One of the most contentious use RNAi in mammalian cells focused on the use of long issues in RNAi is the question of the necessity of siRNA strands of dsRNA. However, these attempts to induce RNAi design, i.e., considering the sequence of the siRNA used. met with limited success, due in part to the induction of the Early work in C. elegans and plants circumvented the issue of interferon response, which results in a general, as opposed to design by introducing long dsRNA (see, for instance, Fire, A. a target-specific, inhibition of protein synthesis. Thus, long etal. (1998) Nature 391:806-811). In this primitive organism, dsRNA is not a viable option for RNAi in mammalian sys long dsRNA molecules are cleaved into siRNA by Dicer, thus temS. generating a diverse population of duplexes that can poten 0007 More recently it has been shown that when short tially cover the entire transcript. While some fraction of these (18-30 bp) RNA duplexes are introduced into mammalian molecules are non-functional (i.e., induce little or no silenc cells in culture, sequence-specific inhibition of target mRNA ing) one or more have the potential to be highly functional, can be realized without inducing an interferon response. Cer thereby silencing the gene of interest and alleviating the need tain of these short dsRNAs, referred to as small inhibitory for siRNA design. Unfortunately, due to the interferon RNAs (“siRNAs), can act catalytically at sub-molar concen response, this same approach is unavailable for mammalian trations to cleave greater than 95% of the target mRNA in the systems. While this effect can be circumvented by bypassing cell. A description of the mechanisms for siRNA activity, as the Dicer cleavage step and directly introducing siRNA, this well as some of its applications are described in Provost et al. tactic carries with it the risk that the chosen siRNA sequence (2002) Ribonuclease Activity and RNA Binding of Recom may be non-functional or semi-functional. US 2008/O 132691 A1 Jun. 5, 2008 0011. A number of researches have expressed the view that siRNA design is not a crucial element of RNAi. On the other hand, others in the field have begun to explore the possibility that RNAi can be made more efficient by paying attention to the design of the siRNA. Unfortunately, none of the reported methods have provided a satisfactory scheme for reliably selecting siRNA with acceptable levels of function ality. Accordingly, there is a need to develop rational criteria by which to select siRNA with an acceptable level of func tionality, and to identify siRNA that have this improved level of functionality, as well as to identify siRNAs that are hyper functional. 6*(number of A+U in position 15-19)-3*(number of G+C in whole siRNA), Formula X SUMMARY OF THE INVENTION wherein position numbering begins at the 5'-most position of 0012. The present invention is directed to increasing the a sense Strand, and efficiency of RNAi, particularly in mammalian systems. A=1 if A is the base at position 1 of the sense strand, other Accordingly, the present invention provides kits, siRNAS and wise its value is 0; methods for increasing siRNA efficacy. 0013. According to a first embodiment, the present inven A=1 if A is the base at position 2 of the sense strand, other tion provides a kit for gene silencing, wherein said kit is wise its value is 0; comprised of a pool of at least two siRNA duplexes, each of A=1 if A is the base at position 3 of the sense strand, other which is comprised of a sequence that is complementary to a wise its value is 0; portion of the sequence of one or more target messenger RNA, and each of which is selected using non-target specific A=1 if A is the base at position 4 of the sense strand, other criteria. wise its value is 0; 0014. According to a second embodiment, the present As 1 if A is the base at position 5 of the sense strand, other invention provides a method for selecting an siRNA, said wise its value is 0; method comprising applying selection criteria to a set of potential siRNA that comprise 18-30 base pairs, wherein said A-1 if A is the base at position 6 of the sense strand, other selection criteria are non-target specific criteria, and said set wise its value is 0; comprises at least two siRNAs and each of said at least two siRNAS contains a sequence that is at least Substantially A, -1 if A is the base at position 7 of the sense strand, other complementary to a target gene; and determining the relative wise its value is 0; functionality of the at least two siRNAs. A-1 if A is the base at position 10 of the sense strand, 0015. According to a third embodiment, the present inven otherwise its value is 0; tion also provides a method for selecting an siRNA wherein A=1 if A is the base at position 11 of the sense strand, said selection criteria are embodied in a formula comprising: otherwise its value is 0; A=1 if A is the base at position 13 of the sense strand, otherwise its value is 0; A=1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; C=1 if C is the base at position 3 of the sense strand, other wise its value is 0; C=1 if C is the base at position 4 of the sense strand, other wise its value is 0; Cs=1 if C is the base at position 5 of the sense strand, other wise its value is 0; C1 if C is the base at position 6 of the sense strand, other wise its value is 0; C, 1 if C is the base at position 7 of the sense strand, other wise its value is 0; C1 if C is the base at position 9 of the sense strand, other wise its value is 0; C=1 if C is the base at position 17 of the sense strand, otherwise its value is 0; US 2008/O 132691 A1 Jun. 5, 2008 Cs-1 if C is the base at position 18 of the sense strand, absence of at least one variable selected from the group con otherwise its value is 0; sisting of the presence or absence of a particular nucleotide at a particular position, the total number of AS and US in posi Co-1 if C is the base at position 19 of the sense strand, tions 15-19, the number of times that the same nucleotide otherwise if another base is present or the sense strand is only repeats within a given sequence, and the total number of Gs 18 base pairs in length, its value is 0; and Cs; and (e) developing an algorithm using the informa G=1 if G is the base at position 1 on the sense strand, tion of step (d). otherwise its value is 0; 0017. According to a fifth embodiment, the present inven tion provides a kit, wherein said kit is comprised of at least G=1 if G is the base at position 2 of the sense strand, other two siRNAs, wherein said at least two siRNAs comprise a wise its value is 0; first optimized siRNA and a second optimized siRNA, Gs=1 if G is the base at position 8 on the sense strand, wherein said first optimized siRNA and said second opti otherwise its value is 0; mized siRNA are optimized according a formula comprising Formula X. Go-1 if G is the base at position 10 on the sense strand, 0018. The present invention also provides a method for otherwise its value is 0; identifying a hyperfunctional siRNA, comprising applying G=1 if G is the base at position 13 on the sense strand, selection criteria to a set of potential siRNA that comprise otherwise its value is 0; G=1 if G is the base at position 19 18-30 base pairs, wherein said selection criteria are non of the sense strand, otherwise if another base is present or the target specific criteria, and said set comprises at least two sense Strand is only 18 base pairs in length, its value is 0; siRNAs and each of said at least two siRNAs contains a sequence that is at least Substantially complementary to a U =1 if U is the base at position 1 on the sense strand, target gene; determining the relative functionality of the at O therwise its value is 0; least two siRNAS and assigning each of the at least two =1 if U is the base at position 2 on the sense strand, siRNAs a functionality score; and selecting siRNAs from the therwise its value is 0; at least two siRNAs that have a functionality score that reflects greater than 80 percent silencing at a concentration in =1 if U is the base at position 3 on the sense strand, the picomolar range, wherein said greater than 80 percent therwise its value is 0; silencing endures for greater than 120 hours. 1 if U is the base at position 4 on the sense Strand, 0019. According to a sixth embodiment, the present inven therwise its value is 0; tion provides a hyperfunctional siRNA that is capable of silencing Bcl2. 7–1 if U is the base at position 7 on the sense strand, 0020. According to a seventh embodiment, the present therwise its value is 0; invention provides a method for developing an siRNA algo =1 if U is the base at position 9 on the sense strand, rithm for selecting functional and hyperfunctional siRNAs therwise its value is 0; for a given sequence. The method comprises: U 1 if U is the base at position 10 on the sense strand, 0021 (a) selecting a set of siRNAs; O therwise its value is 0; 0022 (b) measuring the gene silencing ability of each siRNA from said set; U s=1 if U is the base at position 15 on the sense strand, 0023 (c) determining the relative functionality of each O therwise its value is 0; siRNA; U 1 if U is the base at position 16 on the sense strand, 0024 (d) determining the amount of improved function ality by the presence or absence of at least one variable O therwise its value is 0; selected from the group consisting of the total GC content, U 7–1 if U is the base at position 17 on the sense strand, melting temperature of the siRNA, GC content at positions O therwise its value is 0; 15-19, the presence or absence of a particular nucleotide at a particular position, relative thermodynamic stability at par U 1 if U is the base at position 18 on the sense strand, ticular positions in a duplex, and the number of times that the otherwise its value is 0. same nucleotide repeats within a given sequence; and GCs the number of G and C bases within positions 15-19 0025 (e) developing an algorithm using the information of the sense strand, or within positions 15-18 if the sense of step (d). Strand is only 18 base pairs in length; 0026. According to this embodiment, preferably the set of siRNAs comprises at least 90 siRNAs from at least one gene, GCthetotal number of G and C bases in the sense strand; more preferably at least 180 siRNAs from at least two differ Tm=100 if the siRNA oligo has the internal repeat longer then ent genes, and most preferably at least 270 and 360 siRNAs 4 base pairs, otherwise its value is 0; and from at least three and four different genes, respectively. Additionally, in step (d) the determination is made with pref X=the number of times that the same nucleotide repeats four erably at least two, more preferably at least three, even more or more times in a row. preferably at least four, and most preferably all of the vari 0016. According to a fourth embodiment, the invention ables. The resulting algorithm is not target sequence specific. provides a method for developing an algorithm for selecting 0027. In another embodiment, the present invention pro siRNA, said method comprising: (a) selecting a set of siRNA; vides rationally designed siRNAs identified using the formu (b) measuring gene silencing ability of each siRNA from said las above. set; (c) determining relative functionality of each siRNA; (d) 0028. In yet another embodiment, the present invention is determining improved functionality by the presence or directed to hyperfunctional siRNA. US 2008/O 132691 A1 Jun. 5, 2008 0029. The ability to use the above algorithms, which are 0036. In various embodiments, the duplex of said first not sequence or species specific, allows for the cost-effective siRNA is 19-30 base pairs and said first sense region com selection of optimized siRNAS for specific target sequences. prises a sequence that is identical to at least 18 bases of a Accordingly, there will be both greater efficiency and reli sequence selected from the group consisting of SEQID NO. ability in the use of siRNA technologies. 438 to SEQ ID NO. 622, and said duplex of said second 0030. In various embodiments, siRNAs that target nucle siRNA is 19-30 base pairs and said second region comprises otide sequences for kinase insert domain receptor (KDR, also a sequence that is identical to a sequence selected from the known as VEGFR2) are provided. In various embodiments, group consisting of SEQID NO. 438 to SEQID NO. 622. 0037 For a better understanding of the present invention the siRNAs are rationally designed. In various embodiments, together with other and further advantages and embodiments, the siRNAs are functional or hyperfunctional. reference is made to the following description taken in con 0031. In various embodiments, an siRNA that targets the junction with the examples, the scope of which is set forth in nucleotide sequence for KDR is provided, wherein the siRNA the appended claims. is selected from the group consisting of various siRNA sequences targeting the nucleotide sequences for KDR that are disclosed herein. In various embodiments, the siRNA BRIEF DESCRIPTION OF THE FIGURES sequence is selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 622. 0038 FIG. 1 shows a model for siRNA-RISC interactions. 0032. In various embodiments, siRNA comprising a sense RISC has the ability to interact with either end of the siRNA region and an antisense region are provided, said sense region or miRNA molecule. Following binding, the duplex is and said antisense region together form a duplex region com unwound, and the relevant target is identified, cleaved, and prising 18-30 base pairs, and said sense region comprises a released. sequence that is at least 90% similar to a sequence selected 0039 FIG. 2 is a representation of the functionality of two from the group consisting of siRNA sequences targeting hundred and seventy siRNA duplexes that were generated to nucleotide sequences for KDR that are disclosed herein. In target human cyclophilin, human diazepam-binding inhibitor various embodiments, the siRNA sequence is selected from (DB), and firefly luciferase. the group consisting of SEQID NO. 438 to SEQID NO. 622. 0040 FIG.3a is a representation of the silencing effect of 0033. In various embodiments, an siRNA comprising a 30 siRNAs in three different cells lines, HEK293, DU145, sense region and an antisense region is provided, said sense and Hela. FIG. 3b shows the frequency of different functional region and said antisense region together form a duplex groups (>95% silencing (black), >80% silencing (gray), region comprising 18-30 base pairs, and said sense region >50% silencing (dark gray), and <50% silencing (white)) comprises a sequence that is identical to a contiguous stretch based on GC content. In cases where a given bar is absent of at least 18 bases of a sequence selected from the group from a particular GC percentage, no siRNA were identified consisting of SEQID NO.438 to SEQID NO. 622. In various for that particular group. FIG. 3c shows the frequency of embodiments, the duplex region is 19-30 base pairs, and the different functional groups based on melting temperature sense region comprises a sequence that is identical to a (Tm). sequence selected from the group consisting of SEQID NO. 0041 FIG. 4 is a representation of a statistical analysis that 438 to SEQID NO. 622. revealed correlations between silencing and five sequence 0034. In various embodiments, a pool of at least two siR related properties of siRNA: (A) an A at position 19 of the NAs is provided, wherein said pool comprises a first siRNA sense Strand, (B) an A at position 3 of the sense strand, (C) a and a second siRNA, said first siRNA comprising a duplex Uat position 10 of the sense strand, (D) a base other than Gat region of length 18-30 base pairs that has a first sense region position 13 of the sense strand, and (E) a base other than Cat that is at least 90% similar to 18 bases of a first sequence position 19 of the sense strand. All variables were correlated selected from the group consisting of SEQ ID NO. 438 to with siRNA silencing of firefly luciferase and human cyclo SEQID NO. 622, and said second siRNA comprises a duplex philin. siRNAs satisfying the criterion are grouped on the left region of length 18-30 base pairs that has a second sense (Selected) while those that do not, are grouped on the right region that is at least 90% similar to 18 bases of a second (Eliminated). Y-axis is “96 Silencing of Control. Each posi sequence selected from the group consisting of SEQID NO. tion on the X-axis represents a unique siRNA. 438 to SEQID NO. 622, wherein said first sense region and 0042 FIGS. 5A and 5B are representations of firefly said second sense region are not identical. luciferase and cyclophilin siRNA panels Sorted according to 0035. In various embodiments, the first sense region com functionality and predicted values using Formula VIII. The prises a sequence that is identical to at least 18 bases of a siRNA found within the circle represent those that have For sequence selected from the group consisting of SEQID NO. mula VIII values (SMARTSCORESTM, or siRNA rank) 438 to SEQID NO. 622, and said second sense region com above Zero. siRNA outside the indicated area have calculated prises a sequence that is identical to at least 18 bases of a Formula VIII values that are below zero. Y-axis is “Expres sequence selected from the group consisting of SEQID NO. sion (%. Control). Each position on the X-axis represents a 438 to SEQID NO. 622. In various embodiments, the duplex unique siRNA. of said first siRNA is 19-30 base pairs, and said first sense 0043 FIG. 6A is a representation of the average internal region comprises a sequence that is at least 90% similar to a stability profile (AISP) derived from 270 siRNAs taken from sequence selected from the group consisting of SEQID NO. three separate genes (cyclophilin B, DBI and firefly 438 to SEQ ID NO. 622, and said duplex of said second luciferase). Graphs represent AISP values of highly func siRNA is 19-30 base pairs and comprises a sequence that is at tional, functional, and non-functional siRNA. FIG. 6B is a least 90% similar to a sequence selected from the group comparison between the AISP of naturally derived GFP consisting of SEQID NO. 438 to SEQID NO. 622. siRNA (filled squares) and the AISP of siRNA from cyclo US 2008/O 132691 A1 Jun. 5, 2008 philin B, DBI, and luciferase having >90% silencing proper 0051 FIG. 14 is the knockdown by the top ten Bcl2 siR ties (no fill) for the antisense strand. “DG' is the symbol for NAs at 100 nM concentrations. The Y-axis represents the AG, free energy. amount of expression relative to the non-specific (ins) and 0044 FIG. 7 is a histogram showing the differences in transfection mixture control. duplex functionality upon introduction of base pair mis 0052 FIG. 15 represents a functional walk where siRNA matches. The X-axis shows the mismatch introduced in the beginning on every other base pair of a region of the luciferase gene are tested for the ability to silence the luciferase gene. siRNA and the position it is introduced (e.g., 8C>A reveals The Y-axis represents the percent expression relative to a that position 8 (which normally has a C) has been changed to control. The X-axis represents the position of each individual an A). The Y-axis is “96 Silencing (Normalized to Control).' siRNA. Reading from left to right across the X-axis, the The samples on the X-axis represent siRNAs at 100 nMand position designations are 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21. are, reading from left to right: 1A to C, 1A to G, 1A to U: 2A 23, 25, 27, 29, 31,33,35, 37, 39, 41,43, 45, 47,49, 51,53,55, to C, 2A to G, 2A to U; 3A to C, 3A to G, 3A to U; 4G to A, 57, 59,61,63, 65, 67, 69,71, 73,75, 77, 79,81, 83, 85, 87,89, 4G to C; 4G to U; 5U to A, 5U to C, 5U to G: 6U to A, 6U to and Plasmid. C, 6U to G: 7G to A, 7G to C, 7G to U; 8C to A, 8C to G, 8C 0053 FIGS. 16A and 16B are histograms demonstrating to U; 9G to A, 9G to C, 9G to U; 10C to A, 10C to G, 10C to the inhibition of target gene expression by pools of 2 (16A) U; 11G to A, 11G to C, 11G to U; 12G to A, 12G to C, 12G to and 3 (16B) siRNA duplexes taken from the walk described in U; 13 A to C, 13 A to G, 13A to U; 14G to A, 14G to C, 14G to FIG. 15. The Y-axis in each represents the percent expression U; 15G to A, 15G to C, 15G to U; 16A to C, 16A to G, 16A to relative to control. The X-axis in each represents the position U; 17G to A, 17G to C, 17G to U; 18U to A, 18U to C, 18U to of the first siRNA in paired pools, or trios of siRNAs. For G: 19U to A, 19U to C, 19U to G: 20 wit: Control. instance, the first paired pool contains siRNAs 1 and 3. The 0045 FIG. 8 is histogram that shows the effects of 5'sense second paired pool contains siRNAs3 and 5. Pool 3 (of paired and antisense strand modification with 2'-O-methylation on pools) contains siRNAS 5 and 7, and so on. For each of 16A functionality. and 16B, the X-axis from left to right reads 1,3,5,7,9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45,47, 0046 FIG. 9 shows a graph of SMARTSCORESTM, or 49,51,53,55,57, 59, 61,63, 65, 67, 69,71, 73,75, 77,79, 81, siRNA rank, versus RNAi silencing values for more than 360 83, 85, 87, 89, and Plasmid. siRNA directed against 30 different genes. SiRNA to the right 0054 FIGS. 17A and 17B are histograms demonstrating of the vertical bar represent those siRNA that have desirable the inhibition of target gene expression by pools of 4 (17A) SMARTSCORESTM, or siRNA rank. and 5 (17B) siRNA duplexes. The Y-axis in each represents 0047 FIGS. 10A-E compare the RNAi of five different the percent expression relative to control. The X-axis in each genes (SEAP DBI, PLK, Firefly Luciferase, and Renilla represents the position of the first siRNA in each pool. For Luciferase) by varying numbers of randomly selected siRNA each of 17A and 17B, the X-axis from left to right reads 1, 3, and four rationally designed (SMART-selected) siRNA cho 5, 7,9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33,35, 37,39, sen using the algorithm described in Formula VIII. In addi 41, 43,45, 47,49,51,53,55, 57, 59, 61, 63, 65, 67, 69, 71,73, tion, RNAi induced by a pool of the four SMART-selected 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid. siRNA is reported at two different concentrations (100 and 0055 FIGS. 18A and 18B are histograms demonstrating 400 nM). 10F is a comparison between a pool of randomly the inhibition of target gene expression by siRNAs that are ten selected EGFR siRNA (Pool 1) and a pool of SMART-se (18A) and twenty (18B) base pairs base pairs apart. The lected EGFR siRNA (Pool 2). Pool 1, S1-S4 and Pool 2 S1-S4 Y-axis represents the percent expression relative to a control. represent the individual members that made up each respec The X-axis represents the position of the first siRNA in each tive pool. Note that numbers for random siRNAs represent the pool. For each of 18A and 18B, the X-axis from left to right position of the 5' end of the sense strand of the duplex. The reads 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, Y-axis represents the 96 expression of the control(s). The 35, 37,39, 41,43, 45, 47,49,51,53,55, 57,59, 61,63, 65, 67, X-axis is the percent expression of the control. 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid. 0048 FIG. 11 shows the Western blot results from cells 0056 FIG. 19 shows that pools of siRNAs (dark gray bar) treated with siRNA directed against twelve different genes work as well (or better) than the best siRNA in the pool (light involved in the clathrin-dependent endocytosis pathway gray bar). The Y-axis represents the percent expression rela (CHC, DynII, CALM, CLCa, CLCb, Eps15, Eps15R, Rab5a, tive to a control. The X-axis represents the position of the first Rab5b, Rab5c, B2 subunit of AP-2 and EEA.1). siRNA were siRNA in each pool. The X-axis from left to right reads 1, 3, selected using Formula VIII. “Pool represents a mixture of 5, 7,9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33,35, 37,39, duplexes 1-4. Total concentration of each siRNA in the pool is 41, 43,45, 47,49,51,53,55, 57, 59, 61, 63, 65, 67, 69, 71,73, 25 nM. Total concentration=4x25=100 nM. 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid. 0049 FIG. 12 is a representation of the gene silencing 0057 FIG. 20 shows that the combination of several semi capabilities of rationally-selected siRNA directed against ten functional siRNAs (dark gray) result in a significant improve different genes (human and mouse cyclophilin, C-myc, ment of gene expression inhibition over individual (semi human lamin A/C, QB (ubiquinol-cytochrome c reductase functional; light gray) siRNA. The Y-axis represents the core protein I). MEK1 and MEK2, ATE1 (arginyl-tRNA pro percent expression relative to a control. tein transferase), GAPDH, and Eg5). The Y-axis is the percent 0058 FIGS. 21A, 21B and 21C show both pools (Library, expression of the control. Numbers 1, 2, 3 and 4 represent Lib) and individual siRNAs in inhibition of gene expression individual rationally selected siRNA. “Pool represents a of Beta-Galactosidase, Renilla Luciferase and SEAP (alka mixture of the four individual siRNA. line phosphatase). Numbers on the X-axis indicate the posi 0050 FIG. 13 is the sequence of the top ten Bcl2 siRNAs tion of the 5'-most nucleotide of the sense strand of the as determined by Formula VIII. Sequences are listed 5' to 3'. duplex. The Y-axis represents the percent expression of each US 2008/O 132691 A1 Jun. 5, 2008 gene relative to a control. Libraries contain 19 nucleotide long same example, if 18 base pairs on each strand can hydrogen siRNAS (not including overhangs) that begin at the following bond with each other, the polynucleotide strands exhibit 90% nucleotides: SEAP: Lib 1: 206, 766, 812, 923, Lib 2: 1117, complementarity. 1280, 1300, 1487, Lib 3: 206, 766, 812, 923, 1117, 1280, Deoxynucleotide 1300, 1487, Lib 4: 206, 812, 1117, 1300, Lib 5: 766,923, 1280, 1487, Lib 6: 206, 1487; Bgal: Lib 1:979, 1339, 2029, 0066. The term “deoxynucleotide' refers to a nucleotide 2590, Lib 2: 1087, 1783, 2399, 3257, Lib 3:979, 1783, 2590, or polynucleotide lacking a hydroxyl group (OH group) at the 3257, Lib 4:979, 1087, 1339, 1783, 2029, 2399, 2590,3257, 2" and/or 3' position of a Sugar moiety. Instead, it has a hydro Lib5:979, 1087, 1339, 1783, Lib 6: 2029, 2399, 2590,3257; gen bonded to the 2' and/or 3' carbon. Within an RNA mol ecule that comprises one or more deoxynucleotides, “deoxy Renilla: Lib 1: 174,300, 432,568, Lib 2:592,633, 729, 867, nucleotide' refers to the lack of an OH group at the 2' position Lib 3: 174,300, 432,568,592,633, 729,867, Lib 4: 174,432, of the Sugar moiety, having instead a hydrogen bonded 592, 729, Lib 5:300,568, 633, 867, Lib 6:592,568. directly to the 2 carbon. 0059 FIG. 22 shows the results of an EGFR and TfnR internalization assay when single gene knockdowns are per Deoxyribonucleotide formed. The Y-axis represents percent internalization relative 0067. The terms “deoxyribonucleotide' and “DNA” refer to control. to a nucleotide or polynucleotide comprising at least one 0060 FIG. 23 shows the results of an EGFR and TfnR Sugar moiety that has an H, rather than an OH, at its 2' and/or internalization assay when multiple genes are knocked down 3' position. (e.g., Rab5a, b, c). The Y-axis represents the percent internal ization relative to control. Duplex Region 0061 FIG.24 shows the simultaneous knockdown of four 0068. The phrase “duplex region” refers to the region in different genes. siRNAs directed against G6PD, GAPDH, two complementary or Substantially complementary poly PLK, and UQC were simultaneously introduced into cells. nucleotides that form base pairs with one another, either by Twenty-four hours later, cultures were harvested and assayed Watson-Crick base pairing or any other manner that allows for mRNA target levels for each of the four genes. A com for a stabilized duplex between polynucleotide strands that parison is made between cells transfected with individual are complementary or Substantially complementary. For siRNAs vs. a pool of siRNAs directed against all four genes. example, a polynucleotide strand having 21 nucleotide units can base pair with another polynucleotide of 21 nucleotide 0062 FIG.25 shows the functionality often siRNAs at 0.3 units, yet only 19 bases on each Strand are complementary or nM concentrations. Substantially complementary, Such that the “duplex region' has 19 base pairs. The remaining bases may, for example, DETAILED DESCRIPTION exist as 5' and 3' overhangs. Further, within the duplex region, 100% complementarity is not required; substantial comple Definitions mentarity is allowable within a duplex region. Substantial complementarity refers to 79% or greater complementarity. 0063. Unless stated otherwise, the following terms and For example, a mismatch in a duplex region consisting of 19 phrases have the meanings provided below: base pairs results in 94.7% complementarity, rendering the duplex region Substantially complementary. Complementary Filters 0064. The term “complementary” refers to the ability of 0069. The term “filter” refers to one or more procedures polynucleotides to form base pairs with one another. Base that are performed on sequences that are identified by the pairs are typically formed by hydrogen bonds between nucle algorithm. In some instances, filtering includes in silico pro otide units in antiparallel polynucleotide Strands. Comple cedures where sequences identified by the algorithm can be mentary polynucleotide strands can base pair in the Watson screened to identify duplexes carrying desirable or undesir Crick manner (e.g., A to T. A to U. C to G), or in any other able motifs. Sequences carrying Such motifs can be selected manner that allows for the formation of duplexes. As persons for, or selected against, to obtain a final set with the preferred skilled in the art are aware, when using RNA as opposed to properties. In other instances, filtering includes wet lab DNA, uracil rather than thymine is the base that is considered experiments. For instance, sequences identified by one or to be complementary to adenosine. However, when a U is more versions of the algorithm can be screened using any one denoted in the context of the present invention, the ability to of a number of procedures to identify duplexes that have substitute a T is implied, unless otherwise stated. hyperfunctional traits (e.g., they exhibit a high degree of 0065 Perfect complementarity or 100% complementarity silencing at Subnanomolar concentrations and/or exhibit high refers to the situation in which each nucleotide unit of one degrees of silencing longevity). polynucleotide Strand can hydrogen bond with a nucleotide unit of a second polynucleotide strand. Less than perfect Gene Silencing complementarity refers to the situation in which some, but not 0070 The phrase “gene silencing refers to a process by all, nucleotide units of two strands can hydrogen bond with which the expression of a specific gene product is lessened or each other. For example, for two 20-mers, if only two base attenuated. Gene silencing can take place by a variety of pairs on each Strand can hydrogen bond with each other, the pathways. Unless specified otherwise, as used herein, gene polynucleotide strands exhibit 10% complementarity. In the silencing refers to decreases in gene product expression that US 2008/O 132691 A1 Jun. 5, 2008 results from RNA interference (RNAi), a defined, though and pyrimidines such as N6-methyladenosine, 5-methylcar partially characterized pathway whereby small inhibitory bonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4- RNA (siRNA) act in concert with host proteins (e.g., the RNA one, pyridine-2-one, phenyl and modified phenyl groups such induced silencing complex, RISC) to degrade messenger as aminophenol or 2.4.6-trimethoxy benzene, modified RNA (mRNA) in a sequence-dependent fashion. The level of cytosines that act as G-clamp nucleotides, 8-substituted gene silencing can be measured by a variety of means, includ adenines and guanines, 5-substituted uracils and thymines, ing, but not limited to, measurement of transcript levels by azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxy Northern Blot Analysis, B-DNA techniques, transcription alkylaminoalkyl nucleotides, and alkylcarbonylalkylated sensitive reporter constructs, expression profiling (e.g., DNA nucleotides. Modified nucleotides also include those nucle chips), and related technologies. Alternatively, the level of otides that are modified with respect to the Sugar moiety, as silencing can be measured by assessing the level of the pro well as nucleotides having Sugars or analogs thereof that are tein encoded by a specific gene. This can be accomplished by not ribosyl. For example, the Sugar moieties may be, or be performing a number of studies including Western Analysis, based on, mannoses, arabinoses, glucopyranoses, galactopy measuring the levels of expression of a reporter protein that ranoses, 4'-thioribose, and other Sugars, heterocycles, or car has e.g., fluorescent properties (e.g., GFP) or enzymatic activ bocycles. ity (e.g., alkaline phosphatases), or several other procedures. 0075. The term nucleotide is also meant to include what miRNA are known in the art as universal bases. By way of example, (0071. The term “miRNA refers to microRNA. universal bases include but are not limited to 3-nitropyrrole, 5-nitroindole, or nebularine. The term “nucleotide' is also Nucleotide meant to include the N3' to P5' phosphoramidate, resulting 0072. The term “nucleotide' refers to a ribonucleotide or a from the substitution of a ribosyl 3' oxygen with an amine deoxyribonucleotide or modified form thereof, as well as an group. analog thereof. Nucleotides include species that comprise 0076 Further, the term nucleotide also includes those spe purines, e.g., adenine, hypoxanthine, guanine, and their cies that have a detectable label, such as for example a radio derivatives and analogs, as well as pyrimidines, e.g., cytosine, active or fluorescent moiety, or mass label attached to the uracil, thymine, and their derivatives and analogs. nucleotide. 0073 Nucleotide analogs include nucleotides having modifications in the chemical structure of the base, Sugar Off-Target Silencing and Off-Target Interference and/or phosphate, including, but not limited to, 5-position 0077. The phrases “off-target silencing and “off-target pyrimidine modifications, 8-position purine modifications, interference' are defined as degradation of mRNA other than modifications at cytosine exocyclic amines, and Substitution the intended target mRNA due to overlapping and/or partial of 5-bromo-uracil; and 2'-position Sugar modifications, homology with secondary mRNA messages. including but not limited to, Sugar-modified ribonucleotides in which the 2'-OH is replaced by a group such as an H, OR, Polynucleotide R, halo, SH, SR, NH, NHR, NR, or CN, wherein R is an alkyl moiety. Nucleotide analogs are also meant to include (0078. The term “polynucleotide' refers to polymers of nucleotides with bases such as inosine, queuosine, Xanthine, nucleotides, and includes but is not limited to DNA, RNA, Sugars such as 2'-methyl ribose, non-natural phosphodiester DNA/RNA hybrids including polynucleotide chains of regu linkages such as methylphosphonates, phosphorothioates and larly and/or irregularly alternating deoxyribosyl moieties and peptides. ribosyl moieties (i.e., wherein alternate nucleotide units have 0.074 Modified bases refer to nucleotide bases such as, for an —OH, then and —H, then an —OH, then an —H, and so example, adenine, guanine, cytosine, thymine, uracil, Xan on at the 2' position of a Sugar moiety), and modifications of thine, inosine, and queuosine that have been modified by the these kinds of polynucleotides, wherein the attachment of replacement or addition of one or more atoms or groups. various entities or moieties to the nucleotide units at any Some examples of types of modifications that can comprise position are included. nucleotides that are modified with respect to the base moieties include but are not limited to, alkylated, halogenated, thi Polyribonucleotide olated, aminated, amidated, or acetylated bases, individually (0079. The term “polyribonucleotide” refers to a poly or in combination. More specific examples include, for nucleotide comprising two or more modified or unmodified example, 5-propynyluridine, 5-propynylcytidine, 6-methy ribonucleotides and/or their analogs. The term “polyribo ladenine, 6-methylguanine, N.N.-dimethyladenine, 2-propy nucleotide' is used interchangeably with the term "oligori ladenine, 2-propylguanine, 2-aminoadenine, 1-methyli bonucleotide.” nosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, Ribonucleotide and Ribonucleic Acid 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 0080. The term “ribonucleotide' and the phrase “ribo 3-methylcytidine, 6-methyluridine, 2-methylguanosine, nucleic acid (RNA), refer to a modified or unmodified nucle 7-methylguanosine, 2,2-dimethylguanosine, 5-methylami otide or polynucleotide comprising at least one ribonucle noethyluridine, 5-methyloxyuridine, deaZanucleotides Such otide unit. A ribonucleotide unit comprises anhydroxyl group as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-aZothy attached to the 2' position of a ribosyl moiety that has a midine, 5-methyl-2-thiouridine, other thio bases such as nitrogenous base attached in N-glycosidic linkage at the 1' 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrou position of a ribosyl moiety, and a moiety that either allows ridine, pseudouridine, queuosine, archaeosine, naphthyl and for linkage to another nucleotide or precludes linkage. Substituted naphthyl groups, any O- and N-alkylated purines siRNA US 2008/O 132691 A1 Jun. 5, 2008 I0081. The term “siRNA refers to small inhibitory RNA siRNA is directed. Similarly, “target silencing can refer to duplexes that induce the RNA interference (RNAi) pathway. the state of a gene, or the corresponding mRNA or protein. These molecules can vary in length (generally 18-30 base pairs) and contain varying degrees of complementarity to Transfection their target mRNA in the antisense strand. Some, but not all, I0086. The term “transfection” refers to a process by which siRNA have unpaired overhanging bases on the 5' or 3' end of agents are introduced into a cell. The list of agents that can be the sense Strand and/or the antisense Strand. The term transfected is large and includes, but is not limited to, siRNA, “siRNA’ includes duplexes of two separate strands, as well as sense and/or anti-sense sequences, DNA encoding one or single strands that can form hairpin structures comprising a more genes and organized into an expression plasmid, pro duplex region. teins, protein fragments, and more. There are multiple meth ods for transfecting agents into a cell including, but not lim 0082 siRNA may be divided into five (5) groups (non ited to, electroporation, calcium phosphate-based functional, semi-functional, functional, highly functional, transfections, DEAE-dextran-based transfections, lipid and hyper-functional) based on the level or degree of silenc based transfections, molecular conjugate-based transfections ing that they induce in cultured cell lines. As used herein, (e.g., polylysine-DNA conjugates), microinjection and oth these definitions are based on a set of conditions where the CS. siRNA is transfected into said cell line at a concentration of I0087. The present invention is directed to improving the 100 nM and the level of silencing is tested at a time of roughly efficiency of gene silencing by siRNA. Through the inclusion 24 hours after transfection, and not exceeding 72 hours after of multiple siRNA sequences that are targeted to a particular transfection. In this context, “non-functional siRNA are gene and/or selecting an siRNA sequence based on certain defined as those siRNA that induce less than 50% (<50%) defined criteria, improved efficiency may be achieved. target silencing. "Semi-functional siRNA’ induce 50-79% I0088. The present invention will now be described in con target silencing. "Functional siRNA are molecules that nection with preferred embodiments. These embodiments are induce 80-95% gene silencing. “Highly-functional siRNA presented in order to aid in an understanding of the present are molecules that induce greater than 95% gene silencing. invention and are not intended, and should not be construed, to limit the invention in any way. All alternatives, modifica “Hyperfunctional siRNA are a special class of molecules. tions and equivalents that may become apparent to those of For purposes of this document, hyperfunctional siRNA are ordinary skill upon reading this disclosure are included defined as those molecules that: (1) induce greater than 95% within the spirit and scope of the present invention. silencing of a specific target when they are transfected at I0089. Furthermore, this disclosure is not a primer on RNA Subnanomolar concentrations (i.e., less than one nanomolar); interference. Basic concepts known to persons skilled in the and/or (2) induce functional (or better) levels of silencing for art have not been set forth in detail. greater than 96 hours. These relative functionalities (though 0090 The present invention is directed to increasing the not intended to be absolutes) may be used to compare siRNAs efficiency of RNAi, particularly in mammalian systems. to a particular target for applications such as functional Accordingly, the present invention provides kits, siRNAS and genomics, target identification and therapeutics. methods for increasing siRNA efficacy. 0091. According to a first embodiment, the present inven SMARTSCORETM, or siRNA Rank tion provides a kit for gene silencing, wherein said kit is 0083. The term “SMARTSCORETM, or “siRNA rank” comprised of a pool of at least two siRNA duplexes, each of refers to a number determined by applying any of the formu which is comprised of a sequence that is complementary to a las to a given siRNA sequence. The term “SMART-selected portion of the sequence of one or more target messenger RNA, and each of which is selected using non-target specific or “rationally selected or “rational selection” refers to criteria. Each of the at least two siRNA duplexes of the kit siRNA that have been selected on the basis of their SMART complementary to a portion of the sequence of one or more SCORESTM, or siRNA ranking. target mRNAs is preferably selected using Formula X. 0092. According to a second embodiment, the present Substantially Similar invention provides a method for selecting an siRNA, said 0084. The phrase “substantially similar refers to a simi method comprising applying selection criteria to a set of larity of at least 90% with respect to the identity of the bases potential siRNA that comprise 18-30 base pairs, wherein said of the sequence. selection criteria are non-target specific criteria, and said set comprises at least two siRNAs and each of said at least two siRNAS contains a sequence that is at least Substantially Target complementary to a target gene; and determining the relative I0085. The term “target” is used in a variety of different functionality of the at least two siRNAs. forms throughout this document and is defined by the context 0093. In one embodiment, the present invention also pro in which it is used. “Target mRNA refers to a messenger vides a method wherein said selection criteria are embodied RNA to which a given siRNA can be directed against. “Target in a formula comprising: sequence' and “target site' refer to a sequence within the mRNA to which the sense strand of an siRNA shows varying degrees of homology and the antisense strand exhibits vary ing degrees of complementarity. The phrase “siRNA target' can refer to the gene, mRNA, or protein against which an 19)-30*X; or Formula VIII US 2008/O 132691 A1 Jun. 5, 2008 0096 C=1 if C is the base at position 3 of the sense strand, otherwise its value is 0; C=1 if C is the base at position 4 of the sense strand, other wise its value is 0; C=1 if C is the base at position 5 of the sense strand, other wise its value is 0; C=1 if C is the base at position 6 of the sense strand, other wise its value is 0; C, 1 if C is the base at position 7 of the sense strand, other wise its value is 0; C=1 if C is the base at position 9 of the sense strand, other wise its value is 0; C=1 if C is the base at position 17 of the sense strand, otherwise its value is 0; Cs-1 if C is the base at position 18 of the sense strand, otherwise its value is 0; C=1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; 0097 G=1 if G is the base at position 1 on the sense strand, otherwise its value is 0; G=1 if G is the base at position 2 of the sense strand, other 6* (number of A+U in position 15-19)-3*(number of wise its value is 0; G+C in whole siRNA), Formula X Gs=1 if G is the base at position 8 on the sense strand, 0094 wherein position numbering begins at the 5'-most otherwise its value is 0; position of a sense strand, and Go-1 if G is the base at position 10 on the sense strand, 0095 A=1 if A is the base at position 1 of the sense strand, otherwise its value is 0; otherwise its value is 0; G=1 if G is the base at position 13 on the sense strand, A=1 if A is the base at position 2 of the sense strand, other otherwise its value is 0; wise its value is 0; G-1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only A=1 if A is the base at position 3 of the sense strand, other 18 base pairs in length, its value is 0; wise its value is 0; 0.098 U=1 if U is the base at position 1 on the sense A=1 if A is the base at position 4 of the sense strand, other strand, otherwise its value is 0; wise its value is 0; U=1 if U is the base at position 2 on the sense strand, As=1 if A is the base at position 5 of the sense strand, other otherwise its value is 0; wise its value is 0; U=1 if U is the base at position 3 on the sense strand, A=1 if A is the base at position 6 of the sense strand, other otherwise its value is 0; wise its value is 0; U=1 if U is the base at position 4 on the sense strand, A, -1 if A is the base at position 7 of the sense strand, other otherwise its value is 0; wise its value is 0; U7–1 if U is the base at position 7 on the sense strand, A-1 if A is the base at position 10 of the sense strand, otherwise its value is 0; otherwise its value is 0; U=1 if U is the base at position 9 on the sense strand, A=1 if A is the base at position 11 of the sense strand, otherwise its value is 0; otherwise its value is 0; U 1 if U is the base at position 10 on the sense strand, A=1 if A is the base at position 13 of the sense strand, O therwise its value is 0; otherwise its value is 0; U s=1 if U is the base at position 15 on the sense strand, A=1 if A is the base at position 19 of the sense strand, O therwise its value is 0; otherwise if another base is present or the sense strand is only U 1 if U is the base at position 16 on the sense strand, 18 base pairs in length, its value is 0; otherwise its value is 0; US 2008/O 132691 A1 Jun. 5, 2008 U7–1 if U is the base at position 17 on the sense strand, siRNAs from the at least two siRNAs that have a functionality otherwise its value is 0; score that reflects greater than 80 percent silencing at a con Us–1 if U is the base at position 18 on the sense strand, centration in the picomolar range, wherein said greater than otherwise its value is 0. 80 percent silencing endures for greater than 120 hours. 0109. In other embodiments, the invention provides kits 0099 GC so the number of G and C bases within posi and/or methods wherein the siRNA are comprised of two tions 15-19 of the sense strand, or within positions 15-18 if separate polynucleotide strands; wherein the siRNA are com the sense strand is only 18 base pairs in length; prised of a single contiguous molecule Such as, for example, 0100 GC, the number of G and C bases in the sense a unimolecular siRNA (comprising, for example, either a Strand; nucleotide or non-nucleotide loop); wherein the siRNA are 0101 Tm=100 if the siRNA oligo has the internal repeat expressed from one or more vectors; and whereintwo or more longer then 4 base pairs, otherwise its value is 0; and genes are silenced by a single administration of siRNA. 0102 X=the number of times that the same nucleotide 0110. According to a sixth embodiment, the present inven repeats four or more times in a row. tion provides a hyperfunctional siRNA that is capable of 0103) Any of the methods of selecting siRNA in accor silencing Bcl2. dance with the invention can further comprise comparing the 0111. According to a seventh embodiment, the present internal stability profiles of the siRNAs to be selected, and invention provides a method for developing an siRNA algo selecting those siRNAs with the most favorable internal sta rithm for selecting functional and hyperfunctional siRNAs bility profiles. Any of the methods of selecting siRNA can for a given sequence. The method comprises: further comprise selecting either for or against sequences that 0112 (a) selecting a set of siRNAs; contain motifs that induce cellular stress. Such motifs 0113 (b) measuring the gene silencing ability of each include, for example, toxicity motifs. Any of the methods of siRNA from said set; selecting siRNA can further comprise either selecting for or 0114 (c) determining the relative functionality of each selecting against sequences that comprise stability motifs. siRNA; 0104. In another embodiment, the present invention pro 0115 (d) determining the amount of improved function vides a method of gene silencing, comprising introducing into ality by the presence or absence of at least one variable a cell at least one siRNA selected according to any of the selected from the group consisting of the total GC content, methods of the present invention. The siRNA can be intro melting temperature of the siRNA, GC content at positions duced by allowing passive uptake of siRNA, or through the 15-19, the presence or absence of a particular nucleotide at a use of a vector. particular position, relative thermodynamic stability at par 0105. According to a third embodiment, the invention pro ticular positions in a duplex, and the number of times that the vides a method for developing an algorithm for selecting same nucleotide repeats within a given sequence; and siRNA, said method comprising: (a) selecting a set of siRNA; (b) measuring gene silencing ability of each siRNA from said 0116 (e) developing an algorithm using the information set; (c) determining relative functionality of each siRNA; (d) of step (d). determining improved functionality by the presence or 0117. According to this embodiment, preferably the set of siRNAs comprises at least 90 siRNAs from at least one gene, absence of at least one variable selected from the group con more preferably at least 180 siRNAs from at least two differ sisting of the presence or absence of a particular nucleotide at ent genes, and most preferably at least 270 and 360 siRNAs a particular position, the total number of AS and US in posi from at least three and four different genes, respectively. tions 15-19, the number of times that the same nucleotide Additionally, in step (d) the determination is made with pref repeats within a given sequence, and the total number of Gs erably at least two, more preferably at least three, even more and Cs; and (e) developing an algorithm using the informa preferably at least four, and most preferably all of the vari tion of step (d). ables. The resulting algorithm is not target sequence specific. 0106. In another embodiment, the invention provides a method for selecting an siRNA with improved functionality, 0118. In another embodiment, the present invention pro comprising using the above-mentioned algorithm to identify vides rationally designed siRNAs identified using the formu an siRNA of improved functionality. las above. 0107 According to a fourth embodiment, the present 0119. In yet another embodiment, the present invention is invention provides a kit, wherein said kit is comprised of at directed to hyperfunctional siRNA. least two siRNAs, wherein said at least two siRNAs comprise 0.120. The ability to use the above algorithms, which are a first optimized siRNA and a second optimized siRNA, not sequence or species specific, allows for the cost-effective wherein said first optimized siRNA and said second opti selection of optimized siRNAS for specific target sequences. mized siRNA are optimized according a formula comprising Accordingly, there will be both greater efficiency and reli Formula X. ability in the use of siRNA technologies. 0108. According to a fifth embodiment, the present inven 0.121. The methods disclosed herein can be used in con tion provides a method for identifying a hyperfunctional junction with comparing internal stability profiles of selected siRNA, comprising applying selection criteria to a set of siRNAs, and designing an siRNA with a desirable internal potential siRNA that comprise 18-30 base pairs, wherein said stability profile; and/or in conjunction with a selection either selection criteria are non-target specific criteria, and said set for or against sequences that contain motifs that induce cel comprises at least two siRNAs and each of said at least two lular stress, for example, cellular toxicity. siRNAS contains a sequence that is at least Substantially I0122) Any of the methods disclosed herein can be used to complementary to a target gene; determining the relative silence one or more genes by introducing an siRNA selected, functionality of the at least two siRNAS and assigning each of or designed, in accordance with any of the methods disclosed the at least two siRNAs a functionality score; and selecting herein. The siRNA(s) can be introduced into the cell by any US 2008/O 132691 A1 Jun. 5, 2008 method known in the art, including passive uptake or through (18) An A base at position 8 of the sense strand. the use of one or more vectors. 0123. Any of the methods and kits disclosed herein can (19) A base other than an A at position 9 of the sense strand. employ either unimolecular siRNAs, siRNAs comprised of (20) A base other than an A at position 10 of the sense strand. two separate polynucleotide strands, or combinations thereof. Any of the methods disclosed herein can be used in gene (21) A base other than an A at position 11 of the sense strand. silencing, where two or more genes are silenced by a single (22) A base other than an A at position 12 of the sense strand. administration of siRNA(s). The siRNA(s) can be directed against two or more target genes, and administered in a single (23) An A base at position 13 of the sense strand. dose or single transfection, as the case may be. (24) A base other than an A at position 14 of the sense strand. Optimizing siRNA (25) An A base at position 15 of the sense strand 0.124. According to one embodiment, the present inven (26) An A base at position 16 of the sense strand. tion provides a method for improving the effectiveness of (27) An A base at position 17 of the sense strand. gene silencing for use to silence a particular gene through the selection of an optimal siRNA. An siRNA selected according (28) An A base at position 18 of the sense strand. to this method may be used individually, or in conjunction (29) A base other than a U at position 1 of the sense strand. with the first embodiment, i.e., with one or more other siR NAs, each of which may or may not be selected by this criteria (30) A base other than a U at position 2 of the sense strand. in order to maximize their efficiency. (31) AU base at position 3 of the sense strand. 0.125. The degree to which it is possible to selectan siRNA for a given mRNA that maximizes these criteria will depend (32) A base other than a U at position 4 of the sense strand. on the sequence of the mRNA itself. However, the selection (33) A base other than a U at position 5 of the sense strand. criteria will be independent of the target sequence. According to this method, an siRNA is selected for a given gene by using (34) AU base at position 6 of the sense strand. a rational design. That said, rational design can be described (35) A base other than a U at position 7 of the sense strand. in a variety of ways. Rational design is, in simplest terms, the application of a proven set of criteria that enhance the prob (36) A base other than a U at position 8 of the sense strand. ability of identifying a functional or hyperfunctional siRNA. (37) A base other than a U at position 9 of the sense strand. In one method, rationally designed siRNA can be identified by maximizing one or more of the following criteria: (38) A base other than a U at position 11 of the sense strand. 0126 (1) A low GC content, preferably between about (39) AU base at position 13 of the sense strand. 3O-52%. 0127 (2) At least 2, preferably at least 3A or U bases at (40) A base other than a U at position 14 of the sense strand. positions 15-19 of the siRNA on the sense strand. (41) A base other than a U at position 15 of the sense strand. (3) An A base at position 19 of the sense strand. (42) A base other than a U at position 16 of the sense strand. (4) An A base at position 3 of the sense Strand. (43) AU base at position 17 of the sense strand. (5) AU base at position 10 of the sense strand. (44) AU base at position 18 of the sense strand. (45) AU base at position 19 of the sense strand. (6) An A base at position 14 of the sense Strand. (46) AC base at position 1 of the sense strand. (7) A base other than Cat position 19 of the sense strand. (47) A C base at position 2 of the sense strand. (8) A base other than G at position 13 of the sense strand. (48) A base other than a C at position 3 of the sense strand. 0128 (9) A Tm, which refers to the character of the inter nal repeat that results in inter- or intramolecular structures for (49) AC base at position 4 of the sense strand. one strand of the duplex, that is preferably not stable at greater (50) A base other than a C at position 5 of the sense strand. than 50° C., more preferably not stable at greater than 37°C., even more preferably not stable at greater than 30° C. and (51) A base other than a C at position 6 of the sense strand. most preferably not stable at greater than 20°C. (52) A base other than a C at position 7 of the sense strand. (10) A base other than U at position 5 of the sense strand. (53) A base other than a C at position 8 of the sense strand. (11) A base other than A at position 11 of the sense strand. (54) A C base at position 9 of the sense strand. (12) A base other than an A at position 1 of the sense strand. (55) A C base at position 10 of the sense strand. (13) A base other than an A at position 2 of the sense strand. (56) AC base at position 11 of the sense strand. (14) An A base at position 4 of the sense Strand. (57) A base other than a C at position 12 of the sense strand. (15) An A base at position 5 of the sense strand. (58) A base other than a C at position 13 of the sense strand. (16) An A base at position 6 of the sense strand. (59) A base other than a C at position 14 of the sense strand. (17) An A base at position 7 of the sense strand. (60) A base other than a C at position 15 of the sense strand. US 2008/O 132691 A1 Jun. 5, 2008 (61) A base other than a C at position 16 of the sense strand. additional bases are added at the 5' end of the sense chain and (62) A base other than a C at position 17 of the sense strand. occupy positions 1 to 11. Accordingly, with respect to SEQ. ID NO.0001 NNANANNNNUCNAANNNNA and SEQ. ID (63) A base other than a C at position 18 of the sense strand. NO. 0028 GUCNNANANNNNUCNAANNNNA, both (64) AG base at position 1 of the sense strand. would have A at position 3, A at position 5, U at position 10, C at position 11, A and position 13, A and position 14 and A (65) AG base at position 2 of the sense strand. at position 19. However, SEQ. ID NO. 0028 would also have (66) AG base at position 3 of the sense strand. C at position -1, U at position -2 and G at position -3. (67) A base other than a G at position 4 of the sense strand. 0.135 For a 19 base pair siRNA, an optimal sequence of one of the strands may be represented below, where N is any (68) A base other than a G at position 5 of the sense strand. base, A, C, G, or U: (69) AG base at position 6 of the sense strand. (70) AG base at position 7 of the sense strand. SEO. ID NO. OOO1. INNANANNNNUCNAANNNNA (71) AG base at position 8 of the sense strand. SEO. ID NO. OOO2. INNANANNNNUGNAANNNNA (72) AG base at position 9 of the sense strand. SEO. ID NO. OOO3. INNANANNNNUUNAANNNNA (73) A base other than a G at position 10 of the sense strand. SEO. ID NO. OOO4. INNANANNNNUCNCANNNNA (74) AG base at position 11 of the sense strand. SEO. ID NO. OOO5. INNANANNNNUGNCANNNNA (75) AG base at position 12 of the sense strand. SEO. ID NO. OOO6. INNANANNNNUUNCANNNNA (76) AG base at position 14 of the sense strand. (77) AG base at position 15 of the sense strand. SEO. ID NO. OOO7. INNANANNNNUCNUANNNNA (78) AG base at position 16 of the sense strand. SEO. ID NO. OOO8. INNANANNNNUGNUANNNNA (79) A base other than a G at position 17 of the sense strand. SEO. ID NO. OOO9. INNANANNNNUUNUANNNNA (80) A base other than a G at position 18 of the sense strand. SEQ, ID NO, 0010, INNANCNNNNUCNAANNNNA (81) A base other than a G at position 19 of the sense strand. SEO. ID NO. OO11. INNANCNNNNUGNAANNNNA 0129. The importance of various criteria can vary greatly. SEO. ID NO. OO12. INNANCNNNNUUNAANNNNA For instance, a C base at position 10 of the sense Strand makes a minor contribution to duplex functionality. In contrast, the SEO. ID NO. OO13. INNANCNNNNUCNCANNNNA absence of a C at position 3 of the sense strand is very SEO. ID NO. OO14. INNANCNNNNUGNCANNNNA important. Accordingly, preferably an siRNA will satisfy as many of the aforementioned criteria as possible. SEO. ID NO. OO15. INNANCNNNNUUNCANNNNA 0130. With respect to the criteria, GC content, as well as a high number of AU in positions 15-19 of the sense strand, SEO. ID NO. OO16. INNANCNNNNUCNUANNNNA may be important for easement of the unwinding of double SEO. ID NO. OO17. INNANCNNNNUGNUANNNNA stranded siRNA duplex. Duplex unwinding has been shown to be crucial for siRNA functionality in vivo. SEO. ID NO. OO18. INNANCNNNNUUNUANNNNA 0131 With respect to criterion 9, the internal structure is SEO. ID NO. OO19. INNANGNNNNUCNAANNNNA measured in terms of the melting temperature of the single strand of siRNA, which is the temperature at which 50% of SEO. ID NO. OO2O. INNANGNNNNUGNAANNNNA the molecules will become denatured. With respect to criteria 2-8 and 10-11, the positions refer to sequence positions on the SEO. ID NO. OO21. INNANGNNNNUUNAANNNNA sense strand, which is the strand that is identical to the mRNA. SEO. ID NO. OO22. INNANGNNNNUCNCANNNNA 0.132. In one preferred embodiment, at least criteria 1 and 8 are satisfied. In another preferred embodiment, at least SEO. ID NO. OO23. INNANGNNNNUGNCANNNNA criteria 7 and 8 are satisfied. In still another preferred embodi SEO. ID NO. OO24. INNANGNNNNUUNCANNNNA ment, at least criteria 1, 8 and 9 are satisfied. 0133. It should be noted that all of the aforementioned SEO. ID NO. OO25. INNANGNNNNUCNUANNNNA criteria regarding sequence position specifics are with respect SEO. ID NO. OO26. INNANGNNNNUGNUANNNNA to the 5' end of the sense strand. Reference is made to the sense Strand, because most databases contain information SEO. ID NO. OO27. INNANGNNNNNU.NUANNNNA that describes the information of the mRNA. Because accord ing to the present invention a chain can be from 18 to 30 bases 0.136. In one embodiment, the sequence used as an siRNA in length, and the aforementioned criteria assumes a chain 19 is selected by choosing the siRNA that score highest accord base pairs in length, it is important to keep the aforemen ing to one of the following seven algorithms that are repre tioned criteria applicable to the correct bases. sented by Formulas I-VII: 0134. When there are only 18 bases, the base pair that is Relative functionality of siRNA--(GC/3)+(AU)- not present is the base pair that is located at the 3' of the sense (Tmo)*3-(G1)*3-(Co)+(A)*2+(A)+(Ulo)+ strand. When there are twenty to thirty bases present, then (A)-(U)-(A11) Formula I US 2008/O 132691 A1 Jun. 5, 2008 Relative functionality of siRNA=-(GC/3)-(AUso) all of these algorithms to obtain the best differentiation *3-(G1)*3-(Co)+(Ag)*2+(As) Formula II among sequences. In some instances, application of a given Relative functionality of siRNA=-(GC/3)+(AU is 19 algorithm may identify an unusually large number of poten (Tmoo)*3 Formula III tial siRNA sequences, and in those cases, it may be appropri ate to re-analyze that sequence with a second algorithm that Relative functionality of siRNA-GC/2+(AU)/2- (Tmoo)*2-(G1)*3-(Co)+(Ag)*2+(A)+(Ulo)+ is, for instance, more stringent. Alternatively, it is conceivable (A14)-(US)-(A11) Formula IV that analysis of a sequence with a given formula yields no Relative functionality of siRNA=-(G1)*3-(Co)+ acceptable siRNA sequences (i.e. low SMARTSCORESTM, (Ag)*2+(As)+(Ulo)+(A)-(US)-(A11) Formula V or siRNA ranking). In this instance, it may be appropriate to re-analyze that sequences with a second algorithm that is, for Relative functionality of siRNA=-(G1)*3-(Co)+ (A)*2+(A) Formula VI instance, less stringent. In still other instances, analysis of a single sequence with two separate formulas may give rise to Relative functionality of siRNA--(GC/2)+(AU)/ conflicting results (i.e. one formula generates a set of siRNA 2-(Tm2)*1-(G1)*3-(Co)+(A)*3+(A)*3+ (Ulo)/2+(A)/2-(US)/2-(A)/2 Formula VII with high SMARTSCORESTM, or siRNA ranking, while the other formula identifies a set of siRNA with low SMART 0.137 In Formulas I-VII: I0138 wherein A-1 if A is the base at position 19 on the SCORESTM, or siRNA ranking). In these instances, it may be sense Strand, otherwise its value is 0. necessary to determine which weighted factor(s) (e.g. GC 0139 AU so 0-5 depending on the number of A or U content) are contributing to the discrepancy and assessing the bases on the sense strand at positions 15-19; sequence to decide whether these factors should or should not 0140 G=1 if G is the base at position 13 on the sense be included. Alternatively, the sequence could be analyzed by strand, otherwise its value is 0; a third, fourth, or fifth algorithm to identify a set of rationally 0141 Co-1 if C is the base at position 19 of the sense designed siRNA. strand, otherwise its value is 0; 0142 GC=the number of G and C bases in the entire sense 0146 The above-referenced criteria are particularly Strand; advantageous when used in combination with pooling tech 0143. Tmo. 1 if the Tm is greater than 20°C.; niques as depicted in Table I: A=1 if A is the base at position 3 on the sense strand, TABLE I otherwise its value is 0; FUNCTIONAL PROBABILITY U-1 if U is the base at position 10 on the sense strand, otherwise its value is 0; OLIGOS POOLS A 1 if A is the base at position 14 on the sense Strand, CRITERIA 95% 80% 70% 95% 80% 70% otherwise its value is 0; CURRENT 33.O SO.O 23.0 79.5 97.3 O.3 NEW SO.O 88.5 8.O 93.8 99.98 O.OOS Us=1 if U is the base at position 5 on the sense strand, (GC) 28.0 S8.9 36.0 72.8 97.1 1.6 otherwise its value is 0; and A=1 if A is the base at position 11 of the sense strand, 0147 The term “current used in Table I refers to Tuschl's otherwise its value is 0. conventional siRNA parameters (Elbashir, S. M. et al. (2002) 0144. Formulas I-VII provide relative information regard 'Analysis of gene function in Somatic mammalian cells using ing functionality. When the values for two sequences are small interfering RNAs” Methods 26: 199-213). “New” compared for a given formula, the relative functionality is refers to the design parameters described in Formulas I-VII. ascertained; a higher positive number indicates a greater “GC refers to criteria that select siRNA solely on the basis of functionality. For example, in many applications a value of 5 GC content. or greater is beneficial. 0.148. As Table I indicates, when more functional siRNA 0145 Additionally, in many applications, more than one duplexes are chosen, siRNAs that produce <70% silencing drops from 23% to 8% and the number of siRNA duplexes of these formulas would provide useful information as to the that produce >80% silencing rises from 50% to 88.5%. Fur relative functionality of potential siRNA sequences. How ther, of the siRNA duplexes with >80% silencing, a larger ever, it is beneficial to have more than one type of formula, portion of these siRNAs actually silence >95% of the target because not every formula will be able to help to differentiate expression (the new criteria increases the portion from 33% to among potential siRNA sequences. For example, in particu 50%). Using this new criteria in pooled siRNAs, shows that, larly high GC mRNAs, formulas that take that parameter into with pooling, the amount of silencing >95% increases from account would not be useful and application of formulas that 79.5% to 93.8% and essentially eliminates any siRNA pool lack GC elements (e.g., formulas V and VI) might provide from silencing less than 70%. greater insights into duplex functionality. Similarly, formula 0149 Table II similarly shows the particularly beneficial II might by used in situations where hairpin structures are not results of pooling in combination with the aforementioned observed in duplexes, and formula IV might be applicable for criteria. However, Table II, which takes into account each of sequences that have higher AU content. Thus, one may con the aforementioned variables, demonstrates even a greater sider a particular sequence in light of more than one or even degree of improvement in functionality. US 2008/O 132691 A1 Jun. 5, 2008 14 TABLE II FUNCTIONAL PROBABILITY OLIGOS POOLS NON- NON FUNCTIONAL AVERAGE FUNCTIONAL FUNCTIONAL AVERAGE FUNCTIONAL RANDOM 2O 40 50 67 97 3 CRITERLA 1 52 99 O.1 97 93 O.OO40 CRITERLA 4 89 99 O.1 99 99 O.OOOO 0150. The terms “functional,” “Average,” and “Non-func- 24)*G13+(18)*G14+(11)*G15+(13)*G16+(-7) tional used in Table II, refer to siRNA that exhibit >80%, >50%, and <50% functionality, respectively. Criteria 1 and 4 6*(number of A+U in position 15-19)-3*(number of refer to specific criteria described above. G+C in whole siRNA). 0151. The above-described algorithms may be used with or without a computer program that allows for the inputting of 0153 wherein the sequence of the mRNA and automatically outputs the 0154 A=1 if A is the base at position 1 of the sense strand, optimal siRNA. The computer program may, for example, be otherwise its value is 0; accessible from a local terminal or personal computer, overan A=1 if A is the base at position 2 of the sense strand, other internal network or over the Internet. wise its value is 0; 0152. In addition to the formulas above, more detailed algorithms may be used for selecting siRNA. Preferably, at A=1 if A is the base at position 3 of the sense strand, other least one RNA duplex of 18-30 base pairs is selected such that wise its value is 0; it is optimized according a formula selected from: A=1 if A is the base at position 4 of the sense strand, other wise its value is 0; As=1 if A is the base at position 5 of the sense strand, other wise its value is 0; A=1 if A is the base at position 6 of the sense strand, other wise its value is 0; A, -1 if A is the base at position 7 of the sense strand, other wise its value is 0; A-1 if A is the base at position 10 of the sense strand, otherwise its value is 0; A=1 if A is the base at position 11 of the sense strand, otherwise its value is 0; A=1 if A is the base at position 13 of the sense strand, otherwise its value is 0; A=1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; 0155 C=1 if C is the base at position 3 of the sense strand, otherwise its value is 0; C=1 if C is the base at position 4 of the sense strand, other wise its value is 0; Cs=1 if C is the base at position 5 of the sense strand, other wise its value is 0; C1 if C is the base at position 6 of the sense strand, other wise its value is 0; C, 1 if C is the base at position 7 of the sense strand, other wise its value is 0; C1 if C is the base at position 9 of the sense strand, other wise its value is 0; US 2008/O 132691 A1 Jun. 5, 2008 C7–1 if C is the base at position 17 of the sense strand, to increase the efficiency of gene silencing. A Subset of vari otherwise its value is 0; ables of any of the formulas may be used, though when fewer variables are used, the optimization hierarchy becomes less Cs-1 if C is the base at position 18 of the sense strand, reliable. otherwise its value is 0; (0163 With respect to the variables of the above-refer C-1 if C is the base at position 19 of the sense strand, enced formulas, a single letter of A or C or G or Ufollowed by otherwise if another base is present or the sense strand is only a subscript refers to a binary condition. The binary condition 18 base pairs in length, its value is 0; is that either the particular base is present at that particular position (wherein the value is “1”) or the base is not present 0156 G=1 if G is the base at position 1 on the sense (wherein the value is “0”). Because position 19 is optional, strand, otherwise its value is 0; i.e., there might be only 18 base pairs, when there are only 18 G=1 if G is the base at position 2 of the sense strand, other base pairs, any base with a subscript of 19 in the formulas wise its value is 0; above would have a Zero value for that parameter. Before or after each variable is a number followed by *, which indicates Gs-1 if G is the base at position 8 on the sense strand, that the value of the variable is to be multiplied or weighed by otherwise its value is 0; that number. Go-1 if G is the base at position 10 on the sense strand, 0164. The numbers preceding the variables A, or G, or C, otherwise its value is 0; or U in Formulas VIII, IX, and X (or after the variables in Formula I-VII) were determined by comparing the difference G=1 if G is the base at position 13 on the sense strand, in the frequency of individual bases at different positions in otherwise its value is 0; functional siRNA and total siRNA. Specifically, the fre G-1 if G is the base at position 19 of the sense strand, quency in which a given base was observed at a particular otherwise if another base is present or the sense strand is only position in functional groups was compared with the fre 18 base pairs in length, its value is 0; quency that that same base was observed in the total, ran domly selected siRNA set. If the absolute value of the differ 0157 U=1 if U is the base at position 1 on the sense ence between the functional and total values was found to be strand, otherwise its value is 0; greater than 6%, that parameter was included in the equation. Thus, for instance, if the frequency of finding a “G” at posi U=1 if U is the base at position 2 on the sense strand, tion 13 (G) is found to be 6% in a given functional group, O therwise its value is 0; and the frequency of G in the total population of siRNAs is =1 if U is the base at position 3 on the sense strand, 20%, the difference between the two values is 6%-20%—- therwise its value is 0; 14%. As the absolute value is greater than six (6), this factor (-14) is included in the equation. Thus, in Formula VIII, in 1 if U is the base at position 4 on the sense Strand, cases where the siRNA under study has a G in position 13, the therwise its value is 0; accrued value is (-14)*(1)=–14. In contrast, when a base 7–1 if U is the base at position 7 on the sense strand, other than G is found at position 13, the accrued value is therwise its value is 0; (-14)*(0)=0. 0.165. When developing a means to optimize siRNAs, the =1 if U is the base at position 9 on the sense strand, inventors observed that a bias toward low internal thermody therwise its value is 0; namic stability of the duplex at the 5'-antisense (AS) end is U characteristic of naturally occurring miRNA precursors. The 1 if U is the base at position 10 on the sense strand, inventors extended this observation to siRNAs for which O therwise its value is 0; functionality had been assessed in tissue culture. U s=1 if U is the base at position 15 on the sense strand, 0166 With respect to the parameter GCs, a value of O therwise its value is 0; 0-5 will be ascribed depending on the number of G or C bases U 1 if U is the base at position 16 on the sense strand, at positions 15 to 19. If there are only 18 base pairs, the value is between 0 and 4. O therwise its value is 0; 0167. With respect to the criterion GC content, a num U 7–1 if U is the base at position 17 on the sense strand, ber from 0-30 will be ascribed, which correlates to the total O therwise its value is 0; number of G and C nucleotides on the sense Strand, excluding U 1 if U is the base at position 18 on the sense strand, overhangs. Without wishing to be bound by any one theory, it otherwise its value is 0; is postulated that the significance of the GC content (as well as AU content at positions 15-19, which is a parameter for 0158 GC so the number of G and C bases within posi formulas III-VII) relates to the easement of the unwinding of tions 15-19 of the sense strand, or within positions 15-18 if a double-stranded siRNA duplex. Duplex unwinding is the sense strand is only 18 base pairs in length; believed to be crucial for siRNA functionality in vivo and 0159 GC, the number of G and C bases in the sense overall low internal stability, especially low internal stability Strand; of the first unwound base pair is believed to be important to (0160 Tm=100 if the siRNA oligo has the internal repeat maintain sufficient processivity of RISC complex-induced longer then 4 base pairs, otherwise its value is 0; and duplex unwinding. If the duplex has 19 base pairs, those at 0161 X=the number of times that the same nucleotide positions 15-19 on the sense strand will unwind first if the repeats four or more times in a row. molecule exhibits a sufficiently low internal stability at that 0162 The above formulas VIII, IX, and X, as well as position. As persons skilled in the art are aware, RISC is a formulas I-VII, provide methods for selecting siRNA in order complex of approximately twelve proteins; Dicer is one, but US 2008/O 132691 A1 Jun. 5, 2008 not the only, helicase within this complex. Accordingly, duplexes. Formula X is similar to Formula IX, yet uses a although the GC parameters are believed to relate to activity greater numbers of variables and for that reason, identifies with Dicer, they are also important for activity with other sequences on the basis of slightly different criteria. RISC proteins. 0172. It should also be noted that the output of a particular 0168 The value of the parameter Tm is 0 when there are no algorithm will depend on several of variables including: (1) internal repeats longer than (or equal to) four base pairs the size of the database(s) being analyzed by the algorithm, present in the siRNA duplex; otherwise the value is 1. Thus and (2) the number and stringency of the parameters being for example, if the sequence ACGUACGU, or any other four applied to screen each sequence. Thus, for example, in U.S. nucleotide (or more) palindrome exists within the structure, patent application Ser. No. 10/714,333, entitled “Functional the value will be one (1). Alternatively if the structure ACG and Hyperfunctional siRNA filed Nov. 14, 2003, Formula GACG, or any other 3 nucleotide (or less) palindrome exists, VIII was applied to the known human genome (NCBI REF the value will be zero (0). SEQ database) through ENTREZ (EFETCH). As a result of (0169. The variable “X” refers to the number of times that these procedures, roughly 1.6 million siRNA sequences were the same nucleotide occurs contiguously in a stretch of four or identified. Application of Formula VIII to the same database more units. If there are, for example, four contiguous AS in in March of 2004 yielded roughly 2.2 million sequences, a one part of the sequence and elsewhere in the sequence four difference of approximately 600,000 sequences resulting contiguous Cs, X-2. Further, if there are two separate con from the growth of the database over the course of the months tiguous stretches of four of the same nucleotides or eight or that span this period of time. Application of other formulas more of the same nucleotides in a row, then X=2. However, X (e.g., Formula X) that change the emphasis of, include, or does not increase for five, six or seven contiguous nucle eliminate different variables can yield unequal numbers of otides. siRNAs. Alternatively, in cases where application of one for 0170 Again, when applying Formula VIII, Formula IX, or mula to one or more genes fails to yield Sufficient numbers of Formula X, to a given mRNA, (the “target RNA or “target siRNAs with scores that would be indicative of strong silenc molecule'), one may use a computer program to evaluate the ing, said genes can be reassessed with a second algorithm that criteria for every sequence of 18-30 base pairs or only is, for instance, less Stringent. sequences of a fixed length, e.g., 19 base pairs. Preferably the 0173 siRNA sequences identified using Formula VIII and computer program is designed such that it provides a report Formula X (minus sequences generated by Formula VIII) are ranking of all of the potential siRNAS 18-30 base pairs, contained within the sequence listing. The data included in ranked according to which sequences generate the highest the sequence listing is described more fully below. The value. A higher value refers to a more efficient siRNA for a sequences identified by Formula VIII and Formula X that are particular target gene. The computer program that may be disclosed in the sequence listing may be used in gene silenc used may be developed in any computer language that is ing applications. known to be useful for scoring nucleotide sequences, or it 0.174. It should be noted that for Formulas VIII, IX, and X may be developed with the assistance of commercially avail all of the aforementioned criteria are identified as positions on able product such as Microsoft's PRODUCT.NET. Addition the sense strand when oriented in the 5' to 3' direction as they ally, rather than run every sequence through one and/or are identified in connection with Formulas I-VII unless oth anotherformula, one may compare a Subset of the sequences, erwise specified. which may be desirable if for example only a subset are 0.175 Formulas I-X, may be used to select or to evaluate available. For instance, it may be desirable to first perform a one, or more than one, siRNA in order to optimize silencing. BLAST (Basic Local Alignment Search Tool) search and to Preferably, at least two optimized siRNAs that have been identify sequences that have no homology to other targets. selected according to at least one of these formulas are used to Alternatively, it may be desirable to Scan the sequence and to silence a gene, more preferably at least three and most pref identify regions of moderate GC context, then perform rel erably at least four. The siRNAs may be used individually or evant calculations using one of the above-described formulas together in a pool or kit. Further, they may be applied to a cell on these regions. These calculations can be done manually or simultaneously or separately. Preferably, the at least two siR with the aid of a computer. NAs are applied simultaneously. Pools are particularly ben (0171 As with Formulas I-VII, either Formula VIII, For eficial for many research applications. However, for thera mula IX, or Formula X may be used for a given mRNA target peutics, it may be more desirable to employ a single sequence. However, it is possible that according to one or the hyperfunctional siRNA as described elsewhere in this appli otherformula more than one siRNA will have the same value. cation. Accordingly, it is beneficial to have a second formula by 0176 When planning to conduct gene silencing, and it is which to differentiate sequences. Formulas IX and X were necessary to choose between two or more siRNAs, one should derived in a similar fashion as Formula VIII, yet used a larger do so by comparing the relative values when the siRNA are data set and thus yields sequences with higher statistical Subjected to one of the formulas above. In general a higher correlations to highly functional duplexes. The sequence that scored siRNA should be used. has the highest value ascribed to it may be referred to as a 0177 Useful applications include, but are not limited to, “first optimized duplex. The sequence that has the second target validation, gene functional analysis, research and drug highest value ascribed to it may be referred to as a “second discovery, gene therapy and therapeutics. Methods for using optimized duplex. Similarly, the sequences that have the siRNA in these applications are well known to persons of skill third and fourth highest values ascribed to them may be in the art. referred to as a third optimized duplex and a fourth optimized 0.178 Because the ability of siRNA to function is depen duplex, respectively. When more than one sequence has the dent on the sequence of the RNA and not the species into same value, each of them may, for example, be referred to as which it is introduced, the present invention is applicable first optimized duplex sequences or co-first optimized across abroad range of species, including but not limited to all US 2008/O 132691 A1 Jun. 5, 2008 mammalian species, such as humans, dogs, horses, cats, cows, mice, hamsters, chimpanzees and gorillas, as well as - Continued other species and organisms such as bacteria, viruses, insects, tgtggacgaa gtaccgaaag gtc.ttaccgg aaaacticgac plants and C. elegans. gcaagaaaaa t cagagagat cct cataaag gccaagaagg 0179 The present invention is also applicable for use for silencing a broad range of genes, including but not limited to the roughly 45,000 genes of a human genome, and has par DBI, NM 020548 (202-310) (Every Position) ticular relevance in cases where those genes are associated with diseases such as diabetes, Alzheimer's, cancer, as well as 0185 all genes in the genomes of the aforementioned organisms. 0180. The siRNA selected according to the aforemen SEO. ID NO. OO31: tioned criteria or one of the aforementioned algorithms are acgggcaagg C caagtggga tigcctggaat gagctgaaag also, for example, useful in the simultaneous screening and functional analysis of multiple genes and gene families using ggactitccaa ggaagatgcc atgaaagctt acatcaacaa high throughput strategies, as well as in direct gene Suppres agtagaagag ctaaagaaaa aatacggg sion or silencing. 0186. A list of the siRNAs appears in Table III (see Development of the Algorithms Examples Section, Example II) 0181. To identify siRNA sequence features that promote 0187. The set of duplexes was analyzed to identify corre functionality and to quantify the importance of certain cur lations between siRNA functionality and other biophysical or rently accepted conventional factors—such as G/C content thermodynamic properties. When the siRNA panel was ana and target site accessibility—the inventors synthesized an lyzed in functional and non-functional Subgroups, certain siRNA panel consisting of 270 siRNAs targeting three genes, nucleotides were much more abundant at certain positions in Human Cyclophilin, Firefly Luciferase, and Human DBI. In functional or non-functional groups. More specifically, the all three cases, siRNAs were directed against specific regions frequency of each nucleotide at each position in highly func of each gene. For Human Cyclophilin and Firefly Luciferase, tional siRNA duplexes was compared with that of nonfunc ninety siRNAs were directed against a 199 bp segment of tional duplexes in order to assess the preference for or against each respective mRNA. For DBI, 90 siRNAs were directed any given nucleotide at every position. These analyses were against a smaller, 109 base pair region of the mRNA. The used to determine important criteria to be included in the sequences to which the siRNAs were directed are provided siRNA algorithms (Formulas VIII, IX, and X). below. 0188 The data set was also analyzed for distinguishing biophysical properties of siRNAs in the functional group, 0182. It should be noted that in certain sequences, “t is Such as optimal percent of GC content, propensity for internal present. This is because many databases contain information structures and regional thermodynamic stability. Of the pre in this manner. However, the t denotes a uracil residue in sented criteria, several are involved in duplex recognition, mRNA and siRNA. Any algorithm will, unless otherwise RISC activation/duplex unwinding, and target cleavage specified, process at in a sequence as a u. catalysis. Human Cyclophilin: 193-390, M60857 0189 The original data set that was the source of the statistically derived criteria is shown in FIG. 2. Additionally, 0183) this figure shows that random selection yields siRNA duplexes with unpredictable and widely varying silencing potencies as measured in tissue culture using HEK293 cells. SEO. ID NO. 29: gttccaaaaa cagtggataa ttttgtggcc ttagctacag In the figure, duplexes are plotted Such that each X-axis tick mark represents an individual siRNA, with each subsequent gagagaaagg atttggctac aaaaa.ca.gca aattic catcg siRNA differing in target position by two nucleotides for Human Cyclophilin B and Firefly Luciferase, and by one tgtaatcaag gaCtt catga t cc agggcgg agact tcacc nucleotide for Human DBI. Furthermore, the y-axis denotes aggggagatg gcacaggagg aaagagcatc tacggtgagc the level of target expression remaining after transfection of the duplex into cells and Subsequent silencing of the target. gct tcc.ccga tigaga acttic aaactgaagc act acgggcc 0.190 siRNA identified and optimized in this document tggctggg work equally well in a wide range of cell types. FIG.3a shows the evaluation of thirty siRNAs targeting the DBI gene in three cell lines derived from different tissues. Each DBI Firefly Luciferase: 1434-1631, Uzos (pGL3, Promega) siRNA displays very similar functionality in HEK293 (ATCC, CRL-1573, human embryonic kidney), HeLa 0184 (ATCC, CCL-2, cervical epithelial adenocarcinoma) and DU145 (HTB-81, prostate) cells as determined by the SEO. ID NO. 3O: B-DNA assay. Thus, siRNA functionality is determined by tgaactt CCC gcc.gc.cgttgttgttittgga gcacggaaag the primary sequence of the siRNA and not by the intracel lular environment. Additionally, it should be noted that acgatgacgg aaaaagagat cqtggattac gtc.gc.cagt c although the present invention provides fora determination of aagtaacaac cycgaaaaag ttgcgcggag gagttgttgtt the functionality of siRNA for a given target, the same siRNA may silence more than one gene. For example, the comple mentary sequence of the silencing siRNA may be present in US 2008/O 132691 A1 Jun. 5, 2008 more than one gene. Accordingly, in these circumstances, it otide at each position in a functional siRNA duplex was may be desirable not to use the siRNA with highest SMART compared with that of a nonfunctional duplex in order to SCORETM, or siRNA ranking. In such circumstances, it may assess the preference for or against a certain nucleotide. FIG. be desirable to use the siRNA with the next highest SMART 4 shows the results of these queries and the Subsequent resort SCORETM, or siRNA ranking. ing of the data set (from FIG.2). The data is separated into two (0191 To determine the relevance of G/C content in siRNA sets: those duplexes that meet the criteria, a specific nucle function, the G/C content of each duplex in the panel was otide in a certain position grouped on the left (Selected) and calculated and the functional classes of siRNAs (sF50, those that do not grouped on the right (Eliminated). The 2F50, 2F80,2F95 where Frefers to the percent gene silenc duplexes are further sorted from most functional to least ing) were sorted accordingly. The majority of the highly functional with the y-axis of FIG. 4a-e representing the % functional siRNAs (2F95) fell within the G/C content range expression i.e., the amount of silencing that is elicited by the of 36%-52% (FIG. 3B). Twice as many non-functional duplex (Note: each position on the X-axis represents a differ ( ables and their multipliers. As described above, basic statis TABLE IV-continued tical methods were use to determine the relative values for these multipliers. PERCENT IMPROVEMENT 0203 To determine the value for “Improvement over Ran CRITERION UNCTIONAL OVERRANDOM (%) dom” the difference in the frequency of a given attribute (e.g., F8O 60.4 4.3 GC content, base preference) at a particular position is deter F95 23.9 2.2 II. At least 3 AU bases at FSO 8.2 -1.8 mined between individual functional groups (e.g. tures correlated negatively with siRNA functionality. induce cellular stress and/or cell death (apoptosis) which in Complementary Strands with stable internal repeats are more turn can mislead researchers into associating a particular likely to exist as stable hairpins thus decreasing the effective (e.g., nonessential) gene with, e.g., an essential function. concentration of the functional duplex form. This suggests Alternatively, sequences generated by any of the before men that the duplex is the preferred conformation for initial pre tioned formulas can be filtered to identify and retain duplexes RISC association. Indeed, although single complementary that contain toxic motifs. Such duplexes may be valuable Strands can induce gene silencing, the effective concentration from a variety of perspectives including, for instance, uses as required is at least two orders of magnitude higher than that of therapeutic molecules. A variety of toxic motifs exist and can the duplex form. exert their influence on the cell through RNAi and non-RNAi 0214 siRNA-pre-RISC complex formation is followed by pathways. Examples of toxic motifs are explained more fully an ATP-dependent duplex unwinding step and “activation of in commonly assigned U.S. Provisional Patent Application the RISC. The siRNA functionality was shown to correlate Ser. No. 60/538,874, entitled “Identification of Toxic with overall low internal stability of the duplex and low Sequences.” filed Jan. 23, 2004. Briefly, toxic motifs include internal stability of the 3' sense end (or differential internal A/GUUUA/G/U, G/CAAA G/C, and GCCA, or a comple stability of the 3' sense compare to the 5' sense strand), which ment of any of the foregoing. may reflect strand selection and entry into the RISC. Overall 0218. In another instance, sequences identified by any of duplex stability and low internal stability at the 3' end of the the before mentioned formulas can be filtered to identify sense strand were also correlated with siRNA functionality. duplexes that contain motifs (or general properties) that pro Interestingly, siRNAs with very high and very low overall vide serum stability or induce serum instability. In one envi stability profiles correlate strongly with non-functional Sioned application of siRNA as therapeutic molecules, duplexes. One interpretation is that high internal stability duplexes targeting disease-associated genes will be intro prevents efficient unwinding while very low stability reduces duced into patients intravenously. As the half-life of single siRNA target affinity and subsequent mRNA cleavage by the and double stranded RNA in serum is short, post-algorithm RISC filters designed to select molecules that contain motifs that 0215 Several criteria describe base preferences at specific enhance duplex stability in the presence of serum and/or positions of the sense Strand and are even more intriguing (conversely) eliminate duplexes that contain motifs that when considering their potential mechanistic roles in target destabilize siRNA in the presence of serum, would be ben recognition and mRNA cleavage. Base preferences for A at eficial. position 19 of the sense strand but not C. are particularly 0219. In another instance, sequences identified by any of interesting because they reflect the same base preferences the before mentioned formulas can be filtered to identify observed for naturally occurring miRNA precursors. That is, duplexes that are hyperfunctional. Hyperfunctional among the reported miRNA precursor sequences 75% con sequences are defined as those sequences that (1) induce tain a Uat position 1 which corresponds to an A in position 19 greater than 95% silencing of a specific target when they are of the sense strand of siRNAs, while G was under-represented transfected at Subnanomolar concentrations (i.e., less than in this same position for miRNA precursors. These observa one nanomolar); and/or (2) induce functional (or better) lev tions support the hypothesis that both miRNA precursors and els of silencing for greater than 96 hours. Filters that identify siRNA duplexes are processed by very similar if not identical hyperfunctional molecules can vary widely. In one example, protein machinery. The functional interpretation of the pre the top ten, twenty, thirty, or forty siRNA can be assessed for dominance of a U/A base pair is that it promotes flexibility at the ability to silence a given target at, e.g., concentrations of the 5'antisense ends of both siRNA duplexes and miRNA 1 nM and 0.5 nM to identify hyperfunctional molecules. precursors and facilitates efficient unwinding and selective strand entrance into an activated RISC. Pooling 0216 Among the criteria associated with base preferences 0220 According to another embodiment, the present that are likely to influence mRNA cleavage or possibly prod invention provides a pool of at least two siRNAs, preferably uct release, the preference for U at position 10 of the sense in the form of a kit or therapeutic reagent, wherein one strand Strand exhibited the greatest impact, enhancing the probabil of each of the siRNAS, the sense Strand comprises a sequence ity of selecting an F80 sequence by 13.3%. Activated RISC that is Substantially similar to a sequence within a target preferentially cleaves target mRNA between nucleotides 10 mRNA. The opposite strand, the antisense strand, will pref and 11 relative to the 5' end of the complementary targeting erably comprise a sequence that is Substantially complemen strand. Therefore, it may be that U, the preferred base for most tary to that of the target mRNA. More preferably, one strand endoribonucleases, at this position Supports more efficient of each siRNA will comprise a sequence that is identical to a cleavage. Alternatively, a U/A bp between the targeting sequence that is contained in the target mRNA. Most prefer siRNA strand and its cognate target mRNA may create an ably, each siRNA will be 19 base pairs in length, and one optimal conformation for the RISC-associated "slicing strand of each of the siRNAs will be 100% complementary to activity. a portion of the target mRNA. 0221 By increasing the number of siRNAs directed to a Post Algorithm Filters particular target using a pool or kit, one is able both to 0217. According to another embodiment, the output of any increase the likelihood that at least one siRNA with satisfac one of the formulas previously listed can be filtered to remove tory functionality will be included, as well as to benefit from or select for siRNAs containing undesirable or desirable additive or synergistic effects. Further, when two or more motifs or properties, respectively. In one example, sequences siRNAS directed against a single gene do not have satisfactory identified by any of the formulas can be filtered to remove any levels of functionality alone, if combined, they may satisfac and all sequences that induce toxicity or cellular stress. Intro torily promote degradation of the target messenger RNA and duction of an siRNA containing a toxic motif into a cell can successfully inhibit translation. By including multiple siR US 2008/O 132691 A1 Jun. 5, 2008 22 NAs in the system, not only is the probability of silencing two nucleotides, most often dTdT or UU at the 3' end of the increased, but the economics of operation are also improved sense and antisense strand that are not complementary to the when compared to adding different siRNAs sequentially. This target sequence. The siRNAS may be produced by any effect is contrary to the conventional wisdom that the concur method that is now known or that comes to be known for rent use of multiple siRNA will negatively impact gene synthesizing double stranded RNA that one skilled in the art silencing (e.g., Holen, T. et al. (2003) Similar behavior of would appreciate would be useful in the present invention. single Strand and double strand siRNAS Suggests they act Preferably, the siRNAs will be produced by Dharmacon's through a common RNAi pathway. NA 31: 2401-21407). proprietary ACER) technology. However, other methods for 0222. In fact, when two siRNAs were pooled together, synthesizing siRNAs are well known to persons skilled in the 54% of the pools of two siRNAs induced more than 95% gene art and include, but are not limited to, any chemical synthesis silencing. Thus, a 2.5-fold increase in the percentage of func of RNA oligonucleotides, ligation of shorter oligonucle tionality was achieved by randomly combining two siRNAs. otides, in vitro transcription of RNA oligonucleotides, the use Further, over 84% of pools containing two siRNAs induced of vectors for expression within cells, recombinant Dicer more than 80% gene silencing. products and PCR products. 0223) More preferably, the kit is comprised ofat least three 0227. The siRNA duplexes within the aforementioned siRNAs, wherein one strand of each siRNA comprises a pools of siRNAS may correspond to overlapping sequences sequence that is Substantially similar to a sequence of the within a particular mRNA, or non-overlapping sequences of target mRNA and the other Strand comprises a sequence that the mRNA. However, preferably they correspond to non is substantially complementary to the region of the target overlapping sequences. Further, each siRNA may be selected mRNA. As with the kit that comprises at least two siRNAs, randomly, or one or more of the siRNA may be selected more preferably one strand will comprise a sequence that is according to the criteria discussed above for maximizing the identical to a sequence that is contained in the mRNA and effectiveness of siRNA. another strand that is 100% complementary to a sequence that 0228 Included in the definition of siRNAs are siRNAs is contained in the mRNA. During experiments, when three that contain substituted and/or labeled nucleotides that may, siRNAs were combined together, 60% of the pools induced for example, be labeled by radioactivity, fluorescence or more than 95% gene silencing and 92% of the pools induced mass. The most common Substitutions are at the 2' position of more than 80% gene silencing. the ribose Sugar, where moieties such as H (hydrogen) F. NH 0224 Further, even more preferably, the kit is comprised OCH and other O-alkyl, alkenyl, alkynyl, and orthoesters, of at least four siRNAs, wherein one strand of each siRNA may be substituted, or in the phosphorous backbone, where comprises a sequence that is Substantially similar to a region sulfur, amines or hydrocarbons may be substituted for the of the sequence of the target mRNA, and the other strand bridging of non-bridging atoms in the phosphodiester bond. comprises a sequence that is Substantially complementary to Examples of modified siRNAs are explained more fully in the region of the target mRNA. As with the kit or pool that commonly assigned U.S. patent application Ser. No. 10/613, comprises at least two siRNAs, more preferably one strand of 077, filed Jul. 1, 2003. each of the siRNA duplexes will comprise a sequence that is 0229. Additionally, as noted above, the cell type into identical to a sequence that is contained in the mRNA, and which the siRNA is introduced may affect the ability of the another strand that is 100% complementary to a sequence that siRNA to enter the cell; however, it does not appear to affect is contained in the mRNA. the ability of the siRNA to function once it enters the cell. 0225. Additionally, kits and pools with at least five, at least Methods for introducing double-stranded RNA into various six, and at least seven siRNAs may also be useful with the cell types are well known to persons skilled in the art. present invention. For example, pools of five siRNA induced 0230. As persons skilled in the art are aware, in certain 95% gene silencing with 77% probability and 80% silencing species, the presence of proteins such as RdRP, the RNA with 98.8% probability. Thus, pooling of siRNAs together dependent RNA polymerase, may catalytically enhance the can result in the creation of a target-specific silencing reagent activity of the siRNA. For example, RdRP propagates the with almost a 99% probability of being functional. The fact RNAi effect in C. elegans and other non-mammalian organ that such high levels of Success are achievable using Such isms. In fact, in organisms that contain these proteins, the pools of siRNA, enables one to dispense with costly and siRNA may be inherited. Two other proteins that are well time-consuming target-specific validation procedures. studied and known to be a part of the machinery are members 0226 For this embodiment, as well as the other aforemen of the Argonaute family and Dicer, as well as their homo tioned embodiments, each of the siRNAs within a pool will logues. There is also initial evidence that the RISC complex preferably comprise 18-30 base pairs, more preferably 18-25 might be associated with the ribosome so the more efficiently base pairs, and most preferably 19 base pairs. Within each translated mRNAs will be more susceptible to silencing than siRNA, preferably at least 18 contiguous bases of the anti others. sense strand will be 100% complementary to the target 0231. Another very important factor in the efficacy of mRNA. More preferably, at least 19 contiguous bases of the siRNA is mRNA localization. In general, only cytoplasmic antisense strand will be 100% complementary to the target mRNAs are considered to be accessible to RNAi to any appre mRNA. Additionally, there may be overhangs on either the ciable degree. However, appropriately designed siRNAs, for sense Strand or the antisense Strand, and these overhangs may example, siRNAs modified with internucleotide linkages or beat either the 5' end or the 3' end of either of the strands, for 2'-O-methyl groups, may be able to cause silencing by acting example there may be one or more overhangs of 1-6 bases. in the nucleus. Examples of these types of modifications are When overhangs are present, they are not included in the described in commonly assigned U.S. patent application Ser. calculation of the number of base pairs. The two nucleotide 3' Nos. 10/431,027 and 10/613,077. overhangs mimic natural siRNAS and are commonly used but 0232. As described above, even when one selects at least are not essential. Preferably, the overhangs should consist of two siRNAs at random, the effectiveness of the two may be US 2008/O 132691 A1 Jun. 5, 2008 greater than one would predict based on the effectiveness of used to facilitate delivery. For certain applications it may be two individual siRNAs. This additive or synergistic effect is preferable to deliver molecules without transfection by sim particularly noticeable as one increases to at least three siR ply formulating in a physiological acceptable solution. NAS, and even more noticeable as one moves to at least four 0238. This embodiment may be used in connection with siRNAS. Surprisingly, the pooling of the non-functional and any of the aforementioned embodiments. Accordingly, the semi-functional siRNAs, particularly more than five siRNAs, sequences within any pool may be selected by rational design. can lead to a silencing mixture that is as effective if not more effective than any one particular functional siRNA. Multigene Silencing 0233. Within the kits of the present invention, preferably each siRNA will be present in a concentration of between 0239. In addition to developing kits that contain multiple 0.001 and 200 uM, more preferably between 0.01 and 200 siRNA directed against a single gene, another embodiment nM, and most preferably between 0.1 and 10 nM. includes the use of multiple siRNA targeting multiple genes. 0234. In addition to preferably comprising at least four or Multiple genes may be targeted through the use of high- or five siRNAs, the kits of the present invention will also pref hyper-functional siRNA. High- or hyper-functional siRNA erably comprise a buffer to keep the siRNA duplex stable. that exhibit increased potency, require lower concentrations Persons skilled in the art are aware of buffers suitable for to induce desired phenotypic (and thus therapeutic) effects. keeping siRNA stable. For example, the buffer may be com This circumvents RISC Saturation. It therefore reasons that if prised of 100 mM KC1, 30 mM HEPES-pH 7.5, and 1 mM lower concentrations of a single siRNA are needed for knock MgCl2. Alternatively, kits might contain complementary out or knockdown expression of one gene, then the remaining Strands that contain any one of a number of chemical modi (uncomplexed) RISC will be free and available to interact fications (e.g., a 2'-O-ACE) that protect the agents from deg with siRNA directed against two, three, four, or more, genes. radation by nucleases. In this instance, the user may (or may Thus in this embodiment, the authors describe the use of not) remove the modifying protective group (e.g., deprotect) highly functional or hyper-functional siRNA to knock out before annealing the two complementary Strands together. three separate genes. More preferably, Such reagents could be 0235. By way of example, the kits may be organized such combined to knockout four distinct genes. Even more prefer that pools of siRNA duplexes are provided on an array or ably, highly functional or hyperfunctional siRNA could be microarray of wells or drops for a particular gene set or for used to knockout five distinct genes. Most preferably, siRNA unrelated genes. The array may, for example, be in 96 wells, of this type could be used to knockout or knockdown the 384 wells or 1284 wells arrayed in a plastic plate or on a glass expression of six or more genes. slide using techniques now known or that come to be known to persons skilled in the art. Within an array, preferably there Hyperfunctional siRNA will be controls such as functional anti-lamin A/C, cyclophi 0240. The term hyperfunctional siRNA (hf-siRNA) lin and two siRNA duplexes that are not specific to the gene of describes a subset of the siRNA population that induces RNAi interest. in cells at low- or Sub-nanomolar concentrations for extended 0236. In order to ensure stability of the siRNA pools prior periods of time. These traits, heightened potency and to usage, they may be retained in lyophilized form at minus extended longevity of the RNAi phenotype, are highly attrac twenty degrees (-20°C.) until they are ready for use. Prior to tive from a therapeutic standpoint. Agents having higher usage, they should be resuspended; however, even once resus potency require lesser amounts of the molecule to achieve the pended, for example, in the aforementioned buffer, they desired physiological response, thus reducing the probability should be kept at minus twenty degrees, (-20°C.) until used. of side effects due to “off-target' interference. In addition to The aforementioned buffer, prior to use, may be stored at the potential therapeutic benefits associated with hyperfunc approximately 4°C. or room temperature. Effective tempera tional siRNA, hf-siRNA are also desirable from an economic tures at which to conduct transfections are well known to perspective. Hyperfunctional siRNA may cost less on a per persons skilled in the art and include for example, room treatment basis, thus reducing overall expenditures to both temperature. the manufacturer and the consumer. 0237. The kits may be applied either in vivo or in vitro. 0241 Identification of hyperfunctional siRNA involves Preferably, the siRNA of the pools or kits is applied to a cell multiple steps that are designed to examine an individual through transfection, employing standard transfection proto siRNA agent's concentration- and/or longevity-profiles. In cols. These methods are well known to persons skilled in the one non-limiting example, a population of siRNA directed art and include the use of lipid-based carriers, electropora against a single gene are first analyzed using the previously tion, cationic carriers, and microinjection. Further, one could described algorithm (Formula VIII). Individual siRNA are apply the present invention by synthesizing equivalent DNA then introduced into a test cell line and assessed for the ability sequences (either as two separate, complementary strands, or to degrade the target mRNA. It is important to note that when as hairpin molecules) instead of siRNA sequences and intro performing this step it is not necessary to testall of the siRNA. ducing them into cells through vectors. Once in the cells, the Instead, it is sufficient to test only those siRNA having the cloned DNA could be transcribed, thereby forcing the cells to highest SMARTSCORESTM, or siRNA ranking (i.e., generate the siRNA. Examples of vectors suitable for use with SMARTSCORESTM, or siRNA ranking>-10). Subsequently, the present application include but are not limited to the the gene silencing data is plotted against the SMART standard transient expression vectors, adenoviruses, retrovi SCORESTM, or siRNA rankings (see FIG.9), siRNA that (1) ruses, lentivirus-based vectors, as well as other traditional induce a high degree of gene silencing (i.e., they induce expression vectors. Any vector that has an adequate siRNA greater than 80% gene knockdown) and (2) have Superior expression and procession module may be used. Further SMARTSCORESTM (i.e., a SMARTSCORETM, or siRNA more, certain chemical modifications to siRNAS, including ranking, of >-10, Suggesting a desirable average internal but not limited to conjugations to other molecules, may be stability profile) are selected for further investigations US 2008/O 132691 A1 Jun. 5, 2008 24 designed to better understand the molecule's potency and These methods include, but are not limited to, any manner of longevity. In one, non-limiting study dedicated to under transfection, Such as, for example, transfection employing standing a molecule's potency, an siRNA is introduced into DEAE-Dextran, calcium phosphate, cationic lipids/lipo one (or more) cell types in increasingly diminishing concen Somes, micelles, manipulation of pressure, microinjection, trations (e.g., 3.0->0.3 nM). Subsequently, the level of gene electroporation, immunoporation, use of vectors such as silencing induced by each concentration is examined and viruses, plasmids, cosmids, bacteriophages, cell fusions, and siRNA that exhibit hyperfunctional potency (i.e., those that coupling of the polynucleotides to specific conjugates or induce 80% silencing or greater at, e.g., picomolar concen ligands such as antibodies, antigens, or receptors, passive trations) are identified. In a second study, the longevity pro introduction, adding moieties to the siRNA that facilitate its files of siRNA having high (>-10) SMARTSCORESTM, or uptake, and the like. siRNA rankings and greater than 80% silencing are exam 0245 Having described the invention with a degree of ined. In one non-limiting example of how this is achieved, particularity, examples will now be provided. These examples siRNA are introduced into a test cell line and the levels of are not intended to and should not be construed to limit the RNAi are measured over an extended period of time (e.g., Scope of the claims in any way. 24-168 hrs). siRNAs that exhibit strong RNA interference patterns (i.e. >80% interference) for periods of time greater EXAMPLES than, e.g., 120 hours, are thus identified. Studies similar to those described above can be performed on any and all of the General Techniques and Nomenclatures >10 siRNA included in this document to further define the 0246 siRNA nomenclature. All siRNA duplexes are most functional molecule for any given gene. Molecules pos referred to by sense strand. The first nucleotide of the 5'-end sessing one or both properties (extended longevity and of the sense strand is position 1, which corresponds to posi heightened potency) are labeled “hyperfunctional siRNA. tion 19 of the antisense strand for a 19-mer. In most cases, to and earmarked as candidates for future therapeutic studies. compare results from different experiments, silencing was 0242. While the example(s) given above describe one determined by measuring specific transcript mRNA levels or means by which hyperfunctional siRNA can be isolated, nei enzymatic activity associated with specific transcript levels, ther the assays themselves nor the selection parameters used 24 hours post-transfection, with siRNA concentrations held are rigid and can vary with each family of siRNA. Families of constant at 100 nM. For all experiments, unless otherwise siRNA include siRNAS directed against a single gene, or specified, transfection efficiency was ensured to be over 95%, directed against a related family of genes. and no detectable cellular toxicity was observed. The follow 0243 The highest quality siRNA achievable for any given ing system of nomenclature was used to compare and report gene may vary considerably. Thus, for example, in the case of siRNA-silencing functionality: “F” followed by the degree of one gene (gene X), rigorous studies such as those described minimal knockdown. For example, F50 signifies at least 50% above may enable the identification of an siRNA that, at knockdown, F80 means at least 80%, and so forth. For this picomolar concentrations, induces 99-96 silencing for a study, all sub-F50 siRNAs were considered non-functional. period of 10 days. Yet identical studies of a second gene (gene 0247 Cell culture and transfection. 96-well plates are Y) may yield an siRNA that at high nanomolar concentrations coated with 50 ul of 50 mg/ml poly-L-lysine (Sigma) for 1 hr. (e.g., 100 nM) induces only 75% silencing for a period of 2 and thenwashed 3x with distilled water before being dried for days. Both molecules represent the very optimum siRNA for 20 min. HEK293 cells or HEK293Lucs or any other cell type their respective gene targets and therefore are designated of interest are released from their solid support by trypsiniza “hyperfunctional.” Yet due to a variety of factors including but tion, diluted to 3.5x10 cells/ml, followed by the addition of not limited to target concentration, siRNA stability, cell type, 100 uL of cells/well. Plates are then incubated overnight at off-target interference, and others, equivalent levels of 37° C., 5% CO. Transfection procedures can vary widely potency and longevity are not achievable. Thus, for these depending on the cell type and transfection reagents. In one reasons, the parameters described in the before mentioned non-limiting example, a transfection mixture consisting of 2 assays can vary. While the initial screen selected siRNA that mL Opti-MEM I (Gibco-BRL), 80 ul Lipofectamine 2000 had SMARTSCORESTM above -10 and a gene silencing (Invitrogen), 15 uL SUPERNasin at 20 U/ul (Ambion), and capability of greater than 80%, selections that have stronger 1.5ul of reporter gene plasmid at 1 ug/ul is prepared in 5-ml (or weaker) parameters can be implemented. Similarly, in the polystyrene round bottom tubes. One hundred ul of transfec Subsequent studies designed to identify molecules with high tion reagent is then combined with 100 ul of siRNAs in potency and longevity, the desired cutoff criteria (i.e., the polystyrene deep-well titer plates (Beckman) and incubated lowest concentration that induces a desirable level of inter for 20 to 30 min at room temperature. Five hundred and fifty ference, or the longest period of time that interference can be microliters of Opti-MEM is then added to each well to bring observed) can vary. The experimentation Subsequent to appli the final siRNA concentration to 100 nM. Plates are then cation of the rational criteria of this application is signifi sealed with parafilm and mixed. Media is removed from cantly reduced where one is trying to obtain a Suitable hyper HEK293 cells and replaced with 95 ul of transfection mix functional siRNA for, for example, therapeutic use. When, for ture. Cells are incubated overnight at 37°C., 5% CO. example, the additional experimentation of the type described 0248 Quantification of gene knockdown. A variety of herein is applied by one skilled in the art with this disclosure quantification procedures can be used to measure the level of in hand, a hyperfunctional siRNA is readily identified. silencing induced by siRNA or siRNA pools. In one non 0244. The siRNA may be introduced into a cell by any limiting example: to measure mRNA levels 24hrs post-trans method that is now known or that comes to be known and that fection, QuantiGene branched-DNA (bDNA) kits (Bayer) from reading this disclosure, persons skilled in the art would (Wang, et al. Regulation of insulin preRNA splicing by glu determine would be useful in connection with the present cose. Proc. Natl. Acad. Sci. USA 1997, 94:4360) are used invention in enabling siRNA to cross the cellular membrane. according to manufacturer instructions. To measure US 2008/O 132691 A1 Jun. 5, 2008 TABLE III - continued TABLE III - continued Lull 41 SEQ. O252 AGUUGCGCGGAGGAGUUGU Lull 79 SEO. ID O29 O GUUUUGGAGCACGGAAAGA Lull 42 SEQ. O253 AAAGUUGCGCGGAGGAGUU Lull 8 O SEO. ID O291 UUGUUUUGGAGCACGGAAA Lull 43 SEQ. O254 AAAAAGUUGCGCGGAGGAG Lull 81 SEO. ID O292 UGUUGUUUUGGAGCACGGA Lull 44 SEQ. O255 CGAAAAAGUUGCGCGCAGG Lull 82 SEO. ID O293 GUUGUUGUUUUGGAGCACG Lull 45 SEQ. O256 CGCGAAAAAGUUGCGCGGA Lull 83 SEO. ID O294 CCGUUGUUGUUUUGGAGCA Lull 46 SEQ. O257 ACCGCGAAAAAGUUGCGCG Lull 84 SEO. ID O295 CGCCGUUGUUGUUUUGGAG Lull 47 SEQ. O258 CAACCGCGAAAAAGUUGCG Lull 85 SEO. ID O296 GCCGCCGUUGUUGUUUUGG Lull 48 SEQ. O259 AACAACCGCGAAAAAGUUG. Lull 86 SEO. ID O297 CCGCCGCCGUUGUUGUUUU Lull 49 SEQ. O26 O GUAACAACCGCGAAAAAGU Lull 87 SEO. ID O298 UCCCGCCGCCGUUGUUGUU Lull 50 SEQ. O261 AAGUAACAACCGCGAAAAA Lull 88 SEO. ID O299 CUUCCCGCCGCCGUUGUUG Lull 51 SEQ. O262 UCAAGUAACAACCGCGAAA Lull 89 SEO. ID O3OO AACUUCCCGCCGCCGUUGU Lull 52 SEQ. O263 AGUCAAGUAACAACCGCGA Lull 9 O SEO. ID O301 UGAACUUCCCGCCGCCGUU Lull 53 SEQ. O264 CCAGUCAAGUAACAACCGC Lull 54 SEQ. O265 CGCCAGUCAAGUAACAACC Example II Lull 55 SEO. O266 GUCGCCAGUCAAGUAACAA Validation of the Algorithm Using DBI, Luciferase, Lull 56 SEQ. O267 ACGUCGCCAGUCAAGUAAC PLK, EGFR, and SEAP (0250. The algorithm (Formula VIII) identified siRNAs for Lull 57 SEO. O268 UUACGUCGCCAGUCAAGUA five genes, human DBI, firefly luciferase (fLuc), renilla Lull 58 SEQ. O2.69 GAUUACGUCGCCAGUCAAG luciferase (ruc), human PLK, and human secreted alkaline phosphatase (SEAP). Four individual siRNAs were selected Lull 59 SEQ. O27 O UGGAUUACGUCGCCAGUCA on the basis of their SMARTSCORESTM derived by analysis Lull 6 O SEQ. O271 CGUGGAUUACGUCGCCAGU of their sequence using Formula VIII (all of the siRNAs would be selected with Formula IX as well) and analyzed for Lull 61 SEQ. O272 AUCGUGGAUUACGUCGCCA their ability to silence their targets expression. In addition to the scoring, a BLAST search was conducted for each siRNA. Lull 62 SEO.Q O273 AGAUCGUGGAUUACGUCGC To minimize- - - the potential for off-target silencing- 0 effects, only Lull 63 SEQ. O274 AGAGAUCGUGGAUUACGUC those target sequences with more than three mismatches against un-related sequences were selected. Semizarov, et al. Lull 64 SEQ. O275 AAAGAGAUCGUGGAUUACG (2003) Specificity of short interfering RNA determined Lull 65 SEQ. O276 AAAAAGAGAUCGUGGAUUA. through gene expression signatures, Proc. Natl. Acad. Sci. USA, 100:6347. These duplexes were analyzed individually Lull 66 SEQ. O277 GGAAAAAGAGAUCGUGGAU and in pools of 4 and compared with several siRNAs that were randomly selected. The functionality was measured as a per Lull 67 SEQ. O278 ACGGAAAAAGAGAUCGUGG centage of targeted gene knockdown as compared to controls. Lull 68 SEQ. O279 UGACGGAAAAAGAGAUCGU All siRNAs were transfected as described by the methods above at 100 nM concentration into HEK293 using Lipo Lull 69 SEQ. O28 O GAUGACGGAAAAAGAGAUC fectamine 2000. The level of the targeted gene expression was Lull 7 O SEQ. O281 ACGAUGACGGAAAAAGAGA evaluated by B-DNA as described above and normalized tO the non-specific control. FIG. 10 shows that the siRNAs Lull 71 SEQ. O282 AGACGAUGACGGAAAAAGA selected by the algorithm disclosed herein were significantly more potent than randomly selected siRNAs. The algorithm C 72 SEO.Q O283 AAAGACGAUGACGGAAAAA increased the chances of identifying an F50 siRNA from 48% Lull 73 SEQ. O284 GGAAAGACGAUGACGGAAA to 91%, and an F80 siRNA from 13% to 57%. In addition, pools of SMART siRNA silence the selected target better than Lull 74 SEQ. O285 ACGGAAAGACGAUGACGGA randomly selected pools (see FIG. 10F). Lull 75 SEO. O286 GCACGGAAAGACGAUGACG Example III Lull 76 SEQ. O287 GAGCACGGAAAGACGAUGA Validation- of the Algorithm Using Genes Involved in Lull 77 SEO. O288 UGGAGCACGGAAAGACGAU Clathrin-Dependent Endocytosis Lull 78 SEO. O289 UUUGGAGCACGGAAAGACG 0251 Components of clathrin-mediated endocytosis path way are key to modulating intracellular signaling and play US 2008/O 132691 A1 Jun. 5, 2008 29 important roles in disease. Chromosomal rearrangements that and AlphaImager V5.5 software (Alpha Innotech Corpora result in fusion transcripts between the Mixed-Lineage Leu tion). In experiments with Eps15R-targeted siRNAs, cell kemia gene (MLL) and CALM (clathrin assembly lymphoid lysates were subjected to immunoprecipitation with Ab860, myeloid leukemia gene) are believed to play a role in leuke and Eps15R was detected in immunoprecipitates by Western mogenesis. Similarly, disruptions in Rab7 and Rab9, as well blotting as described above. as HIP1 (Huntingtin-interacting protein), genes that are believed to be involved in endocytosis, are potentially respon 0256 The antibodies to assess the levels of each protein by sible for ailments resulting in lipid storage, and neuronal Western blot were obtained from the following sources: diseases, respectively. For these reasons, siRNA directed monoclonal antibody to clathrin heavy chain (TD.1) was against clathrin and other genes involved in the clathrin obtained from American Type Culture Collection (Rockville, mediated endocytotic pathway are potentially important Md., USA); polyclonal antibody to dynamin II was obtained research and therapeutic tools. from Affinity Bioreagents, Inc. (Golden, C0, USA); mono 0252 siRNAs directed against genes involved in the clath clonal antibodies to EEA.1 and Rab5a were purchased from rin-mediated endocytosis pathways were selected using For BD Transduction Laboratories (Los Angeles, Calif., USA); mula VIII. The targeted genes were clathrin heavy chain the monoclonal antibody to Tsg101 was purchased from (CHC, accession # NM 004859), clathrin light chain A Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif., USA); (CLCa, NM 001833), clathrin light chain B (CLCb, the monoclonal antibody to GFP was from ZYMED Labora NM 001834), CALM (Usa), B2 subunit of AP-2 (B2, tories Inc. (South San Francisco, Calif., USA); the rabbit NM 001282), Eps15 (NM 001981), Eps15R (NM polyclonal antibodies Ab32 specific to C.-adaptins and Ab20 021235), dynamin II (DYNII, NM 004945), Rab5a to CALM were described previously (Sorkin et al. (1995) (BC001267), Rab5b (NM 002868), Rab5c (AF141304), Stoichiometric Interaction of the Epidermal Growth Factor and EEA.1 (XM 018197). Receptor with the Clathrin-associated Protein Complex 0253 For each gene, four siRNAs duplexes with the high AP-2, J. Biol. Chem., 270:619), the polyclonal antibodies to est scores were selected and a BLAST search was conducted clathrin light chains A and B were kindly provided by Dr. F. for each of them using the Human EST database. In order to Brodsky (UCSF); monoclonal antibodies to PP1 (BD Trans minimize the potential for off-target silencing effects, only duction Laboratories) and C.-Actinin (Chemicon) were kindly those sequences with more than three mismatches against provided by Dr. M. Dell’Acqua (University of Colorado); un-related sequences were used. All duplexes were synthe Eps15 Ab577 and Eps15R Ab860 were kindly provided by sized at Dharmacon, Inc. as 21-mers with 3'-UU overhangs Dr. P. P. DiFiore (European Cancer Institute). using a modified method of 2'-ACE chemistry, Scaringe (2000) Advanced 5'-silyl-2'-orthoester approach to RNA oli (0257 FIG. 11 demonstrates the in vivo functionality of 48 gonucleotide synthesis, Methods Enzymol 317:3, and the individual siRNAs, selected using Formula VIII (most of antisense Strand was chemically phosphorylated to insure them will meet the criteria incorporated by Formula IX as maximized activity. well) targeting 12 genes. Various cell lines were transfected 0254. HeLa cells were grown in Dulbecco's modified with siRNA duplexes (Dup1-4) or pools of siRNA duplexes Eagle's medium (DMEM) containing 10% fetal bovine (Pool), and the cells were lysed 3 days after transfection with serum, antibiotics and glutamine. siRNA duplexes were the exception of CALM (2 days) and B2 (4 days). resuspended in 1x siRNA Universal buffer (Dharmacon, Inc.) 0258 Note a 1-adaptin band (part of AP-1 Golgi adaptor to 20 uM prior to transfection. HeLa cells in 12-well plates complex) that runs slightly slower than 32 adaptin. CALM were transfected twice with 4 ul of 20 uM siRNA duplex in 3 has two splice variants, 66 and 72 kD. The full-length Eps 15R ul Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif., (a doublet of -130 kD) and several truncated spliced forms of USA) at 24-hour intervals. For the transfections in which 2 or ~100 kD and ~70 kD were detected in Eps 15R immunopre 3 siRNA duplexes were included, the amount of each duplex cipitates (shown by arrows). The cells were lysed 3 days after was decreased, so that the total amount was the same as in transfection. Equal amounts of lysates were resolved by elec transfections with single siRNAs. Cells were plated into nor trophoresis and blotted with the antibody specific to a targeted mal culture medium 12 hours prior to experiments, and pro protein (GFP antibody forYFP fusion proteins) and the anti tein levels were measured 2 or 4 days after the first transfec body specific to unrelated proteins PP1 phosphatase or C.-ac tion. tinin, and TSG 101. The amount of protein in each specific 0255 Equal amounts of lysates were resolved by electro band was normalized to the amount of non-specific proteins phoresis, blotted, and stained with the antibody specific to in each lane of the gel. Nearly all of them appear to be targeted protein, as well as antibodies specific to unrelated proteins, PP1 phosphatase and Tsg101 (not shown). The cells functional, which establishes that Formula VIII and IX can be were lysed in Triton X-100/glycerol solubilization buffer as used to predict siRNAs functionality in general in a genome described previously. Tebar, Bohlander, & Sorkin (1999) wide manner. Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) 0259. To generate the fusion of yellow fluorescent protein Protein: Localization in Endocytic-coated Pits, Interactions (YFP) with Rab5b or Rab5c (YFP-Rab5b or YFP-Rab5c), a with Clathrin, and the Impact of Overexpression on Clathrin DNA fragment encoding the full-length human Rab5b or mediated Traffic, Mol. Biol. Cell, 10:2687. Cell lysates were Rab5c was obtained by PCR using Pfu polymerase (Strat electrophoresed, transferred to nitrocellulose membranes, agene) with a SacI restriction site introduced into the 5' end and Western blotting was performed with several antibodies and a KipnI site into the 3' end and cloned into pEYFP-C1 followed by detection using enhanced chemiluminescence vector (CLONTECH, Palo Alto, Calif., USA). GFP-CALM system (Pierce, Inc). Several X-ray films were analyzed to and YFP-Rab5a were described previously (Tebar, determine the linear range of the chemiluminescence signals, Bohlander, & Sorkin (1999) Clathrin Assembly Lymphoid and the quantifications were performed using densitometry Myeloid Leukemia (CALM) Protein: Localization in US 2008/O 132691 A1 Jun. 5, 2008 30 Endocytic-coated Pits, Interactions with Clathrin, and the gene function and possibly provide a future therapeutic agent Impact of Overexpression on Clathrin-mediated Traffic, Mol. to battle diseases that result from altered expression or func Biol. Cell 10:2687). tion of this gene. In Silico Identification of Functional siRNA Example IV 0264. To identify functional and hyperfunctional siRNA against the Bcl2 gene, the sequence for Bcl-2 was down Validation of the Algorithm Using EG5, GADPH, loaded from the NCBI Unigene database and analyzed using ATE1, MEK2. MEK1, QB, Lamina/C, c-myc, the Formula VIII algorithm. As a result of these procedures, Human Cyclophilin, and Mouse Cyclophilin both the sequence and SMARTSCORESTM, or siRNA rank ings of the Bcl2 siRNA were obtained and ranked according 0260 A number of genes have been identified as playing to their functionality. Subsequently, these sequences were potentially important roles in disease etiology. Expression BLASTed (database) to insure that the selected sequences profiles of normal and diseased kidneys has implicated Edg5 were specific and contained minimal overlap with unrelated in immunoglobulin A neuropathy, a common renal glomeru genes. The SMARTSCORESTM, or siRNA rankings for the lar disease. Myc1, MEK1/2 and other related kinases have top 10 Bcl-2 siRNA are identified in FIG. 13. been associated with one or more cancers, while lamins have In Vivo Testing of Bcl-2 siRNA been implicated in muscular dystrophy and other diseases. 0265 Bcl-2 siRNAs having the top ten SMART For these reasons, siRNA directed against the genes encoding SCORESTM, or siRNA rankings were selected and tested in a these classes of molecules would be important research and functional assay to determine silencing efficiency. To accom therapeutic tools. plish this, each of the ten duplexes were synthesized using 0261 FIG. 12 illustrates four siRNAs targeting 10 differ 2'-O-ACE chemistry and transfected at 100 nM concentra ent genes (Table V for sequence and accession number infor tions into cells. Twenty-four hours later assays were per mation) that were selected according to the Formula VIII and formed on cell extracts to assess the degree of target silencing. assayed as individuals and pools in HEK293 cells. The level Controls used in these experiments included mock trans of siRNA induced silencing was measured using the B-DNA fected cells, and cells that were transfected with a non-spe assay. These studies demonstrated that thirty-six out of the cific siRNA duplex. forty individual SMART-selected siRNA tested are func 0266 The results of these experiments are presented tional (90%) and all 10 pools are fully functional. below (and in FIG. 14) and show that all ten of the selected siRNA induce 80% or better silencing of the Bcl2 message at 100 nM concentrations. These data verify that the algorithm Example V successfully identified functional Bcl2 siRNA and provide a set of functional agents that can be used in experimental and Validation of the Algorithm Using Bcl2 therapeutic environments. 0262 Bcl-2 is a 25kD. 205-239 amino acid, anti-apoptotic protein that contains considerable homology with other mem siRNA 1 GGGAGAUAGUGAUGAAGUA SEO. ID NO. 3O2 bers of the BCL family including BCLX, MCL1, BAX, BAD, and BIK. The protein exists in at least two forms (Bcl2a, siRNA 2 GAAGUACAUCCAUUAUAAG SEO. ID NO. 303 which has a hydrophobic tail for membrane anchorage, and siRNA 3 GUACGACAACCGGGAGAUA SEO. ID NO. 304 Bcl2b, which lacks the hydrophobic tail) and is predomi nantly localized to the mitochondrial membrane. While Bcl2 siRNA 4 AGAUAGUGAUGAAGUACAU SEO. ID NO. 305 expression is widely distributed, particular interest has siRNA 5 UGAAGACUCUGCUCAGUUU SEO. ID NO. 306 focused on the expression of this molecule in B and T cells. Bcl2 expression is down-regulated in normal germinal center siRNA 6 GCAUGCGGCCUCUGUUUGA SEO. ID NO. 3 O7 B cells yet in a high percentage of follicularlymphomas, Bcl2 siRNA 7 UGCGGCCUCUGUUUGAUUU SEO. ID NO. 3O8 expression has been observed to be elevated. Cytological studies have identified a common translocation (14:18)(q32; siRNA 8 GAGAUAGUGAUGAAGUACA SEO. ID NO. 309 q32)) amongst a high percentage (>70%) of these lympho siRNA 9 GGAGAUAGUGAUGAAGUAC SEO. ID NO. 310 mas. This genetic lesion places the Bcl2 gene injuxtaposition to immunoglobulin heavy chain gene (IgEI) encoding siRNA 1 O GAAGACUCUGCUCAGUUUG SEO. ID NO. 311 sequences and is believed to enforce inappropriate levels of 0267. Bcl2 siRNA: Sense Strand, 5->3' gene expression, and resistance to programmed cell death in the follicle center B cells. In other cases, hypomethylation of Example VI the Bcl2 promoter leads to enhanced expression and again, Sequences Selected by the Algorithm inhibition of apoptosis. In addition to cancer, dysregulated 0268 Sequences of the siRNAs selected using Formulas expression of Bcl-2 has been correlated with multiple sclero (Algorithms) VIII and IX with their corresponding ranking, sis and various neurological diseases. which have been evaluated for the silencing activity in vivo in 0263. The correlation between Bcl-2 translocation and the present study (Formula VIII and IX, respectively) are cancer makes this gene an attractive target for RNAi. Identi shown in Table V. It should be noted that the “t' residues in fication of siRNA directed against the bc12 transcript (or Table V, and elsewhere, when referring to siRNA, should be Bcl2-Igh fusions) would further our understanding Bcl2 replaced by “u' residues. US 2008/O 132691 A1 Jun. 5, 2008 33 TABLE V - continued FORMUL.A. FORMUL.A. GENE Name SEO. ID No. FTLLSEOTENCE VIII IX ATE1-1 NMOO7041 O43 O GAACCCAGCUGGAGAACUU 45 15.5 ATE1-2 NMOO7041 O431 GAUAUACAGUGUGAUCUUA 4 O 12.2 ATE1-3 NMOO7041 O432 GUACUACGAUCCUGAUUAU 37 32.9 ATE1 - 4 NMOO7041 O433 GUGCCGACCUUUACAAUUU 35 18.2 EGFR-1 NMOO5228 O434 GAAGGAAACTGAATTCAAA 68 79.4 EGFR-1 NMOO5228 O435 GGAAATATGTACTACGAAA 49 49.5 EGFR-1 NMOO5228 O436 CCACAAAGCAGTGAATTTA 41 7.6 EGFR-1 NMOO5228 O437 GTAACAAGCTCACGCAGTT 4 O 25.9 0269. Many of the genes to which the described siRNA are siRNA are positioned continuously head to toe (5' end of one directed play critical roles in disease etiology. For this reason, directly adjacent to the 3' end of the others). the siRNAS listed in the sequence listing may potentially act 0274. Additionally, siRNA pools that were tested per as therapeutic agents. A number of prophetic examples follow formed at least as well as the best oligonucleotide in the pool, and should be understood in view of the siRNA that are under the experimental conditions whose results are depicted identified in the sequence listing. To isolate these siRNAs, the in FIG. 19. Moreover, when previously identified non-func appropriate message sequence for each gene is analyzed tional and marginally (semi) functional siRNA duplexes were using one of the before mentioned formulas (preferably for pooled together in groups of five at a time, a significant mula VIII) to identify potential siRNA targets. Subsequently functional cooperative action was observed (see FIG. 20). In these targets are BLASTed to eliminate homology with fact, pools of semi-active oligonucleotides were 5 to 25 times potential off-targets. more functional than the most potent oligonucleotide in the pool. Therefore, pooling several siRNA duplexes together Example VII does not interfere with the functionality of the most potent siRNAs within a pool, and pooling provides an unexpected Evidence for the Benefits of Pooling significant increase in overall functionality 0270. Evidence for the benefits of pooling have been dem Example VIII onstrated using the reporter gene, luciferase. Ninety siRNA duplexes were synthesized using Dharmacon proprietary Additional Evidence of the Benefits of Pooling ACER chemistry against one of the standard reporter genes: 0275 Experiments were performed on the following firefly luciferase. The duplexes were designed to start two genes: B-galactosidase, Renilla luciferase, and Secreted alka base pairs apart and to cover approximately 180 base pairs of line phosphatase, which demonstrates the benefits of pooling. the luciferase gene (see sequences in Table III). Subsequently, (see FIGS. 21A, 21B and 21C). Individual and pools of the siRNA duplexes were co-transfected with a luciferase siRNA (described in Figure legends 21A-C) were transfected expression reporter plasmid into HEK293 cells using stan into cells and tested for silencing efficiency. Approximately dard transfection protocols and luciferase activity was 50% of individual siRNAs designed to silence the above assayed at 24 and 48 hours. specified genes were functional, while 100% of the pools that 0271 Transfection of individual siRNAs showed standard contain the same siRNA duplexes were functional. distribution of inhibitory effect. Some duplexes were active, while others were not. FIG. 15 represents a typical screen of Example IX ninety siRNA duplexes (SEQ. ID NO.0032-0120) positioned two base pairs apart. As the figure Suggests, the functionality Highly Functional siRNA of the siRNA duplex is determined more by a particular 0276 Pools of five siRNAs in which each two siRNAs sequence of the oligonucleotide than by the relative oligo overlap to 10-90% resulted in 98% functional entities (>80% nucleotideposition within a gene or excessively sensitive part silencing). Pools of siRNAs distributed throughout the of the mRNA, which is important for traditional anti-sense mRNA that were evenly spaced, covering an approximate technology. 20-2000 base pair range, were also functional. When the 0272. When two continuous oligonucleotides were pooled pools of siRNA were positioned continuously head to tail together, a significant increase in gene silencing activity was relative to mRNA sequences and mimicked the natural prod observed (see FIGS. 16A and B). A gradual increase in effi ucts of Dicer cleaved long double stranded RNA, 98% of the cacy and the frequency of pools functionality was observed pools evidenced highly functional activity (>95% silencing). when the number of siRNAs increased to 3 and 4 (FIGS. 16A, 16B, 17A, and 17B). Further, the relative positioning of the Example X oligonucleotides within a pool did not determine whether a particular pool was functional (see FIGS. 18A and 18B, in Human Cyclophilin B which 100% of pools of oligonucleotides distanced by 2, 10 (0277 Table III above lists the siRNA sequences for the and 20 base pairs were functional). human cyclophilin B protein. A particularly functional 0273. However, relative positioning may nonetheless have siRNA may be selected by applying these sequences to any of an impact. An increased functionality may exist when the Formula I to VII above. US 2008/O 132691 A1 Jun. 5, 2008 34 0278 Alternatively, one could pool 2, 3, 4, 5 or more of transferrin (Sigma) were iodinated as described previously these sequences to create a kit for silencing a gene. Preferably, (Jiang. X., Huang, F., Marusyk, A. & Sorkin, A. (2003) Mol within the kit there would be at least one sequence that has a Biol Cell 14, 858-70). HeLa cells grown in 12-well dishes relatively high predicted functionality when any of Formulas were incubated with 'I-EGF (1 ng/ml) or 'I-transferrin I-VII is applied. (1 g/ml) in binding medium (DMEM, 0.1% bovine serum albumin) at 37° C., and the ratio of internalized and surface Example XI radioactivity was determined during 5-min time course to calculate specific internalization rate constant k as described Sample Pools of siRNAs and their Application to previously (Jiang, X et al.). The measurements of the uptakes Human Disease of radiolabeled transferrin and EGF were performed using 0279. The genetic basis behind human disease is well short time-course assays to avoid influence of the recycling documented and siRNA may be used as both research or on the uptake kinetics, and using low ligand concentration to diagnostic tools and therapeutic agents, either individually or avoid saturation of the clathrin-dependent pathway (for EGF in pools. Genes involved in signal transduction, the immune Lund, K. A., Opresko, L. K., Strarbuck, C. Walsh, B. J. & response, apoptosis, DNA repair, cell cycle control, and a Wiley, H. S. (1990).J. Biol. Chem. 265, 15713-13723). variety of other physiological functions have clinical rel 0282. The effects of knocking down Rab5a, 5b, 5c, Eps, or evance and therapeutic agents that can modulate expression Eps15R (individually) are shown in FIG.22 and demonstrate of these genes may alleviate Some or all of the associated that disruption of single genes has little or no effect on EGF or symptoms. In some instances, these genes can be described as Tfn internalization. In contrast, simultaneous knock down of a member of a family or class of genes and siRNA (randomly, Rab5a, 5b, and 5c, or Eps and Eps 15R, leads to a distinct conventionally, or rationally designed) can be directed phenotype (note: total concentration of siRNA in these against one or multiple members of the family to induce a experiments remained constant with that in experiments in desired result. which a single siRNA was introduced, see FIG. 23). These 0280. To identify rationally designed siRNA to each gene, experiments demonstrate the effectiveness of using rationally the sequence was analyzed using Formula VIII or Formula X designed siRNA to knockdown multiple genes and validates to identify rationally designed siRNA. To confirm the activity the utility of these reagents to override genetic redundancy. of these sequences, the siRNA are introduced into a cell type of choice (e.g., HeLa cells, HEK293 cells) and the levels of Example XIII the appropriate message are analyzed using one of several art Validation of Multigene Targeting Using G6PD, proven techniques. siRNA having heightened levels of GAPDH, PLK, and UQC potency can be identified by testing each of the before men 0283. Further demonstration of the ability to knock down tioned duplexes at increasingly limiting concentrations. expression of multiple genes using rationally designed Similarly, siRNA having increased levels of longevity can be siRNA was performed using pools of siRNA directed against identified by introducing each duplex into cells and testing four separate genes. To achieve this, siRNA were transfected functionality at 24, 48, 72, 96, 120, 144, 168, and 192 hours into cells (total siRNA concentration of 100 nM) and assayed after transfection. Agents that induce >95% silencing at sub twenty-four hours later by B-DNA. Results shown in FIG. 24 nanomolar concentrations and/or induce functional levels of show that pools of rationally designed molecules are capable silencing for >96 hours are considered hyperfunctional. of simultaneously silencing four different genes. Example XII Example XIV Validation of Multigene Knockout Using Rab5 and Validation of Multigene Knockouts as Demonstrated Eps by Gene Expression Profiling, a Prophetic Example 0281 Two or more genes having similar, overlapping 0284. To further demonstrate the ability to concomitantly functions often leads to genetic redundancy. Mutations that knockdown the expression of multiple gene targets, single knockout only one of, e.g., a pair of Such genes (also referred siRNA or siRNA pools directed against a collection of genes to as homologs) results in little or no phenotype due to the fact (e.g., 4, 8, 16, or 23 different targets) are simultaneously that the remaining intact gene is capable of fulfilling the role transfected into cells and cultured for twenty-four hours. Sub of the disrupted counterpart. To fully understand the function sequently, mRNA is harvested from treated (and untreated) of Such genes in cellular physiology, it is often necessary to cells and labeled with one of two fluorescent probes dyes knockout or knockdown both homologs simultaneously. (e.g., a red fluorescent probe for the treated cells, a green Unfortunately, concomitant knockdown of two or more genes fluorescent probe for the control cells). Equivalent amounts of is frequently difficult to achieve in higher organisms (e.g., labeled RNA from each sample is then mixed together and mice) thus it is necessary to introduce new technologies dis hybridized to sequences that have been linked to a solid sect gene function. One Such approach to knocking down support (e.g., a slide, “DNA CHIP). Following hybridiza multiple genes simultaneously is by using siRNA. For tion, the slides are washed and analyzed to assess changes in example, FIG. 11 showed that rationally designed siRNA the levels of target genes induced by siRNA. directed against a number of genes involved in the clathrin mediated endocytosis pathway resulted in significant levels Example XV of protein reduction (e.g., >80%). To determine the effects of Identifying Hyperfunctional siRNA gene knockdown on clathrin-related endocytosis, internaliza tion assays were performed using epidermal growth factor Identification of Hyperfunctional Bcl-2 siRNA and transferrin. Specifically, mouse receptor-grade EGF (0285. The ten rationally designed Bcl2 siRNA (identified (Collaborative Research Inc.) and iron-saturated human in FIG. 13, 14) were tested to identify hyperpotent reagents. US 2008/O 132691 A1 Jun. 5, 2008 To accomplish this, each of the ten Bcl-2 siRNA were indi vidually transfected into cells at a 300 uM (0.3 nM) concen TABLE XI-continued trations. Twenty-four hours later, transcript levels were assessed by B-DNA assays and compared with relevant con 96-WELL 24-WELL trols. As shown in FIG. 25, while the majority of Bcl-2 siRNA MIXTURE 4 (MEDIA-SIRNATRANSFECTION REAGENT failed to induce functional levels of silencing at this concen MIXTURE) tration, siRNA 1 and 8 induced >80% silencing, and siRNA 6 exhibited greater than 90% silencing at this subnanomolar Mixture 3 20 ul 100 ul concentration. By way of prophetic examples, similar assays Complete media 80 ul 400 ul could be performed with any of the groups of rationally MIXTURE 4 FINAL VOLUME 100 ul 500 ul designed genes described in the Examples. Thus for instance, rationally designed siRNA sequences directed against a gene Incubate 48 hours at 37°C. of interest could be introduced into cells at increasingly lim 0291 Transfection. Create a Mixture 1 by combining the iting concentrations to determine whetherany of the duplexes specified amounts of OPTI-MEM serum free media and are hyperfunctional. transfection reagent in a sterile polystyrene tube. Create a Mixture 2 by combining specified amounts of each siRNA Example XVI with OPTI-MEM media in Sterile 1 ml tubes. Create a Mix Gene Silencing: Prophetic Example ture 3 by combining specified amounts of Mixture 1 and Mixture 2. Mix gently (do not vortex) and incubate at room 0286 Below is an example of how one might transfect a temperature for 20 minutes. Create a Mixture 4 by combining cell. specified amounts of Mixture 3 to complete media. Add 0287 Select a cell line. The selection of a cell line is appropriate volume to each cell culture well. Incubate cells usually determined by the desired application. The most with transfection reagent mixture for 24-72 hours at 37° C. important feature to RNAi is the level of expression of the This incubation time is flexible. The ratio of silencing will gene of interest. It is highly recommended to use cell lines for remain consistent at any point in the time period. Assay for which siRNA transfection conditions have been specified and gene silencing using an appropriate detection method such as validated. RT-PCR, Western blot analysis, immunohistochemistry, phe 0288 Plate the cells. Approximately 24 hours prior to transfection, plate the cells at the appropriate density so that notypic analysis, mass spectrometry, fluorescence, radioac they will be approximately 70-90% confluent, or approxi tive decay, or any other method that is now known or that mately 1x10 cells/ml at the time of transfection. Cell densi comes to be known to persons skilled in the art and that from ties that are too low may lead to toxicity due to excess expo reading this disclosure would useful with the present inven Sure and uptake of transfection reagent-siRNA complexes. tion. The optimal window for observing a knockdown phe Cell densities that are too high may lead to low transfection notype is related to the mRNA turnover of the gene of interest, efficiencies and little or no silencing. Incubate the cells over although 24-72 hours is standard. Final Volume reflects night. Standard incubation conditions for mammalian cells amount needed in each well for the desired cell culture for are 37°C. in 5% CO. Other cell types, such as insect cells, mat. When adjusting volumes for a Stock Mix, an additional require different temperatures and CO concentrations that 10% should be used to accommodate variability in pipetting, are readily ascertainable by persons skilled in the art. Use etc. Duplicate or triplicate assays should be carried out when conditions appropriate for the cell type of interest. possible. 0289 siRNA re-suspension. Add 20 ul siRNA universal buffer to each siRNA to generate a final concentration of 50 Example XVII uM. 0290 siRNA-lipid complex formation. Use RNase-free siRNAs that Target KDR solutions and tubes. Using the following table, Table XI: 0292 siRNAs that target nucleotide sequences for KDR (NCBI accession number NM 002253) and having TABLE XI sequences generated in silico by the algorithms herein, are 96-WELL 24-WELL provided. In various embodiments, the siRNAs are rationally designed. In various embodiments, the siRNAs are functional MIXTURE 1 (TRANSIT-TKO-PLASMID DILUTION MIXTURE) or hyperfunctional. These siRNA that have been generated by Opti-MEM 9.3 46.5 the algorithms of the present invention include: TransIT-TKO (1 gul) O.S 2.5 MIXTURE 1 FINAL VOLUME 1O.O SO.O AAACAAAACUGUGGUGAUU; (SEQ. ID NO. 438) MIXTURE 2 (SIRNA DILUTION MIXTURE) AAAGAAGGAACUAGAAUGA; (SEQ. ID NO. 439) Opti-MEM 9.0 4S.O siRNA (1 M) 1.O S.O AAAGAGACACAGGAAAUUA; (SEQ. ID NO. 440) MIXTURE 2 FINAL VOLUME 1O.O SO.O AAGAAGGGCUUUACUAUUC; (SEQ. ID NO. 441) MIXTURE 3 (SIRNA-TRANSFECTION REAGENT MIXTURE) AAGCAGAUGCCUUUGGAAU; (SEQ. ID NO. 442) Mixture 1 10 50 Mixture 2 10 50 AAGUAAUCCCAGAUGACAA; (SEQ. ID NO. 443) MIXTURE 3 FINAL VOLUME 2O 100 AAUAGAAGGUGCCCAGGAA; (SEQ. ID NO. 444) Incubate 20 minutes at room temperature US 2008/O 132691 A1 Jun. 5, 2008 38 region, wherein said sense region and said antisense region - Continued are at least 90% complementary, said sense region and said GUUUAAAGAUAAUGAGACC; (SEO. ID NO. 599) antisense region together form a duplex region comprising UAACAAAGGUCAUAAUGCU; (SEO. ID NO. 6OO) 19-30 base pairs, and said sense region comprises a sequence that is identical to a contiguous stretch of at least 18 bases of UAGAAGAACCAGAAGUAAA; (SEO. ID NO. 601) a sequence selected from the group consisting of: SEQ. ID UAGCCAGACUUCAAAAUUA; (SEO. ID NO. 6O2) NOS 438-622. 0297. In another embodiment, a pool of at least two siR UAUCAGAGACUUUGAGCAU; (SEO. ID NO. 603) NAs is provided, wherein said pool comprises a first siRNA UCAAGAUCCUCAUUCAUAU; (SEO. ID NO. 604) and a second siRNA, said first siRNA comprises a duplex region of length 18-30 base pairs that has a first sense region UCAGAGUGGCAGUGAGCAA; (SEO. ID NO. 605) that is at least 90% similar to 18 bases of a first sequence UCAGUGAUGUAGAAGAAGA; (SEO. ID NO. 606) selected from the group consisting of: SEQ. ID NOS 438-622 and said second siRNA comprises a duplex region of length UCAUGGAGCUUAAGAAUGC; (SEO. ID NO. 6O7) 18-30 base pairs that has a second sense region that is at least UCAUGGUGAUUGUGGAAUU; (SEO. ID NO. 608) 90% similar to 18 bases of a second sequence selected from the group consisting of: SEQ. ID NOS 438-622 and wherein UCUAAUAGCACAAAUGACA; (SEO. ID NO. 609) said first sense region and said second sense region are not UGAAAGAAGGAACUAGAAU; (SEO. ID NO. 610) identical. 0298. In another embodiment, a pool of at least two siR UGAAAGAGACACAGGAAAU; (SEO. ID NO. 611) NAs is provided, wherein said pool comprises a first siRNA UGAACUAUCUGUUGGAGAA; (SEO. ID NO. 612) and a second siRNA, said first siRNA comprises a duplex region of length 18-30 base pairs that has a first sense region UGAAGCAGAUGCCUUUGGA; (SEO. ID NO. 613) that is identical to at least 18 bases of a sequence selected UGAAUACCCUCAUAUCUGU; (SEO. ID NO. 614) from the group consisting of: SEQ. ID NOS 438-622 and wherein the second siRNA comprises a second sense region UGAUGUAGAAGAAGAGGAA; (SEO. ID NO. 615) that comprises a sequence that is identical to at least 18 bases UGGAAGUGAGUGAAAGAGA; (SEO. ID NO. 616) of a sequence selected from the group consisting of: SEQ. ID NOS 438-622. UGGAGCAUCUCAUCUGUUA; (SEO. ID NO. 617) 0299. In another embodiment, a pool of at least two siR UGGAUACUCUUUGGAAAUU; (SEO. ID NO. 618) NAs is provided, wherein said pool comprises a first siRNA and a second siRNA, said first siRNA comprises a duplex UGGUGGAACAUUUGGGAAA; (SEO. ID NO. 619) region of length 19-30 base pairs and has a first sense region UGUACAUUACUGAGAACAA; (SEO. ID NO. 62O) comprising a sequence that is at least 90% similar to a sequence selected from the group consisting of: SEQ. ID NOS UGUAGAAGACUCAGGCAUU; (SEO. ID NO. 621) 438-622, and said duplex of said second siRNA is 19-30 base and pairs and comprises a second sense region that comprises a UUUCAGAGUUGGUGGAACA (SEO. ID NO. 622) sequence that is at least 90% similar to a sequence selected from the group consisting of: SEQ. ID NOS 438-622. 0293 Thus, consistent with Example XVII, the present invention provides an siRNA that targets a nucleotide 0300. In another embodiment, a pool of at least two siR sequence for KDR, wherein the siRNA is selected from the NAs is provided, wherein said pool comprises a first siRNA group consisting of SEQ. ID NOS. 438-622. and a second siRNA, said first siRNA comprises a duplex 0294. In another embodiment, an siRNA is provided, said region of length 19-30 base pairs and has a first sense region siRNA comprising a sense region and an antisense region, comprising a sequence that is identical to at least 18 bases of wherein said sense region and said antisense region are at a sequence selected the group consisting of: SEQ. ID NOS least 90% complementary, said sense region and said anti 438-622 and said duplex of said second siRNA is 19-30 base sense region together form a duplex region comprising 18-30 pairs and comprises a second sense region comprising a base pairs, and said sense region comprises a sequence that is sequence that is identical to a sequence selected from the at least 90% similar to a sequence selected from the group group consisting of: SEQ. ID NOS 438-622. consisting of: SEQ. ID NOS 438-622. 0301 In each of the aforementioned embodiments, pref 0295. In another embodiment, an siRNA is provided erably the antisense region is at least 90% complementary to wherein the siRNA comprises a sense region and an antisense a contiguous stretch of bases of one of the NCBI sequences region, wherein said sense region and said antisense region identified in Example XVII; each of the recited NCBI are at least 90% complementary, said sense region and said sequences is incorporated by reference as if set forth fully antisense region together form a duplex region comprising herein. In some embodiments, the antisense region is 100% 18-30 base pairs, and said sense region comprises a sequence complementary to a contiguous stretch of bases of one of the that is identical to a contiguous stretch of at least 18 bases of NCBI sequences identified in Example XVII. a sequence selected from the group consisting of SEQ. ID 0302 Further, in some embodiments that are directed to NOS 438-622. siRNA duplexes in which the antisense region is 20-30 bases 0296. In another embodiment, an siRNA is provided in length, preferably there is a stretch of 19 bases that is at wherein the siRNA comprises a sense region and an antisense least 90%, more preferably 100% complementary to the US 2008/O 132691 A1 Jun. 5, 2008 39 recited sequence id number and the entire antisense region is is intended to cover any variations, uses, or adaptations of the at least 90% and more preferably 100% complementary to a invention following, in general, the principles of the invention contiguous stretch of bases of one of the NCBI sequences and including such departure from the present disclosure as identified in Example XVII. come within known or customary practice within the art to 0303 While the invention has been described in connec which the invention pertains and as may be applied to the tion with specific embodiments thereof, it will be understood essential features hereinbefore set forth and as follows in the that it is capable of further modifications and this application Scope of the appended claims. SEQUENCE LISTING <16 Oc NUMBER OF SEO ID NOS: 622 <210 SEQ ID NO 1 <211 LENGTH: 19 &212> TYPE RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 1-2, 4, 6-9, 12, 15-18 <223> OTHER INFORMATION: n is any nucleotide <4 OO SEQUENCE: 1 all Claala 19 <210 SEQ ID NO 2 <211 LENGTH: 19 &212s. TYPE RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 1-2, 4, 6-9, 12, 15-18 <223> OTHER INFORMATION: n is any nucleotide <4 OO SEQUENCE: 2 nnanannnnu gnaannnna 19 <210 SEQ ID NO 3 <211 LENGTH: 19 &212> TYPE RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 1-2, 4, 6-9, 12, 15-18 <223> OTHER INFORMATION: n is any nucleotide <4 OO SEQUENCE: 3 ala Laala 19 <210 SEQ ID NO 4 <211 LENGTH: 19 &212> TYPE RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 1-2, 4, 6-9, 12, 15-18 <223> OTHER INFORMATION: n is any nucleotide <4 OO SEQUENCE: 4 US 2008/O 132691 A1 Jun. 5, 2008 40 - Continued all Clcala 19 SEO ID NO 5 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 5 nnanannnnu gncannnna 19 SEQ ID NO 6 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 6 ala Calla 19 SEO ID NO 7 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 7 ala Calla 19 SEQ ID NO 8 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 8 nnanannnnu gnuannnna 19 SEO ID NO 9 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 US 2008/O 132691 A1 Jun. 5, 2008 41 - Continued <223> OTHER INFORMATION: n is any nucleotide <4 OO SEQUENCE: 9 ala Lala 19 <210 SEQ ID NO 10 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 1-2, 4, 6-9, 12, 15-18 <223> OTHER INFORMATION: n is any nucleotide <4 OO SEQUENCE: 10 accl. Claala 19 <210 SEQ ID NO 11 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 1-2, 4, 6-9, 12, 15-18 <223> OTHER INFORMATION: n is any nucleotide <4 OO SEQUENCE: 11 nnancinnnnu gnaannnna 19 <210 SEQ ID NO 12 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 1-2, 4, 6-9, 12, 15-18 <223> OTHER INFORMATION: n is any nucleotide <4 OO SEQUENCE: 12 ac. Laala 19 <210 SEQ ID NO 13 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 1-2, 4, 6-9, 12, 15-18 <223> OTHER INFORMATION: n is any nucleotide <4 OO SEQUENCE: 13 lac Coala 19 <210 SEQ ID NO 14 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: US 2008/O 132691 A1 Jun. 5, 2008 42 - Continued OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 14 nnancinnnnu gncannnna 19 SEO ID NO 15 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 15 accl. Calla 19 SEQ ID NO 16 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 16 accl. Calla 19 SEO ID NO 17 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 17 nnancinnnnu gnuannnna 19 SEQ ID NO 18 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 18 ac. Lala 19 SEQ ID NO 19 US 2008/O 132691 A1 Jun. 5, 2008 43 - Continued LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 19 nnangnnnnu cina annnna 19 SEQ ID NO 2 O LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 2O nnangnnnnu gnaannnna 19 SEQ ID NO 21 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 21 nnangnnnnu unaannnna 19 SEQ ID NO 22 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 22 nnangnnnnu cincannnna 19 SEQ ID NO 23 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 23 US 2008/O 132691 A1 Jun. 5, 2008 44 - Continued nnangnnnnu gncannnna 19 SEQ ID NO 24 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 24 nnangnnnnu uncannnna 19 SEO ID NO 25 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 25 nnangnnnnu Cnuannnna 19 SEQ ID NO 26 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 26 nnangnnnnu gnuannnna 19 SEO ID NO 27 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 27 nnangnnnnu unuannnna 19 SEQ ID NO 28 LENGTH: 22 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic FEATURE: NAMEAKEY: misc feature LOCATION: 4-5, 7, 9-12, 15, 18-21 US 2008/O 132691 A1 Jun. 5, 2008 45 - Continued <223> OTHER INFORMATION: n is any nucleotide <4 OO SEQUENCE: 28 gucinnanann innucnaannn na 22 <210 SEQ ID NO 29 <211 LENGTH: 2O8 &212> TYPE: DNA <213> ORGANISM: Homo sapiens &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (1) . . . (208) <223> OTHER INFORMATION: human cyclophilin fragment <4 OO SEQUENCE: 29 gttcCaaaaa cagtggataa ttttgttggcc ttagctacag gagagaaagg atttggctac 6 O aaaaac agca aattic catcg togtaatcaag gactt catga t coaggg.cgg agactitcacc 12 O aggggagatg gcacaggagg aaagagcatc tacggtgagc gct tcc.ccga tigaga acttic 18O aaactgaagc act acgggcc tigctggg 2O8 <210 SEQ ID NO 3 O <211 LENGTH: 2OO &212> TYPE: DNA <213> ORGANISM: Photinus pyralis &220s FEATURE: <221 NAME/KEY: misc feature <222> LOCATION: (1) . . . (2OO) <223> OTHER INFORMATION: firefly luciferase fragment <4 OO SEQUENCE: 30 tgaact tccc gcc.gc.cgttgttgttittgga gcacggaaag acgatgacgg aaaaagagat 6 O cgtggattac gtc.gc.cagtic aagtaacaac cqcgaaaaag ttgcgcggag gagttgttgtt 12 O tgtggacgaa gtaccgaaag gtc.ttaccgg aaaacticgac gcaagaaaaa t cagagagat 18O Cct cataaag gccaagaagg 2OO <210 SEQ ID NO 31 <211 LENGTH: 108 &212> TYPE: DNA <213> ORGANISM: Homo sapiens &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (1) . . . (108) <223> OTHER INFORMATION: human DBL fragment <4 OO SEQUENCE: 31 acgggcaagg C caagtggga tigcctggaat gagctgaaag ggact tccala ggalagatgcc 6 O atgaaagctt acatcaacaa agtagaagag ctaaagaaaa aatacggg 108 <210 SEQ ID NO 32 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 32 guluccaaaaa Cagluggalla 19 <210 SEQ ID NO 33 US 2008/O 132691 A1 Jun. 5, 2008 46 - Continued <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 33 Cccaaaaa.ca gluggaluaalu 19 <210 SEQ ID NO 34 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 34 caaaaacagul ggaluaaluulu. 19 <210 SEQ ID NO 35 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 35 aaaacagugg aluaaluuluug 19 <210 SEQ ID NO 36 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 36 alacagluggau aaluuluugug 19 <210 SEQ ID NO 37 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 37 Cagluggallaa luluuluguggc 19 <210 SEQ ID NO 38 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 38 gluggallalalull luluguggc.cul 19 <210 SEQ ID NO 39 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: US 2008/O 132691 A1 Jun. 5, 2008 47 - Continued <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 39 ggaluaaluuluu gluggccuula 19 <210 SEQ ID NO 4 O <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 40 aluaaluuluugu ggccuulagc 19 <210 SEQ ID NO 41 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 41 aaluuluugugg CCuluagcula 19 <210 SEQ ID NO 42 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 42 uuuuguggcc uluagculaca 19 <210 SEQ ID NO 43 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 43 uluguggccuu agcuacagg 19 <210 SEQ ID NO 44 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 44 gluggcCullag Clacaggag 19 <210 SEQ ID NO 45 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 45 US 2008/O 132691 A1 Jun. 5, 2008 48 - Continued ggccuulag cu acaggaga.g 19 <210 SEQ ID NO 46 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 46 Ccullagculac aggagagaa 19 <210 SEQ ID NO 47 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 47 uluagculacag gagagaaag 19 <210 SEQ ID NO 48 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 48 agculacagga gagaaagga 19 <210 SEQ ID NO 49 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 49 culacaggaga gaaaggaulu. 19 <210 SEQ ID NO 50 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 5 O acaggagaga aaggaululug 19 <210 SEQ ID NO 51 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 51 aggagaga aa galuuluggc 19 <210 SEQ ID NO 52 US 2008/O 132691 A1 Jun. 5, 2008 49 - Continued <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 52 gagagaaagg aluuluggcula 19 <210 SEQ ID NO 53 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 53 gagaaaggau uluggculaca 19 <210 SEQ ID NO 54 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 54 gaaaggaululu ggculacaaa 19 <210 SEQ ID NO 55 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 55 aaggaululugg cluacaaaaa 19 <210 SEQ ID NO 56 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 56 ggaluuluggcu acaaaaa.ca 19 <210 SEQ ID NO 57 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 57 aluuluggculac aaaaac agc 19 <210 SEQ ID NO 58 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: US 2008/O 132691 A1 Jun. 5, 2008 50 - Continued <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 58 uluggculacaa aaa.ca.gcaa. 19 <210 SEQ ID NO 59 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 59 ggculacaaaa acagcaaalu 19 <210 SEQ ID NO 60 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 60 culacaaaaac agcaaauuc 19 <210 SEQ ID NO 61 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 61 acaaaaacag caaaulucca 19 <210 SEQ ID NO 62 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 62 aaaaac agca aauluccaulic 19 <210 SEQ ID NO 63 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 63 aaac agcaaa luluccalucgu. 19 <210 SEQ ID NO 64 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 64 US 2008/O 132691 A1 Jun. 5, 2008 51 - Continued acagcaaaluul C caucgugu. 19 <210 SEQ ID NO 65 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 65 agcaaalulu.cc aucguguaa 19 <210 SEQ ID NO 66 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 66 caaauluccalu cquguaaluc 19 <210 SEQ ID NO 67 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 67 alauluccalucg lugulaaucaia 19 <210 SEQ ID NO 68 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 68 uluccalucgug ulaaluca agg 19 <210 SEQ ID NO 69 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 69 cCaucgugua aucaaggac 19 <210 SEQ ID NO 70 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 7 O aucguguaalu Caaggacuu. 19 <210 SEQ ID NO 71 US 2008/O 132691 A1 Jun. 5, 2008 52 - Continued <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 71 cguguaaluca aggaculuca 19 <210 SEQ ID NO 72 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 72 uguaalucaag gacuucaug 19 <210 SEQ ID NO 73 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 73 ulaauca agga culucaugau. 19 <210 SEQ ID NO 74 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 74 aucaaggacul ulcalugalucc 19 <210 SEQ ID NO 75 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 75 Caaggacuuc alugauccag 19 <210 SEQ ID NO 76 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 76 aggacuucau gaucCaggg 19 <210 SEO ID NO 77 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: US 2008/O 132691 A1 Jun. 5, 2008 53 - Continued <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 77 gacuucauga luccagggcg 19 <210 SEQ ID NO 78 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 78 cuucaugauc Cagggcgga 19 <210 SEQ ID NO 79 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 79 ulcalugalucca gggcggaga 19 <210 SEQ ID NO 8O <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 80 alugaluccagg gcggagacul 19 <210 SEQ ID NO 81 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 81 gaucCagggc ggagacuuc 19 <210 SEQ ID NO 82 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 82 luccagggcgg agacuucac 19 <210 SEQ ID NO 83 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 83 US 2008/O 132691 A1 Jun. 5, 2008 54 - Continued Cagggcggag acuucacca 19 <210 SEQ ID NO 84 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 84 gggcggagac ulcacCagg 19 <210 SEQ ID NO 85 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 85 gcggagacull CacCagggg 19 <210 SEQ ID NO 86 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 86 ggagaculuca CCaggggag 19 <210 SEQ ID NO 87 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 87 agacuucacic aggggagalu. 19 <210 SEQ ID NO 88 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 88 acuucaccag gggagalugg 19 <210 SEQ ID NO 89 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 89 uucacCaggg gagaluggca 19 <210 SEQ ID NO 90 US 2008/O 132691 A1 Jun. 5, 2008 55 - Continued <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 9 O Caccagggga gauggcaca 19 <210 SEQ ID NO 91 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 91 cCaggggaga luggcac agg 19 <210 SEQ ID NO 92 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 92 aggggagalug gCacaggag 19 <210 SEQ ID NO 93 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 93 gggagaluggc acaggagga 19 <210 SEQ ID NO 94 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 94 gagaluggcac aggaggaala 19 <210 SEQ ID NO 95 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 95 galuggcacag gaggaaaga 19 <210 SEQ ID NO 96 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: US 2008/O 132691 A1 Jun. 5, 2008 56 - Continued OTHER INFORMATION: Synthetic SEQUENCE: 96 luggcacagga gaaagagc 19 SEO ID NO 97 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic SEQUENCE: 97 gcacaggagg aaagagcau, 19 SEO ID NO 98 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic SEQUENCE: 98 acaggaggaa agagcaucu. 19 SEO ID NO 99 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic SEQUENCE: 99 aggaggaaag agcaucuac 19 SEQ ID NO 100 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic SEQUENCE: 1.OO gaggaaagag caucuacgg 19 SEQ ID NO 101 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic SEQUENCE: 101 ggaaagagca ulculacggug 19 SEQ ID NO 102 LENGTH 19 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic SEQUENCE: 1 O2