(12) Patent Application Publication (10) Pub. No.: US 2009/0088563 A1 Khvorova Et Al

US 20090088,563A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0088563 A1 KhVOrOVa et al. (43) Pub. Date: Apr. 2, 2009

(54) SIRNA TARGETING TRANSDUCIN Related U.S. Application Data (BETA)-LIKE 3 (TBL3) (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 (US) (57) ABSTRACT Efficient sequence specific gene silencing is possible through (73) Assignee: Dharmacon, Inc., Lafayette, CO the use of siRNA technology. By selecting particular siRNAs (US) by rational design, one can maximize the generation of an effective gene silencing reagent, as well as methods for (21) Appl. No.: 12/287,757 silencing genes. Methods, compositions, and kits generated through rational design of siRNAS are disclosed including (22) Filed: Oct. 14, 2008 those directed to nucleotide sequences for TBL3. Patent Application Publication Apr. 2, 2009 Sheet 1 of 43 US 2009/0088,563 A1

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SRNA TARGETING TRANSDUCN RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in (BETA)-LIKE 3 (TBL3) C. elegans, Cell 109(7):861-71; Ketting et al. (2002) Dicer Functions in RNA Interference and in Synthesis of Small CROSS-REFERENCE TO RELATED RNA Involved in Developmental Timing in C. elegans; Mar APPLICATIONS tinez et al., Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi, Cell 110(5):563; Hutvagner & 0001. This application is a continuation-in-part of U.S. Zamore (2002) A microRNA in a multiple-turnover RNAi Ser. No. 10/714,333, filed Nov. 14, 2003, which claims the enzyme complex, Science 297:2056. benefit of U.S. Provisional Application No. 60/426,137, filed 0008. From a mechanistic perspective, introduction of Nov. 14, 2002, and also claims the benefit of U.S. Provisional long double stranded RNA into plants and invertebrate cells is Application No. 60/502,050, filed Sep. 10, 2003; this appli broken down into siRNA by a Type II endonuclease known as cation is also a continuation-in-part of U.S. Ser. No. 10/940, Dicer. Sharp, RNA interference 2001, Genes Dev. 2001, 892, filed Sep. 14, 2004, which is a continuation of PCT 15:485. Dicer, a ribonuclease-III-like enzyme, processes the Application No. PCT/US04/14885, international filing date dsRNA into 19-23 base pair short interfering RNAs with May 12, 2004. The disclosures of the priority applications, characteristic two base 3' overhangs. Bernstein, Caudy, Ham including the sequence listings and tables Submitted in elec mond, & Hannon (2001) Role for abidentate ribonuclease in tronic form in lieu of paper, are incorporated by reference into the initiation step of RNA interference, Nature 409:363. The the instant specification. siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the SEQUENCE LISTING siRNA duplex, enabling the complementary antisense Strand 0002 The sequence listing for this application has been to guide target recognition. Nykanen, Haley, & Zamore submitted in accordance with 37 CFRS1.52(e) and 37 CFR (2001) ATP requirements and small interfering RNA struc S1.821 on CD-ROM in lieu of paper on a disk containing the ture in the RNA interference pathway, Cell 107:309. Upon sequence listing file entitled “DHARMA 2100-US103 binding to the appropriate target mRNA, one or more endo CRF.txt created May. 21, 2008, 88 kb. Applicants hereby nucleases within the RISC cleaves the target to induce silenc incorporate by reference the sequence listing provided on ing. Elbashir, Lendeckel, & Tuschl (2001) RNA interference CD-ROM in lieu of paper into the instant specification. is mediated by 21- and 22-nucleotide RNAs, Genes Dev. 15:188, FIG.1. FIELD OF INVENTION 0009. The interference effect can be long lasting and may be detectable after many cell divisions. Moreover, RNAi 0003. The present invention relates to RNA interference exhibits sequence specificity. Kisielow, M. et al., (2002) Iso (“RNAi). form-specific knockdown and expression of adaptor protein ShcA using small interfering RNA. J. Biochem. 363: 1-5. BACKGROUND OF THE INVENTION Thus, the RNAi machinery can specifically knock down one 0004 Relatively recently, researchers observed that type of transcript, while not affecting closely related mRNA. double stranded RNA (dsRNA) could be used to inhibit These properties make siRNA a potentially valuable tool for protein expression. This ability to silence a gene has broad inhibiting gene expression and studying gene function and potential for treating human diseases, and many researchers drug target validation. Moreover, siRNAs are potentially use and commercial entities are currently investing considerable ful as therapeutic agents against: (1) diseases that are caused resources in developing therapies based on this technology. by over-expression or misexpression of genes; and (2) dis 0005 Double stranded RNA induced gene silencing can eases brought about by expression of genes that contain muta occur on at least three different levels: (i) transcription inac tions. tivation, which refers to RNA guided DNA or histone methy 0010. Successful siRNA-dependent gene silencing lation; (ii) siRNA induced mRNA degradation; and (iii) depends on a number of factors. One of the most contentious mRNA induced transcriptional attenuation. issues in RNAi is the question of the necessity of siRNA 0006. It is generally considered that the major mechanism design, i.e., considering the sequence of the siRNA used. of RNA induced silencing (RNA interference, or RNAi) in Early work in C. elegans and plants circumvented the issue of mammalian cells is mRNA degradation. Initial attempts to design by introducing long dsRNA (see, for instance, Fire, A. use RNAi in mammalian cells focused on the use of long etal. (1998) Nature 391:806-811). In this primitive organism, strands of dsRNA. However, these attempts to induce RNAi long dsRNA molecules are cleaved into siRNA by Dicer, thus met with limited success, due in part to the induction of the generating a diverse population of duplexes that can poten interferon response, which results in a general, as opposed to tially cover the entire transcript. While some fraction of these a target-specific, inhibition of protein synthesis. Thus, long molecules are non-functional (i.e., induce little or no silenc dsRNA is not a viable option for RNAi in mammalian sys ing) one or more have the potential to be highly functional, temS. thereby silencing the gene of interest and alleviating the need 0007 More recently it has been shown that when short for siRNA design. Unfortunately, due to the interferon (18-30 bp) RNA duplexes are introduced into mammalian response, this same approach is unavailable for mammalian cells in culture, sequence-specific inhibition of target mRNA systems. While this effect can be circumvented by bypassing can be realized without inducing an interferon response. Cer the Dicer cleavage step and directly introducing siRNA, this tain of these short dsRNAs, referred to as small inhibitory tactic carries with it the risk that the chosen siRNA sequence RNAs (“siRNAs), can act catalytically at sub-molar concen may be non-functional or semi-functional. trations to cleave greater than 95% of the target mRNA in the 0011. A number of researches have expressed the view cell. A description of the mechanisms for siRNA activity, as that siRNA design is not a crucial element of RNAi. On the well as some of its applications are described in Provost et al. other hand, others in the field have begun to explore the (2002) Ribonuclease Activity and RNA Binding of Recom possibility that RNAi can be made more efficient by paying binant Human Dicer, EMBO.J. 21 (21): 5864-5874: Tabara et attention to the design of the siRNA. Unfortunately, none of al. (2002) The dsRNA Binding Protein RDE-4 Interacts with the reported methods have provided a satisfactory scheme for US 2009/0088,563 A1 Apr. 2, 2009

reliably selecting siRNA with acceptable levels of function A=1 if A is the base at position 1 of the sense strand, other ality. Accordingly, there is a need to develop rational criteria wise its value: is 0; by which to select siRNA with an acceptable level of func A=1 if A is the base at position 2 of the sense strand, other tionality, and to identify siRNA that have this improved level wise its value: is 0; of functionality, as well as to identify siRNAs that are hyper A=1 if A is the base at position 3 of the sense strand, other functional. wise its value: is 0; A=1 if A is the base at position 4 of the sense strand, other SUMMARY OF THE INVENTION wise its value is 0; 0012. The present invention is directed to increasing the As=1 if A is the base at position 5 of the sense strand, other efficiency of RNAi, particularly in mammalian systems. wise its value is 0; Accordingly, the present invention provides kits, siRNAS and A=1 if A is the base at position 6 of the sense strand, other methods for increasing siRNA efficacy. wise its value is 0; 0013. According to a first embodiment, the present inven A, -1 if A is the base at position 7 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 10 of the sense strand, which is comprised of a sequence that is complementary to a otherwise its value is 0; portion of the sequence of one or more target messenger A=1 if A is the base at position 11 of the sense strand, RNA, and each of which is selected using non-target specific otherwise its value is 0; criteria. A=1 if A is the base at position 13 of the sense strand, 0014. According to a second embodiment, the present otherwise its value is 0; invention provides a method for selecting an siRNA, said A=1 if A is the base at position 19 of the sense strand, method comprising applying selection criteria to a set of otherwise if another base is present or the sense strand is only potential siRNA that comprise 18-30 base pairs, wherein said 18 base pairs in length, its value is 0; selection criteria are non-target specific criteria, and said set C=1 if C is the base at position 3 of the sense strand, other comprises at least two siRNAs and each of said at least two wise its value is 0; siRNAS contains a sequence that is at least Substantially C=1 if C is the base at position 4 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. Cs=1 if C is the base at position 5 of the sense strand, other 0015 According to a third embodiment, the present inven wise its value is 0; tion also provides a method for selecting an siRNA wherein C=1 if C is the base at position 6 of the sense strand, other said selection criteria are embodied in a formula comprising: wise its value is 0; C, 1 if C is the base at position 7 of the sense strand, other (-14): G13 - 13: A - 12: U7 - 11 : U2 - 10: A 1.1 - Formula VIII wise its value: is 0; 10 : U - 10: C3 - 10: C5 - 10: C – 9: A 10 - C1 if C is the base at position 9 of the sense strand, other 9: U9 - 9: C18 - 8: G10 - 7: U - 7: U16 - wise its value is 0; 7: C17 - 7: Co -- 7: U17 + 8: A2 + 8: A + C=1 if C is the base at position 17 of the sense strand, 8 : As + 8: C+9: G8 + 10: A7 + 10: U18 + otherwise its value is 0; 11 : A19 - 11: Co -- 15: G -- 18: A3 - 19: U10 - Cs-1 if C is the base at position 18 of the sense strand, Tin - 3: (GC) - 6: (GCs 19) - 30 : X; or otherwise its value is 0; C-1 if C is the base at position 19 of the sense strand, (-8): A 1 + (-1): A2 + (12): A3 + (7): A4 + (18): A5 + Formula X otherwise if another base is present or the sense strand is only (12): A6 + (19): A7+ (6): A8 + (-4): A9 -- (-5): A 10 + 18 base pairs in length, its value is 0; (-2): A11 + (-5): A 12 + (17): A 13 + (-3): A14 + G=1 if G is the base at position 1 on the sense strand, (4): A 15 + (2): A 16 + (8): A 17--(11): A 18 + otherwise its value is 0; (30): A 19-- (-13) : U 1 + (-10): U2 + (2): U3 + G=1 if G is the base at position 2 of the sense strand, other (-2) : U 4 + (-5): U.5 + (5) : U 6 -- (-2): U7 + wise its value is 0; (-10): U8 + (-5): U9 + (15) : U 10 + (-1) : U 11 + Gs=1 if G is the base at position 8 on the sense strand, (O) : U 12 + (10): U13 + (-9) : U 14-- (-13) : U 15 + otherwise its value is 0; (–10): U16+ (3) : U 17+ (9): U 18 + (9): U19 + Go-1 if G is the base at position 10 on the sense strand, (7): C1 + (3): C2 + (-21): C3 + (5): C4 -- (-9): C5 + otherwise its value is 0; (-20): C6 -- (-18): C7 -- (-5): C8 + (5): C9 + G=1 if G is the base at position 13 on the sense strand, (1): C10 + (2): C11 + (-5): C12 + (-3): C13 + otherwise its value is 0; (-6): C14 -- (-2): C15 + (-5): C16 + (-3): C17+ G-1 if G is the base at position 19 of the sense strand, (-12): C18 + (-18): C19 + (14): G1 + (8): G2 + otherwise if another base is present or the sense strand is only (7): G3 + (-10): G4 + (-4): G5 + (2): G6 + (1) : G7 -- 18 base pairs in length, its value is 0; (9): G8 + (5): G9 -- (-11) : G10 + (1) : G11 + (9): G12 -- (-24) : G13 + (18): G14 + (11): G15 + U=1 if U is the base at position 1 on the sense strand, (13) : G16 -- (-7): G17 -- (-9): G18 + (-22): G19 + otherwise its value is 0; 68 (number of A + U in position 15 - 19) - U=1 if U is the base at position 2 on the sense strand, 3: (number of G + C in whole siRNA), otherwise its value is 0; U=1 if U is the base at position 3 on the sense strand, otherwise its value is 0; wherein position numbering begins at the 5'-most position of U=1 if U is the base at position 4 on the sense strand, a sense Strand, and otherwise its value is 0; US 2009/0088,563 A1 Apr. 2, 2009

7–1 if U is the base at position 7 on the sense strand, 0023 (c) determining the relative functionality of each therwise its value is 0; siRNA; =1 if U is the base at position 9 on the sense strand, 0024 (d) determining the amount of improved function therwise its value is 0; ality by the presence or absence of at least one variable 1 if U is the base at position 10 on the sense strand, selected from the group consisting of the total GC content, therwise its value is 0; melting temperature of the siRNA, GC content at positions s=1 if U is the base at position 15 on the sense strand, 15-19, the presence or absence of a particular nucleotide at a therwise its value is 0; particular position, relative thermodynamic stability at par 1 if U is the base at position 16 on the sense strand, ticular positions in a duplex, and the number of times that the therwise its value is 0; same nucleotide repeats within a given sequence; and 7–1 if U is the base at position 17 on the sense strand, 0025 (e) developing an algorithm using the information therwise its value is 0; of step (d). U is 1 if U is the base at position 18 on the sense strand, otherwise its value is 0. 0026. According to this embodiment, preferably the set of GCs the number of G and C bases within positions 15-19 siRNAs comprises at least 90 siRNAs from at least one gene, of the sense strand, or within positions 15-18 if the sense more preferably at least 180 siRNAs from at least two differ Strand is only 18 base pairs in length; ent genes, and most preferably at least 270 and 360 siRNAs GC, the number of G and C bases in the sense Strand; from at least three and four different genes, respectively. Tm=100 if the siRNA oligo has the internal repeat longer Additionally, in step (d) the determination is made with pref then 4 base pairs, otherwise its value is 0; and erably at least two, more preferably at least three, even more X=the number of times that the same nucleotide repeats four preferably at least four, and most preferably all of the vari or more times in a row. ables. The resulting algorithm is not target sequence specific. 0016. According to a fourth embodiment, the invention 0027. In another embodiment, the present invention pro provides a method for developing an algorithm for selecting vides rationally designed siRNAs identified using the formu siRNA, said method comprising: (a) selecting a set of siRNA; las above. (b) measuring gene silencing ability of each siRNA from said 0028. In yet another embodiment, the present invention is set; (c) determining relative functionality of each siRNA; (d) directed to hyperfunctional siRNA. determining improved functionality by the presence or 0029. The ability to use the above algorithms, which are absence of at least one variable selected from the group con sisting of the presence or absence of a particular nucleotide at not sequence or species specific, allows for the cost-effective a particular position, the total number of AS and US in posi selection of optimized siRNAS for specific target sequences. tions 15-19, the number of times that the same nucleotide Accordingly, there will be both greater efficiency and reli repeats within a given sequence, and the total number of Gs ability in the use of siRNA technologies. and Cs; and (e) developing an algorithm using the informa 0030. In various embodiments, siRNAs that target nucle tion of step (d). otide sequences for Homo sapiens transducin (beta)-like 3 0017. According to a fifth embodiment, the present inven (TBL3) are provided. In various embodiments, the siRNAs tion provides a kit, wherein said kit is comprised of at least are rationally designed. In various embodiments, the siRNAs two siRNAs, wherein said at least two siRNAs comprise a are functional or hyperfunctional. first optimized siRNA and a second optimized siRNA, 0031. In various embodiments, an siRNA that targets the wherein said first optimized siRNA and said second opti nucleotide sequence for TBL3 is provided, wherein the mized siRNA are optimized according a formula comprising siRNA is selected from the group consisting of various Formula X. siRNA sequences targeting the nucleotide sequences for 0018. The present invention also provides a method for TBL3 that are disclosed herein. In various embodiments, the identifying a hyperfunctional siRNA, comprising applying siRNA sequence is selected from the group consisting of SEQ selection criteria to a set of potential siRNA that comprise ID NO. 438 to SEQID NO. 502. 18-30 base pairs, wherein said selection criteria are non target specific criteria, and said set comprises at least two 0032. In various embodiments, siRNA comprising a sense siRNAs and each of said at least two siRNAs contains a region and an antisense region are provided, said sense region sequence that is at least Substantially complementary to a and said antisense region together form a duplex region com target gene; determining the relative functionality of the at prising 18-30 base pairs, and said sense region comprises a least two siRNAS and assigning each of the at least two sequence that is at least 90% similar to a sequence selected siRNAs a functionality score; and selecting siRNAs from the from the group consisting of siRNA sequences targeting at least two siRNAs that have a functionality score that nucleotide sequences for TBL3 that are disclosed herein. In reflects greater than 80 percent silencing at a concentration in various embodiments, the siRNA sequence is selected from the picomolar range, wherein said greater than 80 percent the group consisting of SEQID NO. 438 to SEQID NO. 502. silencing endures for greater than 120 hours. 0033. In various embodiments, an siRNA comprising a 0019. According to a sixth embodiment, the present inven sense region and an antisense region is provided, said sense tion provides a hyperfunctional siRNA that is capable of region and said antisense region together form a duplex silencing Bcl2. region comprising 18-30 base pairs, and said sense region 0020. According to a seventh embodiment, the present comprises a sequence that is identical to a contiguous stretch invention provides a method for developing an siRNA algo of at least 18 bases of a sequence selected from the group rithm for selecting functional and hyperfunctional siRNAs consisting of SEQID NO.438 to SEQID NO. 502. In various for a given sequence. The method comprises: embodiments, the duplex region is 19-30 base pairs, and the 0021 (a) selecting a set of siRNAs; sense region comprises a sequence that is identical to a 0022 (b) measuring the gene silencing ability of each sequence selected from the group consisting of SEQID NO. siRNA from said set; 438 to SEQID NO. 502. US 2009/0088,563 A1 Apr. 2, 2009

0034. In various embodiments, a pool of at least two siR Uat position 10 of the sense strand, (D) a base other than Gat NAs is provided, wherein said pool comprises a first siRNA position 13 of the sense strand, and (E) a base other than Cat and a second siRNA, said first siRNA comprising a duplex position 19 of the sense strand. All variables were correlated region of length 18-30 base pairs that has a first sense region with siRNA silencing of firefly luciferase and human cyclo that is at least 90% similar to 18 bases of a first sequence philin. siRNAs satisfying the criterion are grouped on the left selected from the group consisting of SEQ ID NO. 438 to (Selected) while those that do not, are grouped on the right SEQID NO. 502, and said second siRNA comprises a duplex (Eliminated). Y-axis is “96 Silencing of Control. Each posi region of length 18-30 base pairs that has a second sense tion on the X-axis represents a unique siRNA. region that is at least 90% similar to 18 bases of a second 0042 FIGS. 5A and 5B are representations of firefly sequence selected from the group consisting of SEQID NO. luciferase and cyclophilin siRNA panels Sorted according to 438 to SEQID NO. 502, wherein said first sense region and functionality and predicted values using Formula VIII. The said second sense region are not identical. siRNA found within the circle represent those that have For 0035. In various embodiments, the first sense region com mula VIII values (SMARTSCORESTM, or siRNA rank) prises a sequence that is identical to at least 18 bases of a above Zero. siRNA outside the indicated area have calculated sequence selected from the group consisting of SEQID NO. Formula VIII values that are below zero. Y-axis is “Expres 438 to SEQID NO. 502, and said second sense region com sion (%. Control). Each position on the X-axis represents a prises a sequence that is identical to at least 18 bases of a unique siRNA. sequence selected from the group consisting of SEQID NO. 0043 FIG. 6A is a representation of the average internal 438 to SEQID NO. 502. In various embodiments, the duplex stability profile (AISP) derived from 270 siRNAs taken from of said first siRNA is 19-30 base pairs, and said first sense three separate genes (cyclophilin B, DBI and firefly region comprises a sequence that is at least 90% similar to a luciferase). Graphs represent AISP values of highly func sequence selected from the group consisting of SEQID NO. tional, functional, and non-functional siRNA. FIG. 6B is a 438 to SEQ ID NO. 502, and said duplex of said second comparison between the AISP of naturally derived GFP siRNA is 19-30 base pairs and comprises a sequence that is at siRNA (filled squares) and the AISP of siRNA from cyclo least 90% similar to a sequence selected from the group philin B, DBI, and luciferase having >90% silencing proper consisting of SEQID NO. 438 to SEQID NO. 502. ties (no fill) for the antisense strand. “DG' is the symbol for 0036. In various embodiments, the duplex of said first AG, free energy. siRNA is 19-30 base pairs and said first sense region com 0044 FIG. 7 is a histogram showing the differences in prises a sequence that is identical to at least 18 bases of a duplex functionality upon introduction of base pair mis sequence selected from the group consisting of SEQID NO. matches. The X-axis shows the mismatch introduced in the 438 to SEQ ID NO. 502, and said duplex of said second siRNA and the position it is introduced (e.g., 8C>A reveals siRNA is 19-30 base pairs and said second region comprises that position 8 (which normally has a C) has been changed to a sequence that is identical to a sequence selected from the an A). The Y-axis is “96 Silencing (Normalized to Control).' group consisting of SEQID NO. 438 to SEQID NO. 502. The samples on the X-axis represent siRNAs at 100 nMand 0037 For a better understanding of the present invention are, reading from left to right: 1A to C, 1A to G, 1A to U: 2A together with other and further advantages and embodiments, to C, 2A to G, 2A to U; 3A to C, 3A to G, 3A to U; 4G to A, reference is made to the following description taken in con 4G to C; 4G to U; 5U to A, 5U to C, 5U to G: 6U to A, 6U to junction with the examples, the scope of which is set forth in C, 6U to G: 7G to A, 7G to C, 7G to U; 8C to A, 8C to G, 8C the appended claims. to U; 9G to A, 9G to C, 9G to U; 10C to A, 10C to G, 10C to U; 11G to A, 11G to C, 11G to U; 12G to A, 12G to C, 12G to BRIEF DESCRIPTION OF THE FIGURES U; 13 A to C, 13 A to G, 13 A to U; 14G to A, 14G to C, 14G to 0038 FIG. 1 shows a model for siRNA-RISC interactions. U; 15G to A, 15G to C, 15G to U; 16A to C, 16A to G, 16A to RISC has the ability to interact with either end of the siRNA U; 17G to A, 17G to C, 17G to U; 18U to A, 18U to C, 18U to or miRNA molecule. Following binding, the duplex is G: 19U to A, 19U to C, 19U to G: 20 wit: Control. unwound, and the relevant target is identified, cleaved, and 0045 FIG. 8 is histogram that shows the effects of 5' sense released. and antisense strand modification with 2'-O-methylation on 0039 FIG. 2 is a representation of the functionality of two functionality. hundred and seventy siRNA duplexes that were generated to 0046 FIG. 9 shows a graph of SMARTSCORESTM, or target human cyclophilin, human diazepam-binding inhibitor siRNA rank, versus RNAi silencing values for more than 360 (DB), and firefly luciferase. siRNA directed against 30 different genes. SiRNA to the right 0040 FIG.3a is a representation of the silencing effect of of the vertical bar represent those siRNA that have desirable 30 siRNAs in three different cells lines, HEK293, DU145, SMARTSCORESTM, or siRNA rank. and Hela. FIG. 3b shows the frequency of different functional 0047 FIGS. 10A-E compare the RNAi of five different groups (>95% silencing (black), >80% silencing (gray), genes (SEAP DBI, PLK, Firefly Luciferase, and Renilla >50% silencing (dark gray), and <50% silencing (white)) Luciferase) by varying numbers of randomly selected siRNA based on GC content. In cases where a given bar is absent and four rationally designed (SMART-selected) siRNA cho from a particular GC percentage, no siRNA were identified sen using the algorithm described in Formula VIII. In addi for that particular group. FIG. 3c shows the frequency of tion, RNAi induced by a pool of the four SMART-selected different functional groups based on melting temperature siRNA is reported at two different concentrations (100 and (Tm). 400 nM). 10F is a comparison between a pool of randomly 0041 FIG. 4 is a representation of a statistical analysis that selected EGFR siRNA (Pool 1) and a pool of SMART-se revealed correlations between silencing and five sequence lected EGFR siRNA (Pool 2). Pool 1, S1-S4 and Pool 2 S1-S4 related properties of siRNA: (A) an A at position 19 of the represent the individual members that made up each respec sense Strand, (B) an A at position 3 of the sense strand, (C) a tive pool. Note that numbers for random siRNAs represent the US 2009/0088,563 A1 Apr. 2, 2009

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, Dynil, 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 1), MEK1 and MEK2, ATE1 (arginyl-tRNA percent expression relative to a control. protein transferase), GAPDH, and Eg5). The Y-axis is the 0058 FIGS. 21A, 21B and 21C show both pools (Library, percent expression of the control. Numbers 1, 2, 3 and 4 Lib) and individual siRNAs in inhibition of gene expression represent individual rationally selected siRNA. “Pool repre of Beta-Galactosidase, Renilla Luciferase and SEAP (alka sents a 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 0051 FIG. 14 is the knockdown by the top ten Bcl2 siR gene relative to a control. Libraries contain 19 nucleotide long NAs at 100 nM concentrations. The Y-axis represents the siRNAS (not including overhangs) that begin at the following amount of expression relative to the non-specific (ins) and nucleotides: SEAP: Lib 1: 206, 766, 812, 923, Lib 2: 1117, transfection mixture control. 1280, 1300, 1487, Lib 3: 206, 766, 812, 923, 1117, 1280, 0052 FIG. 15 represents a functional walk where siRNA 1300, 1487, Lib 4: 206, 812, 1117, 1300, Lib 5: 766,923, beginning on every other base pair of a region of the luciferase 1280, 1487, Lib 6: 206, 1487; Bgal: Lib 1:979, 1339, 2029, gene are tested for the ability to silence the luciferase gene. 2590, Lib 2: 1087, 1783,2399, 3257, Lib 3:979, 1783, 2590, The Y-axis represents the percent expression relative to a 3257, Lib 4:979, 1087, 1339, 1783, 2029, 2399, 2590,3257, control. The X-axis represents the position of each individual Lib5:979, 1087, 1339, 1783, Lib 6: 2029, 2399, 2590,3257; siRNA. Reading from left to right across the X-axis, the Renilla: Lib 1: 174, 300, 432,568, Lib 2:592,633, 729,867, position designations are 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21. Lib 3: 174,300, 432,568,592,633, 729,867, Lib 4: 174,432, 23, 25, 27, 29, 31,33,35, 37, 39, 41,43, 45, 47,49, 51,53,55, 592, 729, Lib 5:300,568, 633, 867, Lib 6:592,568. 57, 59,61,63, 65, 67, 69,71, 73,75, 77, 79,81, 83, 85, 87,89, 0059 FIG. 22 shows the results of an EGFR and TfnR and Plasmid. internalization assay when single gene knockdowns are per 0053 FIGS. 16A and 16B are histograms demonstrating formed. The Y-axis represents percent internalization relative the inhibition of target gene expression by pools of 2 (16A) to control. and 3 (16B) siRNA duplexes taken from the walk described in 0060 FIG. 23 shows the results of an EGFR and TfnR FIG. 15. The Y-axis in each represents the percent expression internalization assay when multiple genes are knocked down relative to control. The X-axis in each represents the position (e.g., Rab5a, b, c). The Y-axis represents the percent internal of the first siRNA in paired pools, or trios of siRNAs. For ization relative to control. instance, the first paired pool contains siRNAs 1 and 3. The 0061 FIG. 24 shows the simultaneous knockdown of four second paired pool contains siRNAs3 and 5. Pool 3 (of paired different genes. siRNAs directed against G6PD, GAPDH, pools) contains siRNAS 5 and 7, and so on. For each of 16A PLK, and UQC were simultaneously introduced into cells. and 16B, the X-axis from left to right reads 1,3,5,7,9,11, 13, Twenty-four hours later, cultures were harvested and assayed 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45,47, for mRNA target levels for each of the four genes. A com 49,51,53,55,57, 59, 61,63, 65, 67, 69,71, 73,75, 77,79, 81, parison is made between cells transfected with individual 83, 85, 87, 89, and Plasmid. siRNAs vs. a pool of siRNAs directed against all four genes. 0054 FIGS. 17A and 17B are histograms demonstrating 0062 FIG.25 shows the functionality often siRNAs at 0.3 the inhibition of target gene expression by pools of 4 (17A) nM concentrations. and 5 (17B) siRNA duplexes. The Y-axis in each represents the percent expression relative to control. The X-axis in each DETAILED DESCRIPTION represents the position of the first siRNA in each pool. For Definitions each of 17A and 17B, 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, 0063. Unless stated otherwise, the following terms and 41, 43,45, 47,49,51,53,55, 57, 59, 61, 63, 65, 67, 69, 71,73, phrases have the meanings provided below: 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid. Complementary 0055 FIGS. 18A and 18B are histograms demonstrating the inhibition of target gene expression by siRNAs that are ten 0064. The term “complementary” refers to the ability of (18A) and twenty (18B) base pairs base pairs apart. The polynucleotides to form base pairs with one another. Base Y-axis represents the percent expression relative to a control. pairs are typically formed by hydrogen bonds between nucle The X-axis represents the position of the first siRNA in each otide units in antiparallel polynucleotide strands. Comple pool. For each of 18A and 18B, the X-axis from left to right mentary polynucleotide strands can base pair in the Watson US 2009/0088,563 A1 Apr. 2, 2009

Crick manner (e.g., A to T. A to U. C to G), or in any other for, or selected against, to obtain a final set with the preferred manner that allows for the formation of duplexes. As persons properties. In other instances, filtering includes wet lab skilled in the art are aware, when using RNA as opposed to experiments. For instance, sequences identified by one or DNA, uracil rather than thymine is the base that is considered more versions of the algorithm can be screened using any one to be complementary to adenosine. However, when a U is of a number of procedures to identify duplexes that have denoted in the context of the present invention, the ability to hyperfunctional traits (e.g., they exhibit a high degree of substitute a T is implied, unless otherwise stated. silencing at Subnanomolar concentrations and/or exhibit high 0065 Perfect complementarity or 100% complementarity degrees of silencing longevity). refers to the situation in which each nucleotide unit of one polynucleotide Strand can hydrogen bond with a nucleotide Gene Silencing unit of a second polynucleotide strand. Less than perfect 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 same example, if 18 base pairs on each strand can hydrogen results from RNA interference (RNAi), a defined, though bond with each other, the polynucleotide strands exhibit 90% partially characterized pathway whereby small inhibitory complementarity. RNA (siRNA) act in concert with host proteins (e.g., the RNA induced silencing complex, RISC) to degrade messenger Deoxynucleotide RNA (mRNA) in a sequence-dependent fashion. The level of gene silencing can be measured by a variety of means, includ 0066. The term “deoxynucleotide' refers to a nucleotide ing, but not limited to, measurement of transcript levels by or polynucleotide lacking a hydroxyl group (OH group) at the Northern Blot Analysis, B-DNA techniques, transcription 2" and/or 3' position of a Sugar moiety. Instead, it has a hydro sensitive reporter constructs, expression profiling (e.g., DNA gen bonded to the 2' and/or 3' carbon. Within an RNA mol chips), and related technologies. Alternatively, the level of ecule that comprises one or more deoxynucleotides, “deoxy silencing can be measured by assessing the level of the pro nucleotide' refers to the lack of an OH group at the 2' position tein encoded by a specific gene. This can be accomplished by of the Sugar moiety, having instead a hydrogen bonded performing a number of studies including Western Analysis, directly to the 2 carbon. measuring the levels of expression of a reporter protein that has e.g., fluorescent properties (e.g., GFP) or enzymatic activ Deoxyribonucleotide ity (e.g., alkaline phosphatases), or several other procedures. 0067. The terms “deoxyribonucleotide' and “DNA” refer miRNA to a nucleotide or polynucleotide comprising at least one (0071. The term “miRNA refers to microRNA. Sugar moiety that has an H, rather than an OH, at its 2' and/or 3'position. Nucleotide Duplex Region 0072. The term “nucleotide' refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an 0068. The phrase “duplex region” refers to the region in analog thereof. Nucleotides include species that comprise two complementary or Substantially complementary poly purines, e.g., adenine, hypoxanthine, guanine, and their nucleotides that form base pairs with one another, either by derivatives and analogs, as well as pyrimidines, e.g., cytosine, Watson-Crick base pairing or any other manner that allows uracil, thymine, and their derivatives and analogs. for a stabilized duplex between polynucleotide strands that 0073 Nucleotide analogs include nucleotides having are complementary or Substantially complementary. For modifications in the chemical structure of the base, Sugar example, a polynucleotide strand having 21 nucleotide units and/or phosphate, including, but not limited to, 5-position can base pair with another polynucleotide of 21 nucleotide pyrimidine modifications, 8-position purine modifications, units, yet only 19 bases on each Strand are complementary or modifications at cytosine exocyclic amines, and Substitution Substantially complementary, such that the “duplex region' of 5-bromo-uracil; and 2'-position Sugar modifications, has 19 base pairs. The remaining bases may, for example, including but not limited to, Sugar-modified ribonucleotides exist as 5' and 3' overhangs. Further, within the duplex region, in which the 2'-OH is replaced by a group such as an H, OR, 100% complementarity is not required; substantial comple R, halo, SH, SR, NH, NHR, NR, or CN, wherein R is an mentarity is allowable within a duplex region. Substantial alkyl moiety. Nucleotide analogs are also meant to include complementarity refers to 79% or greater complementarity. nucleotides with bases such as inosine, queuosine, Xanthine, For example, a mismatch in a duplex region consisting of 19 Sugars such as 2'-methyl ribose, non-natural phosphodiester base pairs results in 94.7% complementarity, rendering the linkages such as methylphosphonates, phosphorothioates and duplex region Substantially complementary. peptides. 0074 Modified bases refer to nucleotide bases such as, for Filters example, adenine, guanine, cytosine, thymine, uracil, Xan 0069. The term “filter” refers to one or more procedures thine, inosine, and queuosine that have been modified by the that are performed on sequences that are identified by the replacement or addition of one or more atoms or groups. algorithm. In some instances, filtering includes in silico pro Some examples of types of modifications that can comprise cedures where sequences identified by the algorithm can be nucleotides that are modified with respect to the base moieties screened to identify duplexes carrying desirable or undesir include but are not limited to, alkylated, halogenated, thi able motifs. Sequences carrying Such motifs can be selected olated, aminated, amidated, or acetylated bases, individually US 2009/0088,563 A1 Apr. 2, 2009

or in combination. More specific examples include, for ribonucleotides and/or their analogs. The term “polyribo example, 5-propynyluridine, 5-propynylcytidine, 6-methy nucleotide' is used interchangeably with the term "oligori ladenine, 6-methylguanine, N.N.-dimethyladenine, 2-propy bonucleotide.” ladenine, 2-propylguanine, 2-aminoadenine, 1-methyli nosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine Ribonucleotide and Ribonucleic Acid and other nucleotides having a modification at the 5 position, 0080. The term “ribonucleotide' and the phrase “ribo 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine, nucleic acid (RNA), refer to a modified or unmodified nucle 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, otide or polynucleotide comprising at least one ribonucle 3-methylcytidine, 6-methyluridine, 2-methylguanosine, otide unit. A ribonucleotide unit comprises anhydroxyl group 7-methylguanosine, 2,2-dimethylguanosine, 5-methylami attached to the 2' position of a ribosyl moiety that has a noethyluridine, 5-methyloxyuridine, deaZanucleotides Such nitrogenous base attached in N-glycosidic linkage at the 1' as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-aZothy position of a ribosyl moiety, and a moiety that either allows midine, 5-methyl-2-thiouridine, other thio bases such as for linkage to another nucleotide or precludes linkage. 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrou siRNA ridine, pseudouridine, queuosine, archaeosine, naphthyl and I0081. The term “siRNA” refers to small inhibitory RNA Substituted naphthyl groups, any O- and N-alkylated purines duplexes that induce the RNA interference (RNAi) pathway. and pyrimidines Such as N6-methyladenosine, 5-methylcar These molecules can vary in length (generally 18-30 base bonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4- pairs) and contain varying degrees of complementarity to one, pyridine-2-one, phenyl and modified phenyl groups such their target mRNA in the antisense strand. Some, but not all, as aminophenol or 2.4.6-trimethoxy benzene, modified siRNA have unpaired overhanging bases on the 5' or 3' end of cytosines that act as G-clamp nucleotides, 8-substituted the sense Strand and/or the antisense Strand. The term adenines and guanines, 5-substituted uracils and thymines, “siRNA’ includes duplexes of two separate strands, as well as azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxy single strands that can form hairpin structures comprising a alkylaminoalkyl nucleotides, and alkylcarbonylalkylated duplex region. nucleotides. Modified nucleotides also include those nucle I0082 siRNA may be divided into five (5) groups (non otides that are modified with respect to the Sugar moiety, as functional, semi-functional, functional, highly functional, well as nucleotides having Sugars or analogs thereof that are and hyper-functional) based on the level or degree of silenc not ribosyl. For example, the Sugar moieties may be, or be ing that they induce in cultured cell lines. As used herein, based on, mannoses, arabinoses, glucopyranoses, galactopy these definitions are based on a set of conditions where the ranoses, 4'-thioribose, and other Sugars, heterocycles, or car siRNA is transfected into said cell line at a concentration of bocycles. 100 nM and the level of silencing is tested at a time of roughly 0075. The term nucleotide is also meant to include what 24 hours after transfection, and not exceeding 72 hours after are known in the art as universal bases. By way of example, transfection. In this context, “non-functional siRNA are universal bases include but are not limited to 3-nitropyrrole, defined as those siRNA that induce less than 50% (<50%) 5-nitroindole, or nebularine. The term “nucleotide' is also target silencing. "Semi-functional siRNA’ induce 50-79% meant to include the N3' to P5' phosphoramidate, resulting target silencing. "Functional siRNA are molecules that from the substitution of a ribosyl 3' oxygen with an amine induce 80-95% gene silencing. “Highly-functional siRNA group. are molecules that induce greater than 95% gene silencing. 0076 Further, the term nucleotide also includes those spe “Hyperfunctional siRNA are a special class of molecules. cies that have a detectable label, such as for example a radio For purposes of this document, hyperfunctional siRNA are active or fluorescent moiety, or mass label attached to the defined as those molecules that: (1) induce greater than 95% nucleotide. silencing of a specific target when they are transfected at Subnanomolar concentrations (i.e., less than one nanomolar); Off-Target Silencing and Off-Target Interference and/or (2) induce functional (or better) levels of silencing for 0077. The phrases “off-target silencing and “off-target greater than 96 hours. These relative functionalities (though interference' are defined as degradation of mRNA other than not intended to be absolutes) may be used to compare siRNAs the intended target mRNA due to overlapping and/or partial to a particular target for applications such as functional homology with secondary mRNA messages. genomics, target identification and therapeutics. SMARTSCORETM, or siRNA Rank Polynucleotide 0083. The term “SMARTSCORETM, or “siRNA rank” 0078. The term “polynucleotide' refers to polymers of refers to a number determined by applying any of the formu nucleotides, and includes but is not limited to DNA, RNA, las to a given siRNA sequence. The term “SMART-selected DNA/RNA hybrids including polynucleotide chains of regu or “rationally selected or “rational selection” refers to larly and/or irregularly alternating deoxyribosyl moieties and siRNA that have been selected on the basis of their SMART ribosyl moieties (i.e., wherein alternate nucleotide units have SCORESTM, or siRNA ranking. an —OH, then and —H, then an —OH, then an —H, and so on at the 2' position of a Sugar moiety), and modifications of Substantially Similar these kinds of polynucleotides, wherein the attachment of I0084. The phrase “substantially similar refers to a simi various entities or moieties to the nucleotide units at any larity of at least 90% with respect to the identity of the bases position are included. of the sequence. Polyribonucleotide Target 0079. The term “polyribonucleotide” refers to a poly I0085. The term “target” is used in a variety of different nucleotide comprising two or more modified or unmodified forms throughout this document and is defined by the context US 2009/0088,563 A1 Apr. 2, 2009 in which it is used. “Target mRNA refers to a messenger RNA to which a given siRNA can be directed against. “Target sequence' and “target site' refer to a sequence within the (-14): G13 - 13: A 1 - 12: U7 - 11 : U2 - 10: A 1.1 - Formula VIII mRNA to which the sense strand of an siRNA shows varying 10 : U - 10: C3 - 10: C5 - 10: C – 9: A 10 - degrees of homology and the antisense strand exhibits vary 9: U9 - 9: C18 - 8: G10 - 7: U1 - 7: U16 - ing degrees of complementarity. The phrase “siRNA target' 7: C7 - 7: Co -- 7: U17 + 8: A2 + 8: A + can refer to the gene, mRNA, or protein against which an 8 : As + 8: C+9: G8 + 10: A7 + 10: U18 + siRNA is directed. Similarly, “target silencing can refer to 11 : A19 - 11: Co -- 15: G -- 18: A3 - 19: U10 - the state of a gene, or the corresponding mRNA or protein. Tin - 3: (GC) - 6: (GCs 19) - 30: X; or Transfection (-8): A 1 + (-1): A2 + (12): A3 + (7): A4 + (18): A5 + Formula X (12): A6 + (19): A7+ (6): A8 + (-4): A9 -- (-5): A 10 + I0086. The term “transfection” refers to a process by which (-2): A11 + (-5): A 12 + (17): A 13 + (-3): A14 + agents are introduced into a cell. The list of agents that can be (4): A 15 + (2): A 16 + (8): A 17--(11): A 18 + transfected is large and includes, but is not limited to, siRNA, (30): A 19-- (-13) : U 1 + (-10): U2 + (2): U3 + sense and/or anti-sense sequences, DNA encoding one or (-2) : U 4 + (-5): U.5 + (5): U6 -- (-2): U7+ more genes and organized into an expression plasmid, pro (-10): U8 + (-5): U9 + (15) : U 10+ (-1) : U 11 + teins, protein fragments, and more. There are multiple meth (O) : U 12 + (10): U13 + (-9) : U 14-- (-13) : U 15 + ods for transfecting agents into a cell including, but not lim (–10): U16+ (3): U 17+ (9): U 18 + (9): U19 + ited to, electroporation, calcium phosphate-based (7): C1 + (3): C2 + (-21): C3 + (5): C4 -- (-9): C5 + transfections, DEAE-dextran-based transfections, lipid (-20): C6 -- (-18): C7 -- (-5): C8 + (5): C9 + based transfections, molecular conjugate-based transfections (1): C10 + (2): C11 + (-5): C12 -- (-3): C13 + (e.g., polylysine-DNA conjugates), microinjection and oth (-6): C14 -- (-2): C15 + (-5): C16 + (-3): C17+ CS. (-12): C18 + (-18): C19 + (14): G1 + (8): G2 + 0087. The present invention is directed to improving the (7) : G3 + (-10): G4 + (-4): G5 + (2): G6 + (1) : G7 -- efficiency of gene silencing by siRNA. Through the inclusion (9): G8 + (5): G9 -- (-11) : G10 + (1) : G11 + of multiple siRNA sequences that are targeted to a particular (9): G12 -- (-24) : G13 + (18): G14 + (11): G15 + gene and/or selecting an siRNA sequence based on certain (13) : G16 -- (-7): G17 -- (-9): G18 + (-22): G19 + defined criteria, improved efficiency may be achieved. 68 (number of A + U in position 15 - 19) - I0088. The present invention will now be described in con 3: (number of G + C in whole siRNA), nection with preferred embodiments. These embodiments are presented in order to aid in an understanding of the present invention and are not intended, and should not be construed, 0094 wherein position numbering begins at the 5'-most to limit the invention in any way. All alternatives, modifica position of a sense strand, and tions and equivalents that may become apparent to those of 0.095 A=1 if A is the base at position 1 of the sense strand, ordinary skill upon reading this disclosure are included otherwise its value is 0; within the spirit and scope of the present invention. 0096 A=1 if A is the base at position 2 of the sense strand, 0089. Furthermore, this disclosure is not a primer on RNA otherwise its value is 0; interference. Basic concepts known to persons skilled in the I0097 A=1 if A is the base at position 3 of the sense strand, art have not been set forth in detail. otherwise its value is 0; 0090 The present invention is directed to increasing the I0098 A-1 if A is the base at position 4 of the sense strand, efficiency of RNAi, particularly in mammalian systems. otherwise its value is 0; Accordingly, the present invention provides kits, siRNAS and 0099 As=1 if A is the base at position 5 of the sense strand, methods for increasing siRNA efficacy. otherwise its value is 0; 0091. According to a first embodiment, the present inven 0100 A–1 if A is the base at position 6 of the sense strand, tion provides a kit for gene silencing, wherein said kit is otherwise its value is 0; comprised of a pool of at least two siRNA duplexes, each of 0101 A-1 if A is the base at position 7 of the sense strand, which is comprised of a sequence that is complementary to a otherwise its value is 0; portion of the sequence of one or more target messenger 0102 A-1 if A is the base at position 10 of the sense RNA, and each of which is selected using non-target specific strand, otherwise its value is 0; criteria. Each of the at least two siRNA duplexes of the kit (0103) A =1 if A is the base at position 11 of the sense complementary to a portion of the sequence of one or more strand, otherwise its value is 0; target mRNAs is preferably selected using Formula X. I0104 A-1 if A is the base at position 13 of the sense 0092. According to a second embodiment, the present strand, otherwise its value is 0; invention provides a method for selecting an siRNA, said 0105 A1 if A is the base at position 19 of the sense method comprising applying selection criteria to a set of Strand, otherwise if another base is present or the sense Strand potential siRNA that comprise 18-30 base pairs, wherein said is only 18 base pairs in length, its value is 0; selection criteria are non-target specific criteria, and said set I0106 C=1 ifC is the base at position 3 of the sense strand, comprises at least two siRNAs and each of said at least two otherwise its value is 0; siRNAS contains a sequence that is at least Substantially 0107 C-1 if C is the base at position 4 of the sense strand, complementary to a target gene; and determining the relative otherwise its value is 0; functionality of the at least two siRNAs. 0.108 C-1 if C is the base at position 5 of the sense strand, 0093. In one embodiment, the present invention also pro otherwise its value is 0; vides a method wherein said selection criteria are embodied 0109) C-1 if C is the base at position 6 of the sense strand, in a formula comprising: otherwise its value is 0; US 2009/0088,563 A1 Apr. 2, 2009

0110 C, 1 if C is the base at position 7 of the sense strand, selecting siRNA can further comprise either selecting for or otherwise its value is 0; selecting against sequences that comprise stability motifs. 0111 C-1 if C is the base at position 9 of the sense strand, 0.137 In another embodiment, the present invention pro otherwise its value is 0; vides a method of gene silencing, comprising introducing into 0112 C, 1 if C is the base at position 17 of the sense a cell at least one siRNA selected according to any of the strand, otherwise its value is 0; methods of the present invention. The siRNA can be intro 0113. Cs-1 if C is the base at position 18 of the sense duced by allowing passive uptake of siRNA, or through the strand, otherwise its value is 0; use of a vector. 0114 Co-1 if C is the base at position 19 of the sense 0.138 According to a third embodiment, the invention pro Strand, otherwise if another base is present or the sense Strand vides a method for developing an algorithm for selecting is only 18 base pairs in length, its value is 0; siRNA, said method comprising: (a) selecting a set of siRNA; 0115 G=1 if G is the base at position 1 on the sense (b) measuring gene silencing ability of each siRNA from said strand, otherwise its value is 0; set; (c) determining relative functionality of each siRNA; (d) 0116 G=1 if G is the base at position 2 of the sense strand, determining improved functionality by the presence or otherwise its value is 0; absence of at least one variable selected from the group con 0117 G-1 if G is the base at position 8 on the sense sisting of the presence or absence of a particular nucleotide at strand, otherwise its value is 0; a particular position, the total number of AS and US in posi 0118 Go-1 if G is the base at position 10 on the sense tions 15-19, the number of times that the same nucleotide strand, otherwise its value is 0; repeats within a given sequence, and the total number of Gs 0119 G=1 if G is the base at position 13 on the sense and Cs; and (e) developing an algorithm using the informa strand, otherwise its value is 0; tion of step (d). 0.139. In another embodiment, the invention provides a I0120 G =1 if G is the base at position 19 of the sense method for selecting an siRNA with improved functionality, Strand, otherwise if another base is present or the sense Strand comprising using the above-mentioned algorithm to identify is only 18 base pairs in length, its value is 0; an siRNA of improved functionality. I0121 U=1 if U is the base at position 1 on the sense 0140. According to a fourth embodiment, the present strand, otherwise its value is 0; invention provides a kit, wherein said kit is comprised of at I0122 U-1 if U is the base at position 2 on the sense least two siRNAs, wherein said at least two siRNAs comprise strand, otherwise its value is 0; a first optimized siRNA and a second optimized siRNA, 0123 U-1 if U is the base at position 3 on the sense wherein said first optimized siRNA and said second opti strand, otherwise its value is 0; mized siRNA are optimized according a formula comprising 0.124 U-1 if U is the base at position 4 on the sense Formula X. strand, otherwise its value is 0; 0.141. According to a fifth embodiment, the present inven 0.125 U7–1 if U is the base at position 7 on the sense tion provides a method for identifying a hyperfunctional strand, otherwise its value is 0; siRNA, comprising applying selection criteria to a set of I0126 U-1 if U is the base at position 9 on the sense potential siRNA that comprise 18-30 base pairs, wherein said strand, otherwise its value is 0; selection criteria are non-target specific criteria, and said set 0127 U-1 if U is the base at position 10 on the sense comprises at least two siRNAs and each of said at least two strand, otherwise its value is 0; siRNAS contains a sequence that is at least Substantially 0128. Us=1 if U is the base at position 15 on the sense complementary to a target gene; determining the relative strand, otherwise its value is 0; functionality of the at least two siRNAS and assigning each of 0129. U-1 if U is the base at position 16 on the sense the at least two siRNAs a functionality score; and selecting strand, otherwise its value is 0; siRNAs from the at least two siRNAs that have a functionality I0130 U–1 if U is the base at position 17 on the sense score that reflects greater than 80 percent silencing at a con strand, otherwise its value is 0; centration in the picomolar range, wherein said greater than 0131 U-1 if U is the base at position 18 on the sense 80 percent silencing endures for greater than 120 hours. strand, otherwise its value is 0. 0142. In other embodiments, the invention provides kits 0132 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, 0.133 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 0134) 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. 0135 X=the number of times that the same nucleotide 0.143 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 0.136 Any of the methods of selecting siRNA in accor silencing Bcl2. dance with the invention can further comprise comparing the 0144. 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 0145 (a) selecting a set of siRNAs; contain motifs that induce cellular stress. Such motifs 0146 (b) measuring the gene silencing ability of each include, for example, toxicity motifs. Any of the methods of siRNA from said set; US 2009/0088,563 A1 Apr. 2, 2009

0147 (c) determining the relative functionality of each to this method, an siRNA is selected for a given gene by using siRNA; a rational design. That said, rational design can be described 0148 (d) determining the amount of improved function in a variety of ways. Rational design is, in simplest terms, the ality by the presence or absence of at least one variable application of a proven set of criteria that enhance the prob selected from the group consisting of the total GC content, ability of identifying a functional or hyperfunctional siRNA. melting temperature of the siRNA, GC content at positions In one method, rationally designed siRNA can be identified 15-19, the presence or absence of a particular nucleotide at a by maximizing one or more of the following criteria: particular position, relative thermodynamic stability at par 0159 (1) A low GC content, preferably between about ticular positions in a duplex, and the number of times that the 3O-52%. same nucleotide repeats within a given sequence; and 0160 (2) At least 2, preferably at least 3A or U bases at 0149 (e) developing an algorithm using the information positions 15-19 of the siRNA on the sense strand. of step (d). 0.161 (3) An Abase at position 19 of the sense strand. 0150. According to this embodiment, preferably the set of 0162 (4) An Abase at position 3 of the sense strand. siRNAs comprises at least 90 siRNAs from at least one gene, 0163 (5) AU base at position 10 of the sense strand. more preferably at least 180 siRNAs from at least two differ 0164 (6) An Abase at position 14 of the sense strand. ent genes, and most preferably at least 270 and 360 siRNAs 0.165 (7) A base other than Cat position 19 of the sense from at least three and four different genes, respectively. Strand. Additionally, in step (d) the determination is made with pref 0166 (8) A base other than G at position 13 of the sense erably at least two, more preferably at least three, even more Strand. preferably at least four, and most preferably all of the vari 0.167 (9) A Tm, which refers to the character of the inter ables. The resulting algorithm is not target sequence specific. nal repeat that results in inter- or intramolecular structures for 0151. In another embodiment, the present invention pro one strand of the duplex, that is preferably not stable at greater vides rationally designed siRNAs identified using the formu than 50° C., more preferably not stable at greater than 37°C., las above. even more preferably not stable at greater than 30° C. and 0152. In yet another embodiment, the present invention is most preferably not stable at greater than 20°C. directed to hyperfunctional siRNA. 0168 (10) A base other than U at position 5 of the sense 0153. The ability to use the above algorithms, which are Strand. not sequence or species specific, allows for the cost-effective 0169 (11) A base other than A at position 11 of the sense selection of optimized siRNAs for specific target sequences. strand. Accordingly, there will be both greater efficiency and reli 0170 (12) A base other than an A at position 1 of the sense ability in the use of siRNA technologies. Strand. 0154 The methods disclosed herein can be used in con 0171 (13) A base other than an A at position 2 of the sense junction with comparing internal stability profiles of selected Strand. siRNAs, and designing an siRNA with a desirable internal 0172 (14) An A base at position 4 of the sense strand. stability profile; and/or in conjunction with a selection either 0173 (15) An A base at position 5 of the sense strand. for or against sequences that contain motifs that induce cel 0.174 (16) An A base at position 6 of the sense strand. lular stress, for example, cellular toxicity. 0.175 (17) An A base at position 7 of the sense strand. 0155 Any of the methods disclosed herein can be used to 0176 (18) An A base at position 8 of the sense strand. silence one or more genes by introducing an siRNA selected, 0177 (19) A base other than an A at position 9 of the sense or designed, in accordance with any of the methods disclosed Strand. herein. The siRNA(s) can be introduced into the cell by any method known in the art, including passive uptake or through 0.178 (20) A base other than an A at position 10 of the the use of one or more vectors. sense Strand. 0156 Any of the methods and kits disclosed herein can 0179 (21) A base other than an A at position 11 of the employ either unimolecular siRNAs, siRNAs comprised of sense Strand. two separate polynucleotide strands, or combinations thereof. 0180 (22) A base other than an A at position 12 of the Any of the methods disclosed herein can be used in gene sense Strand. silencing, where two or more genes are silenced by a single 0181 (23) An A base at position 13 of the sense strand. administration of siRNA(s). The siRNA(s) can be directed 0182 (24) A base other than an A at position 14 of the against two or more target genes, and administered in a single sense Strand. dose or single transfection, as the case may be. 0183 (25) An A base at position 15 of the sense strand Optimizing siRNA 0.184 (26) An A base at position 16 of the sense strand. 0157 According to one embodiment, the present inven 0185 (27) An A base at position 17 of the sense strand. tion provides a method for improving the effectiveness of 0186 (28) An A base at position 18 of the sense strand. gene silencing for use to silence a particular gene through the 0187 (29) A base other than a U at position 1 of the sense selection of an optimal siRNA. An siRNA selected according Strand. to this method may be used individually, or in conjunction 0188 (30) A base other than a U at position 2 of the sense with the first embodiment, i.e., with one or more other siR Strand. NAs, each of which may or may not be selected by this criteria 0189 (31) AU base at position 3 of the sense strand. in order to maximize their efficiency. 0.190 (32) A base other than a U at position 4 of the sense 0158. The degree to which it is possible to selectan siRNA Strand. for a given mRNA that maximizes these criteria will depend 0191 (33) A base other than a U at position 5 of the sense on the sequence of the mRNA itself. However, the selection Strand. criteria will be independent of the target sequence. According 0.192 (34) AU base at position 6 of the sense strand. US 2009/0088,563 A1 Apr. 2, 2009

0193 (35) A base other than a U at position 7 of the sense 0235 (77) AG base at position 15 of the sense strand. Strand. 0236 (78) AG base at position 16 of the sense strand. 0194 (36) A base other than a U at position 8 of the sense 0237 (79) A base other thana Gat position 17 of the sense Strand. Strand. 0195 (37) A base other than a U at position 9 of the sense 0238 (80) A base other thana Gat position 18 of the sense Strand. Strand. 0196) (38) A base other thana Uat position 11 of the sense 0239 (81) A base other thana Gat position 19 of the sense Strand. Strand. 0197) (39) AU base at position 13 of the sense strand. 0240. The importance of various criteria can vary greatly. 0198 (40) A base other thana Uat position 14 of the sense For instance, a C base at position 10 of the sense Strand makes Strand. a minor contribution to duplex functionality. In contrast, the 0199. (41) A base other thana Uat position 15 of the sense absence of a C at position 3 of the sense strand is very Strand. important. Accordingly, preferably an siRNA will satisfy as 0200 (42) A base other thana Uat position 16 of the sense many of the aforementioned criteria as possible. Strand. 0241. With respect to the criteria, GC content, as well as a 0201 (43) AU base at position 17 of the sense strand. high number of AU in positions 15-19 of the sense strand, 0202 (44) AU base at position 18 of the sense strand. may be important for easement of the unwinding of double 0203 (45) AU base at position 19 of the sense strand. stranded siRNA duplex. Duplex unwinding has been shown 0204 (46) AC base at position 1 of the sense strand. to be crucial for siRNA functionality in vivo. 0205 (47) A C base at position 2 of the sense strand. 0242. With respect to criterion 9, the internal structure is 0206 (48) A base other than a C at position 3 of the sense measured in terms of the melting temperature of the single Strand. strand of siRNA, which is the temperature at which 50% of 0207 (49) AC base at position 4 of the sense strand. the molecules will become denatured. With respect to criteria 0208 (50) A base other than a C at position 5 of the sense 2-8 and 10-11, the positions refer to sequence positions on the Strand. sense strand, which is the strand that is identical to the mRNA. 0209 (51) A base other than a C at position 6 of the sense 0243 In one preferred embodiment, at least criteria 1 and Strand. 8 are satisfied. In another preferred embodiment, at least 0210 (52) A base other than a C at position 7 of the sense criteria 7 and 8 are satisfied. Instill another preferred embodi Strand. ment, at least criteria 1, 8 and 9 are satisfied. 0211 (53) A base other than a C at position 8 of the sense 0244. It should be noted that all of the aforementioned Strand. criteria regarding sequence position specifics are with respect 0212 (54) A C base at position 9 of the sense strand. to the 5' end of the sense strand. Reference is made to the 0213 (55) A C base at position 10 of the sense strand. sense Strand, because most databases contain information 0214 (56) AC base at position 11 of the sense strand. that describes the information of the mRNA. Because accord 0215 (57) A base other thana Cat position 12 of the sense ing to the present invention a chain can be from 18 to 30 bases Strand. in length, and the aforementioned criteria assumes a chain 19 0216 (58) A base other thana Cat position 13 of the sense base pairs in length, it is important to keep the aforemen Strand. tioned criteria applicable to the correct bases. 0217 (59) A base other thana Cat position 14 of the sense 0245. When there are only 18 bases, the base pair that is Strand. not present is the base pair that is located at the 3' of the sense 0218 (60) A base other thana Cat position 15 of the sense strand. When there are twenty to thirty bases present, then Strand. additional bases are added at the 5' end of the sense chain and 0219) (61) A base other thana Cat position 16 of the sense occupy positions 1 to 11. Accordingly, with respect to SEQ. Strand. ID NO.0001 NNANANNNNUCNAANNNNA and SEQ. ID 0220 (62) A base other thana Cat position 17 of the sense NO. 0028 GUCNNANANNNNUCNAANNNNA, both Strand. would have A at position 3, A at position 5, U at position 10, 0221) (63) A base other thana Cat position 18 of the sense C at position 11, A and position 13, A and position 14 and A Strand. at position 19. However, SEQ. ID NO. 0028 would also have 0222 (64) AG base at position 1 of the sense strand. C at position -1, U at position -2 and G at position -3. 0223 (65) AG base at position 2 of the sense strand. 0246 For a 19 base pair siRNA, an optimal sequence of 0224 (66) AG base at position 3 of the sense strand. one of the strands may be represented below, where N is any 0225 (67) A base other than a Gat position 4 of the sense base, A, C, G, or U: Strand. 0226 (68) A base other than a Gat position 5 of the sense Strand. SEO. ID NO. OOO1. INNANANNNNUCNAANNNNA 0227 (69) AG base at position 6 of the sense strand. SEO. ID NO. OOO2. INNANANNNNUGNAANNNNA 0228 (70) AG base at position 7 of the sense strand. 0229 (71) AG base at position 8 of the sense strand. SEO. ID NO. OOO3. INNANANNNNUUNAANNNNA 0230 (72) AG base at position 9 of the sense strand. SEO. ID NO. OOO4. INNANANNNNUCNCANNNNA 0231 (73) A base other thana Gat position 10 of the sense Strand. SEO. ID NO. OOO5. INNANANNNNUGNCANNNNA 0232 (74) AG base at position 11 of the sense strand. SEO. ID NO. OOO6. INNANANNNNUUNCANNNNA 0233 (75) AG base at position 12 of the sense strand. 0234 (76) AG base at position 14 of the sense strand. US 2009/0088,563 A1 Apr. 2, 2009 12

- Continued -continued SEO. ID NO. OOO7. INNANANNNNUCNUANNNNA Relative functionality of siRNA = Formula VII SEO. ID NO. OOO8. INNANANNNNUGNUANNNNA -(GCf2) + (A U1519) f2 - (Tnzoo C.) : 1 - (G13): 3 - (C19) + (A19) : 3 + (A3): 3 + SEO. ID NO. OOO9. INNANANNNNUUNUANNNNA (U10) f2 + (A14)/2 - (Us) f2 - (A11)/2

SEO. ID NO. OO10. INNANCNNNNUCNAANNNNA 0248. In Formulas I-VII: SEO. ID NO. OO11. INNANCNNNNUGNAANNNNA 0249 wherein A-1 if A is the base at position 19 on the sense Strand, otherwise its value is 0. SEO. ID NO. OO12. INNANCNNNNUUNAANNNNA 0250 AU so-0-5 depending on the number of A or U SEO. ID NO. OO13. INNANCNNNNUCNCANNNNA bases on the sense strand at positions 15-19; 0251 G=1 if G is the base at position 13 on the sense SEO. ID NO. OO14. INNANCNNNNUGNCANNNNA strand, otherwise its value is 0; 0252 C-1 if C is the base at position 19 of the sense SEO. ID NO. OO15. INNANCNNNNUUNCANNNNA strand, otherwise its value is 0; 0253 GC=the number of G and C bases in the entire sense SEO. ID NO. OO16. INNANCNNNNUCNUANNNNA Strand; SEO. ID NO. OO17. INNANCNNNNUGNUANNNNA 0254 Tmo. 1 if the Tm is greater than 20° C.; 0255 A=1 if A is the base at position 3 on the sense SEO. ID NO. OO18. INNANCNNNNUUNUANNNNA strand, otherwise its value is 0; 0256 U-1 if U is the base at position 10 on the sense SEO. ID NO. OO19. INNANGNNNNUCNAANNNNA strand, otherwise its value is 0; 0257 A-1 if A is the base at position 14 on the sense SEO. ID NO. OO2O. INNANGNNNNUGNAANNNNA strand, otherwise its value is 0; SEO. ID NO. OO21. INNANGNNNNUUNAANNNNA 0258 Us=1 if U is the base at position 5 on the sense strand, otherwise its value is 0; and SEO. ID NO. OO22. NNANGNNNNUCNCANNNNA 0259 A=1 if A is the base at position 11 of the sense strand, otherwise its value is 0. SEO. ID NO. OO23. INNANGNNNNUGNCANNNNA 0260 Formulas I-VII provide relative information regard ing functionality. When the values for two sequences are SEO. ID NO. OO24. INNANGNNNNUUNCANNNNA compared for a given formula, the relative functionality is ascertained; a higher positive number indicates a greater SEO. ID NO. OO25. INNANGNNNNUCNUANNNNA functionality. For example, in many applications a value of 5 SEO. ID NO. OO26. INNANGNNNNUGNUANNNNA or greater is beneficial. 0261 Additionally, in many applications, more than one SEO. ID NO. OO27. INNANGNNNNNU.NUANNNNA of these formulas would provide useful information as to the relative functionality of potential siRNA sequences. How 0247. In one embodiment, the sequence used as an siRNA ever, it is beneficial to have more than one type of formula, is selected by choosing the siRNA that score highest accord because not every formula will be able to help to differentiate ing to one of the following seven algorithms that are repre among potential siRNA sequences. For example, in particu sented by Formulas I-VI: larly high GC mRNAs, formulas that take that parameter into account would not be useful and application of formulas that lack GC elements (e.g., formulas V and VI) might provide Relative functionality of siRNA = Formula I greater insights into duplex functionality. Similarly, formula -(GC/3) + (A U1519) - (Tnzoo C.): 3 - (G13): 3 II might by used in situations where hairpin structures are not (C19) + (A19): 2 + (A3) + (U10) + (A14) - (Us) - (A11) observed in duplexes, and formula IV might be applicable for sequences that have higher AU content. Thus, one may con Relative functionality of siRNA = -(GC/3) - Formula II sider a particular sequence in light of more than one or even (AU1519) : 3 - (G13): 3 - (C.19) + (A19): 2 + (A3) all of these algorithms to obtain the best differentiation among sequences. In some instances, application of a given Relative functionality of siRNA = Formula III algorithm may identify an unusually large number of poten - (GC/3) + (AU1519) - (Tnzoo C.): 3 tial siRNA sequences, and in those cases, it may be appropri Relative functionality of siRNA = Formula IV ate to re-analyze that sequence with a second algorithm that - GCf2 + (AU1519) f2 - (Tnzoo C.): 2 - (G13): 3 is, for instance, more stringent. Alternatively, it is conceivable (C19) + (A19): 2 + (A3) + (U10) + (A14) - (Us) - (A11) that analysis of a sequence with a given formula yields no acceptable siRNA sequences (i.e. low SMARTSCORESTM, Relative functionality of siRNA = -(G13): 3- (C.19) + Formula W or siRNA ranking). In this instance, it may be appropriate to (A19): 2 + (A3) + (U10) + (A14) - (Us) - (A11) re-analyze that sequences with a second algorithm that is, for instance, less stringent. In still other instances, analysis of a Relative functionality of siRNA = Formula VI single sequence with two separate formulas may give rise to -(G13): 3 - (C19) + (A19): 2 + (A3) conflicting results (i.e. one formula generates a set of siRNA with high SMARTSCORESTM, or siRNA ranking, while the US 2009/0088,563 A1 Apr. 2, 2009

other formula identifies a set of siRNA with low SMART the sequence of the mRNA and automatically outputs the SCORESTM, or siRNA ranking). In these instances, it may be optimal siRNA. The computer program may, for example, be necessary to determine which weighted factor(s) (e.g. GC accessible from a local terminal or personal computer, overan content) are contributing to the discrepancy and assessing the internal network or over the Internet. sequence to decide whether these factors should or should not 0268. In addition to the formulas above, more detailed be included. Alternatively, the sequence could be analyzed by algorithms, may be used for selecting siRNA. Preferably, at a third, fourth, or fifth algorithm to identify a set of rationally least one RNA duplex of 18-30 base pairs is selected such that designed siRNA. it is optimized according a formula selected from: 0262 The above-referenced criteria are particularly advantageous when used in combination with pooling tech (-14): G13 - 13: A 1 - 12: U7 - 11 : U2 - 10: A 1.1 - Formula VIII niques as depicted in Table I: 10: U4 - 10: C3 - 10: C5 - 10: C6 - 9: A 10 - 9: Ug-9: C18 - 8: Go - 7: U1 - 7: U16 - TABLE I 7: C7 - 7: Co -- 7: U17 + 8: A2 + 8: A + 8: A5 + 8: C+9: G8 + 10: A7 - 10: U18 + FUNCTIONAL PROBABILITY 11 : A19 - 11: Co. + 15: G + 18: A3 - 19: U10 - Tin - 3: (GC) - 6: (GCs 19) - 30 : X; and OLIGOS POOLS (14.1): A3 + (14.9): A6 + (17.6): A 13 + Formula IX CRITERIA 95% 80% 70% 95% 80% 70% (24.7) : A 19 + (14.2): U10 + (10.5): Co. + (23.9): G + CURRENT 33.0 SO.O 23.0 79.5 97.3 O.3 (16.3): G -- (-12.3): A 1 + (-19.3) : U + NEW SO.O 88.5 8.O 93.8 99.98 O.OOS (-12.1): U2 + (-11): U3 + (-15.2): U15 + (GC) 28.0 S8.9 36.0 72.8 97.1 1.6 (-11.3) : U + (-11.8): C3 + (-17.4): C + (-10.5): C7 -- (-13.7) : G3 + (-25.9) : Go 0263. The term “current used in Table I refers to Tuschl's Tin - 3: (GCit) - 6: (GC15-19) - 30 : X; and conventional siRNA parameters (Elbashir, S. M. et al. (2002) 'Analysis of gene function in Somatic mammalian cells using (-8): A 1 + (-1): A2 + (12): A3 + (7): A4 + (18): A5 + Formula X small interfering RNAs Methods 26: 199-213). “New” (12): A6 + (19): A7+ (6): A8 + (-4): A9 -- (-5): A 10 + refers to the design parameters described in Formulas I-VII. (-2): A11 + (-5): A 12 + (17): A 13 + (-3): A14 + “GC refers to criteria that select siRNA solely on the basis of (4): A 15 + (2): A 16 + (8): A 17--(11): A 18 + GC content. (30): A 19-- (-13) : U 1 + (-10): U2 + (2): U3 + 0264. As Table I indicates, when more functional siRNA (-2) : U 4 + (-5): U.5 + (5): U6 -- (-2): U7+ duplexes are chosen, siRNAs that produce <70% silencing (-10): U8 + (-5): U9 + (15) : U 10+ (-1) : U 11 + drops from 23% to 8% and the number of siRNA duplexes (O) : U 12 + (10): U13 + (-9) : U 14-- (-13) : U 15 + that produce >80% silencing rises from 50% to 88.5%. Fur (–10): U16+ (3): U 17+ (9): U 18 + (9): U19 + ther, of the siRNA duplexes with >80% silencing, a larger (7): C1 + (3): C2 + (-21): C3 + (5): C4 -- (-9): C5 + portion of these siRNAs actually silence >95% of the target (-20): C6 -- (-18): C7 -- (-5): C8 + (5): C9 + expression (the new criteria increases the portion from 33% to (1): C10 + (2): C11 + (-5): C12 -- (-3): C13 + 50%). Using this new criteria in pooled siRNAs, shows that, (-6): C14 -- (-2): C15 + (-5): C16 + (-3): C17+ with pooling, the amount of silencing >95% increases from (-12): C18 + (-18): C19 + (14): G1 + (8): G2 + 79.5% to 93.8% and essentially eliminates any siRNA pool (7) : G3 + (-10): G4 + (-4): G5 + (2): G6 + (1) : G7 -- from silencing less than 70%. (9): G8 + (5): G9 -- (-11) : G10 + (1) : G11 + 0265 Table II similarly shows the particularly beneficial (9): G12 -- (-24) : G13 + (18): G14 + (11): G15 + results of pooling in combination with the aforementioned (13) : G16 -- (-7): G17 -- (-9): G18 + (-22): G19 + criteria. However, Table II, which takes into account each of 68 (number of A + U in position 15 - 19) - the aforementioned variables, demonstrates even a greater 3: (number of G + C in whole siRNA). degree of improvement in functionality.

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

0266 The terms “functional.” “Average.” and “Non-func- 0269 wherein tional 99 used in Table II, refer to siRNA that exhibit- 0 >80%,O (0270 A=1 ifY A is the base at position 1 of the sense strand, >50%, and <50% functionality, respectively. Criteria 1 and 4 refer to specific criteria described above. otherwise its value is 0; 0267. The above-described algorithms may be used with 0271 A=1 if A is the base at position 2 of the sense strand, or without a computer program that allows for the inputting of otherwise its value is 0; US 2009/0088,563 A1 Apr. 2, 2009 14

0272 A=1 if A is the base at position 3 of the sense strand, 0302 U-1 if U is the base at position 10 on the sense otherwise its value is 0; strand, otherwise its value is 0; 0273 A-1 if A is the base at position 4 of the sense strand, 0303 Uls=1 if U is the base at position 15 on the sense otherwise its value is 0; strand, otherwise its value is 0; 0274 As=1 if A is the base at position 5 of the sense strand, 0304 U-1 if U is the base at position 16 on the sense otherwise its value is 0; strand, otherwise its value is 0; 0275 A-1 if A is the base at position 6 of the sense strand, 0305 U-1 if U is the base at position 17 on the sense otherwise its value is 0; strand, otherwise its value is 0; 0276 A =1 if A is the base at position 7 of the sense strand, 0306 UF1 if U is the base at position 18 on the sense otherwise its value is 0; strand, otherwise its value is 0; 0277 A-1 if A is the base at position 10 of the sense 0307 GC so the number of G and C bases within posi strand, otherwise its value is 0; tions 15-19 of the sense strand, or within positions 15-18 if 0278 A=1 if A is the base at position 11 of the sense the sense Strand is only 18 base pairs in length; strand, otherwise its value is 0; 0308 GC, the number of G and C bases in the sense 0279 A-1 if A is the base at position 13 of the sense Strand; strand, otherwise its value is 0; (0309 Tm=100 if the siRNA oligo has the internal repeat 0280 A-1 if A is the base at position 19 of the sense longer then 4 base pairs, otherwise its value is 0; and Strand, otherwise if another base is present or the sense Strand 0310 X=the number of times that the same nucleotide is only 18 base pairs in length, its value is 0; repeats four or more times in a row. (0281 C=1 ifC is the base at position 3 of the sense strand, 0311. The above formulas VIII, IX, and X, as well as otherwise its value is 0; formulas I-VII, provide methods for selecting siRNA in order 0282 C-1 ifC is the base at position 4 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 0283 C-1 if C is the base at position 5 of the sense strand, variables are used, the optimization hierarchy becomes less otherwise its value is 0; reliable. 0284 C=1 if C is the base at position 6 of the sense strand, 0312. With respect to the variables of the above-refer otherwise its value is 0; enced formulas, a single letter of A or C or G or Ufollowed by 0285 C, 1 if C is the base at position 7 of the sense strand, a subscript refers to a binary condition. The binary condition otherwise its value is 0; is that either the particular base is present at that particular 0286 C-1 ifC is the base at position 9 of the sense strand, position (wherein the value is “1”) or the base is not present otherwise its value is 0; (wherein the value is “0”). Because position 19 is optional, 0287 C7-1 if C is the base at position 17 of the sense i.e., there might be only 18 base pairs, when there are only 18 strand, otherwise its value is 0; base pairs, any base with a subscript of 19 in the formulas 0288 C1 if C is the base at position 18 of the sense above would have a Zero value for that parameter. Before or strand, otherwise its value is 0; after each variable is a number followed by *, which indicates 0289 Co-1 if C is the base at position 19 of the sense that the value of the variable is to be multiplied or weighed by Strand, otherwise if another base is present or the sense Strand that number. is only 18 base pairs in length, its value is 0; 0313 The numbers preceding the variables A, or G, or C, 0290 G=1 if G is the base at position 1 on the sense or U in Formulas VIII, IX, and X (or after the variables in Formula I-VII) were determined by comparing the difference strand, otherwise its value is 0; in the frequency of individual bases at different positions in 0291 G=1 if G is the base at position 2 of the sense strand, functional siRNA and total siRNA. Specifically, the fre otherwise its value is 0; quency in which a given base was observed at a particular 0292 Gs-1 if G is the base at position 8 on the sense position in functional groups was compared with the fre strand, otherwise its value is 0; quency that that same base was observed in the total, ran 0293 Go-1 if G is the base at position 10 on the sense domly selected siRNA set. If the absolute value of the differ strand, otherwise its value is 0; ence between the functional and total values was found to be 0294 G=1 if G is the base at position 13 on the sense greater than 6%, that parameter was included in the equation. strand, otherwise its value is 0; Thus, for instance, if the frequency of finding a “G” at posi 0295 G-1 if G is the base at position 19 of the sense tion 13 (G) is found to be 6% in a given functional group, Strand, otherwise if another base is present or the sense Strand and the frequency of G in the total population of siRNAs is is only 18 base pairs in length, its value is 0; 20%, the difference between the two values is 6%-20%=- 0296 U=1 if U is the base at position 1 on the sense 14%. As the absolute value is greater than six (6), this factor strand, otherwise its value is 0; (-14) is included in the equation. Thus, in Formula VIII, in 0297 U-1 if U is the base at position 2 on the sense cases where the siRNA under study has a G in position 13, the strand, otherwise its value is 0; accrued value is (-14)*(1)=–14. In contrast, when a base 0298 U-1 if U is the base at position 3 on the sense other than G is found at position 13, the accrued value is strand, otherwise its value is 0; (-14)*(0)=0. 0299 U =1 if U is the base at position 4 on the sense 0314. When developing a means to optimize siRNAs, the strand, otherwise its value is 0; inventors observed that a bias toward low internal thermody 0300 U7–1 if U is the base at position 7 on the sense namic stability of the duplex at the 5'-antisense (AS) end is strand, otherwise its value is 0; characteristic of naturally occurring miRNA precursors. The 0301 U-1 if U is the base at position 9 on the sense inventors extended this observation to siRNAs for which strand, otherwise its value is 0; functionality had been assessed in tissue culture. US 2009/0088,563 A1 Apr. 2, 2009

0315. With respect to the parameter GCs, a value of evant calculations using one of the above-described formulas 0-5 will be ascribed depending on the number of G or C bases on these regions. These calculations can be done manually or at positions 15 to 19. If there are only 18 base pairs, the value with the aid of a computer. is between 0 and 4. 0320. As with Formulas I-VII, either Formula VIII, For 0316. With respect to the criterion GC content, a num mula IX, or Formula X may be used for a given mRNA target ber from 0-30 will be ascribed, which correlates to the total sequence. However, it is possible that according to one or the number of G and C nucleotides on the sense strand, excluding otherformula more than one siRNA will have the same value. overhangs. Without wishing to be bound by any one theory, it Accordingly, it is beneficial to have a second formula by is postulated that the significance of the GC content (as well which to differentiate sequences. Formulas IX and X were as AU content at positions 15-19, which is a parameter for derived in a similar fashion as Formula VIII, yet used a larger formulas III-VII) relates to the easement of the unwinding of data set and thus yields sequences with higher statistical a double-stranded siRNA duplex. Duplex unwinding is correlations to highly functional duplexes. The sequence that believed to be crucial for siRNA functionality in vivo and has the highest value ascribed to it may be referred to as a overall low internal stability, especially low internal stability “first optimized duplex. The sequence that has the second of the first unwound base pair is believed to be important to highest value ascribed to it may be referred to as a “second maintain sufficient processivity of RISC complex-induced optimized duplex. Similarly, the sequences that have the duplex unwinding. If the duplex has 19 base pairs, those at third and fourth highest values ascribed to them may be positions 15-19 on the sense strand will unwind first if the referred to as a third optimized duplex and a fourth optimized molecule exhibits a sufficiently low internal stability at that duplex, respectively. When more than one sequence has the position. As persons skilled in the art are aware, RISC is a same value, each of them may, for example, be referred to as complex of approximately twelve proteins; Dicer is one, but first optimized duplex sequences or co-first optimized 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. 0321. It should also be noted that the output of a particular 0317. 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 0318. 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 0319 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 0322 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 0323. 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 0324 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 US 2009/0088,563 A1 Apr. 2, 2009 together in a pool or kit. Further, they may be applied to a cell simultaneously or separately. Preferably, the at least two siR - Continued NAs are applied simultaneously. Pools are particularly ben eficial for many research applications. However, for thera tgtaat Caag gaCtt catga t cc agggcgg agact tcacc peutics, it may be more desirable to employ a single aggggagatg gcacaggagg aaagagcatc tacggtgagc hyperfunctional siRNA as described elsewhere in this appli cation. gct tcc.ccga tigaga acttic aaactgaagc act acgggcc 0325 When planning to conduct gene silencing, and it is tggctggg necessary to choose between two or more siRNAs, one should do so by comparing the relative values when the siRNA are Firefly luciferase: 1434-1631, U47298 Subjected to one of the formulas above. In general a higher (p.GL3, Promega) scored siRNA should be used. SEO. ID NO. 3O: 0326 Useful applications include, but are not limited to, tgaact tccc gcc.gc.cgttgttgttittgga gcacggaaag target validation, gene functional analysis, research and drug acgatgacgg aaaaagagat cqtggattac gtc.gc.cagt c discovery, gene therapy and therapeutics. Methods for using siRNA in these applications are well known to persons of skill aagtaacaac cycgaaaaag ttgcgcggag gagttgttgtt in the art. tgtggacgaa gtaccgaaag gtc.ttaccgg aaaacticgac 0327 Because the ability of siRNA to function is depen dent on the sequence of the RNA and not the species into gcaagaaaaa toaga.gagat cct cataaag gccaagaagg which it is introduced, the present invention is applicable DBI, NM O2O548 (202-310) (every position) across abroad range of species, including but not limited to all SEO. ID NO. OO31: mammalian species, such as humans, dogs, horses, cats, acgggcaagg C caagtggga tigcctggaat gagctgaaag cows, mice, hamsters, chimpanzees and gorillas, as well as other species and organisms such as bacteria, viruses, insects, ggactitccaa ggaagatgcc atgaaagctt acatcaacaa plants and C. elegans. agtagaagag ctaaagaaaa aatacggg 0328. The present invention is also applicable for use for silencing a broad range of genes, including but not limited to 0332 A list of the siRNAs appears in Table III (see the roughly 45,000 genes of a human genome, and has par Examples Section, Example II) ticular relevance in cases where those genes are associated 0333. The set of duplexes was analyzed to identify corre with diseases such as diabetes, Alzheimer's, cancer, as well as lations between siRNA functionality and other biophysical or all genes in the genomes of the aforementioned organisms. thermodynamic properties. When the siRNA panel was ana 0329. The siRNA selected according to the aforemen lyzed in functional and non-functional Subgroups, certain tioned criteria or one of the aforementioned algorithms are nucleotides were much more abundant at certain positions in also, for example, useful in the simultaneous screening and functional or non-functional groups. More specifically, the functional analysis of multiple genes and gene families using frequency of each nucleotide at each position in highly func high throughput strategies, as well as in direct gene Suppres tional siRNA duplexes was compared with that of nonfunc sion or silencing. tional duplexes in order to assess the preference for or against any given nucleotide at every position. These analyses were Development of the Algorithms used to determine important criteria to be included in the 0330. To identify siRNA sequence features that promote siRNA algorithms (Formulas VIII, IX, and X). functionality and to quantify the importance of certain cur 0334. The data set was also analyzed for distinguishing rently accepted conventional factors—such as G/C content biophysical properties of siRNAs in the functional group, and target site accessibility—the inventors synthesized an Such as optimal percent of GC content, propensity for internal siRNA panel consisting of 270 siRNAs targeting three genes, structures and regional thermodynamic stability. Of the pre Human Cyclophilin, Firefly Luciferase, and Human DBI. In sented criteria, several are involved in duplex recognition, all three cases, siRNAs were directed against specific regions RISC activation/duplex unwinding, and target cleavage of each gene. For Human Cyclophilin and Firefly Luciferase, catalysis. ninety siRNAs were directed against a 199 bp segment of 0335 The original data set that was the source of the each respective mRNA. For DBI, 90 siRNAs were directed statistically derived criteria is shown in FIG. 2. Additionally, against a smaller, 109 base pair region of the mRNA. The this figure shows that random selection yields siRNA sequences to which the siRNAs were directed are provided duplexes with unpredictable and widely varying silencing below. potencies as measured in tissue culture using HEK293 cells. 0331. It should be noted that in certain sequences, “t is In the figure, duplexes are plotted Such that each X-axis tick present. This is because many databases contain information mark represents an individual siRNA, with each subsequent in this manner. However, the t denotes a uracil residue in siRNA differing in target position by two nucleotides for mRNA and siRNA. Any algorithm will, unless otherwise Human Cyclophilin B and Firefly Luciferase, and by one specified, process at in a sequence as a u. nucleotide for Human DBI. Furthermore, the y-axis denotes the level of target expression remaining after transfection of the duplex into cells and Subsequent silencing of the target. Human cyclophilin: 193-390, M60857 0336 siRNA identified and optimized in this document SEO. ID NO. 29: work equally well in a wide range of cell types. FIG.3a shows gttccaaaaa cagtggataa ttttgtggcc ttagctacag the evaluation of thirty siRNAs targeting the DBI gene in gagagaaagg atttggctac aaaaa.ca.gca aattic catcg three cell lines derived from different tissues. Each DBI siRNA displays very similar functionality in HEK293 US 2009/0088,563 A1 Apr. 2, 2009

(ATCC, CRL-1573, human embryonic kidney), HeLa the most potent silencers (no F95 duplex with predicted hair (ATCC, CCL-2, cervical epithelial adenocarcinoma) and pin structure T-60° C.). In contrast, about 60% of the DU145 (HTB-81, prostate) cells as determined by the duplexes in the groups having internal hairpins with calcu B-DNA assay. Thus, siRNA functionality is determined by lated T values less than 20°C. were F80. Thus, the stability the primary sequence of the siRNA and not by the intracel of internal repeats is inversely proportional to the silencing lular environment. Additionally, it should be noted that effect and defines Criterion III (predicted hairpin structure although the present invention provides fora determination of the functionality of siRNA for a given target, the same siRNA Ts20° C.). may silence more than one gene. For example, the comple Sequence-Based Determinants of siRNA Functionality mentary sequence of the silencing siRNA may be present in 0340 When the siRNA panel was sorted into functional more than one gene. Accordingly, in these circumstances, it and non-functional groups, the frequency of a specific nucle may be desirable not to use the siRNA with highest SMART otide at each position in a functional siRNA duplex was SCORETM, or siRNA ranking. In such circumstances, it may compared with that of a nonfunctional duplex in order to be desirable to use the siRNA with the next highest SMART assess the preference for or against a certain nucleotide. FIG. SCORETM, or siRNA ranking. 4 shows the results of these queries and the Subsequent resort 0337 To determine the relevance of G/C content in siRNA ing of the data set (from FIG.2). The data is separated into two function, the G/C content of each duplex in the panel was sets: those duplexes that meet the criteria, a specific nucle calculated and the functional classes of siRNAs (57% GC content) compared to the 36%-52% group. The ent duplex). Statistical analysis revealed correlations between group with extremely low GC content (26% or less) contained silencing and several sequence-related properties of siRNAS. a higher proportion of non-functional siRNAS and no highly FIG. 4 and Table IV show quantitative analysis for the fol functional siRNAs. The G/C content range of 30%-52% was lowing five sequence-related properties of siRNA: (A) an A at therefore selected as Criterion I for siRNA functionality, con position 19 of the sense strand; (B) an A at position 3 of the sistent with the observation that a G/C range 30%-70% pro sense strand; (C) a U at position 10 of the sense strand; (D) a motes efficient RNAi targeting. Application of this criterion base other than Gat position 13 of the sense strand; and (E) a alone provided only a marginal increase in the probability of base other than Cat position 19 of the sense strand. selecting functional siRNAs from the panel: selection of F50 (0341 When the siRNAs in the panel were evaluated for and F95 siRNAs was improved by 3.6% and 2.2%, respec the presence of an A at position 19 of the sense strand, the tively. The siRNA panel presented here permitted a more percentage of non-functional duplexes decreased from 20% systematic analysis and quantification of the importance of to 11.8%, and the percentage of F95 duplexes increased from this criterion than that used previously. 21.7% to 29.4% (Table IV). Thus, the presence of an A in this 0338 A relative measure of local internal stability is the position defined Criterion IV. A/U base pair (bp) content; therefore, the frequency of A/Ubp 0342 Another sequence-related property correlated with was determined for each of the five terminal positions of the silencing was the presence of an A in position 3 of the sense duplex (5' sense (S)/5' antisense (AS)) of all siRNAs in the strand (FIG. 4b). Of the siRNAs with A3, 34.4% were F95, panel. Duplexes were then categorized by the number of A/U compared with 21.7% randomly selected siRNAs. The pres bp in positions 1-5 and 15-19 of the sense strand. The ther ence of a Ubase in position 10 of the sense strand exhibited an modynamic flexibility of the duplex 5'-end (positions 1-5; S) even greater impact (FIG. 4c). Of the duplexes in this group, did not appear to correlate appreciably with silencing 41.7% were F95. These properties became criteria V and VI, potency, while that of the 3'-end (positions 15-19; S) corre respectively. lated with efficient silencing. No duplexes lacking A/Ubp in 0343. Two negative sequence-related criteria that were positions 15-19 were functional. The presence of one A/Ubp identified also appear on FIG. 4. The absence of a G at in this region conferred some degree of functionality, but the position 13 of the sense Strand, conferred a marginal increase presence of three or more A/Us was preferable and therefore in selecting functional duplexes (FIG. 4d). Similarly, lack of defined as Criterion II. When applied to the test panel, only a a C at position 19 of the sense strand also correlated with marginal increase in the probability of functional siRNA functionality (FIG. 4e). Thus, among functional duplexes, selection was achieved: a 1.8% and 2.3% increase for F50 and position 19 was most likely occupied by A, and rarely occu F95 duplexes, respectively (Table IV). pied by C. These rules were defined as criteria VII and VIII, 0339. The complementary strands of siRNAs that contain respectively. internal repeats or palindromes may form internal fold-back 0344) Application of each criterion individually provided structures. These hairpin-like structures exist in equilibrium marginal but statistically significant increases in the probabil with the duplexed form effectively reducing the concentra ity of selecting a potent siRNA. Although the results were tion of functional duplexes. The propensity to form internal informative, the inventors sought to maximize potency and hairpins and their relative stability can be estimated by pre therefore consider multiple criteria or parameters. Optimiza dicted melting temperatures. High Tm reflects a tendency to tion is particularly important when developing therapeutics. form hairpin structures. Lower Tm values indicate a lesser Interestingly, the probability of selecting a functional siRNA tendency to form hairpins. When the functional classes of based on each thermodynamic criteria was 2%-4% higher siRNAs were sorted by T., (FIG. 3c), the following trends than random, but 4%-8% higher for the sequence-related were identified: duplexes lacking stable internal repeats were determinates. Presumably, these sequence-related increases US 2009/0088,563 A1 Apr. 2, 2009

reflect the complexity of the RNAi mechanism and the mul 0348. The methods for obtaining the seven criteria embod titude of protein-RNA interactions that are involved in RNAi ied in Table IV are illustrative of the results of the process mediated silencing. used to develop the information for Formulas VIII, IX, and X. Thus similar techniques were used to establish the other vari TABLE IV ables and their multipliers. As described above, basic statis tical methods were use to determine the relative values for IMPROVEMENT these multipliers. PERCENT OVER 0349 To determine the value for “Improvement over Ran CRITERION FUNCTIONAL RANDOM (%) dom” the difference in the frequency of a given attribute (e.g., I. 30%-52% GC Content FSO 6.4 -3.6 GC content, base preference) at a particular position is deter FSO 83.6 3.6 F8O 60.4 4.3 mined between individual functional groups (e.g. F95 group contained a “U” at this F95 29.4 7.7 V. An Abase at FSO 7.2 -2.8 position 41.7% of the time. In contrast, the total group of 270 position 3 of FSO 82.8 2.8 siRNAs had a “U” at this position 21.7% of the time, thus the the sense strand F8O 62.5 6.4 improvement over random is calculated to be 20% (or 41.7%- F95 34.4 12.7 21.7%). VI. AU base at FSO 3.9 -6.1 Identifying the Average Internal Stability Profile of Strong position 10 of FSO 86.1 6.1 the sense strand F8O 69.4 13.3 siRNA F95 41.7 2O 0350. In order to identify an internal stability profile that is VII. A base other than FSO 8.8 -1.2 characteristic of strong siRNA, 270 different siRNAs derived C at position 19 FSO 81.2 1.2 of the sense strand F8O 59.7 3.6 from the cyclophilin B, the diazepam binding inhibitor (DBI), F95 24.2 2.5 and the luciferase gene were individually transfected into VIII. A base other than FSO 5.2 -4.8 HEK293 cells and tested for their ability to induce RNAi of Gat position 13 FSO 84.8 4.8 the respective gene. Based on their performance in the in vivo of the sense strand F8O 61.4 5.3 assay, the sequences were then Subdivided into three groups, F95 26.5 4.8 (i) >95% silencing; (ii) 80-95% silencing; and (iii) less than 50% silencing. Sequences exhibiting 51-84% silencing were The siRNA Selection Algorithm eliminated from further consideration to reduce the difficul 0345. In an effort to improve selection further, all identi ties in identifying relevant thermodynamic patterns. fied criteria, including but not limited to those listed in Table 0351. Following the division of siRNA into three groups, IV were combined into the algorithms embodied in Formula a statistical analysis was performed on each member of each VIII, Formula IX, and Formula X. Each siRNA was then group to determine the average internal stability profile assigned a score (referred to as a SMARTSCORETM, or (AISP) of the siRNA. To accomplish this the Oligo 5.0 Primer siRNA ranking) according to the values derived from the Analysis Software and other related Statistical packages (e.g., formulas. Duplexes that scored higher than 0 or -20 (unad Excel) were exploited to determine the internal stability of justed), for Formulas VIII and IX, respectively, effectively pentamers using the nearest neighbor method described by selected a set of functional siRNAs and excluded all non Freier et al., (1986) Improved free-energy parameters for functional siRNAs. Conversely, all duplexes scoring lower predictions of RNA duplex stability, Proc Natl. Acad. Sci. than 0 and -20 (minus 20) according to formulas VIII and IX, USA 83 (24): 9373-7. Values for each group at each position respectively, contained some functional siRNAs but included were then averaged, and the resulting data were graphed on a all non-functional siRNAs. A graphical representation of this linear coordinate system with the Y-axis expressing the AG selection is shown in FIG.5. It should be noted that the scores (free energy) values in kcal/mole and the X-axis identifying derived from the algorithm can also be provided as “adjusted the position of the base relative to the 5' end. scores. To convert Formula VIII unadjusted scores into 0352. The results of the analysis identified multiple key adjusted scores it is necessary to use the following equation: regions in siRNA molecules that were critical for successful gene silencing. At the 3'-most end of the sense Strand (5'anti (160+unadjusted score)/2.25 sense), highly functional siRNA (>95% gene silencing, see 0346 When this takes place, an unadjusted score of “O'” FIG. 6a, >F95) have a low internal stability (AISP of position (Zero) is converted to 75. Similarly, unadjusted scores for 19=~-7.6 kcal/mol). In contrast low-efficiency siRNA (i.e., Formula X can be converted to adjusted scores. In this those exhibiting less than 50% silencing,

mately -8.1 kcal/mol for the 5' terminus. siRNA with poor can be defined as being those molecules that induce 20%, silencing capabilities show a distinctly different profile. 30%, 40%, 50%, 60%, or 70% silencing at 100 nM transfec While the AISP value at position 12 is nearly identical with tion conditions. Similarly, non-functional siRNA can be that of strong siRNAs, the values at positions 7 and 8 rise defined as being those molecules that silence gene expression considerably, peaking at a high of ~-9.0 kcal/mol. In addi by less than 70%, 60%, 50%, 40%, 30%, or less. Nonetheless, tion, at the 5' end of the molecule the AISP profile of strong unless otherwise stated, the descriptions stated in the “Defi and weak siRNA differ dramatically. Unlike the relatively nitions' section of this text should be applied. strong values exhibited by siRNA in the >95% silencing 0355 Functional attributes can be assigned to each of the group, siRNAS that exhibit poor silencing activity have weak key positions in the AISP of strong siRNA. The low 5' (sense AISP values (-7.6, -7.5, and -7.5 kcal/mol for positions 1, 2 strand) AISP values of strong siRNAs may be necessary for and 3 respectively). determining which end of the molecule enters the RISC com 0353. Overall the profiles of both strong and weak siRNAs plex. In contrast, the high and low AISP values observed in form distinct sinusoidal shapes that are roughly 180° out-of the central regions of the molecule may be critical for siRNA phase with each other. While these thermodynamic descrip target mRNA interactions and product release, respectively. tions define the archetypal profile of a strong siRNA, it will 0356. If the AISP values described above accurately define likely be the case that neither the AG values given for key the thermodynamic parameters of strong siRNA, it would be positions in the profile or the absolute position of the profile expected that similar patterns would be observed in strong along the Y-axis (i.e., the AG-axis) are absolutes. Profiles that siRNA isolated from nature. Natural siRNAs existina harsh, are shifted upward or downward (i.e., having on an average, RNase-rich environment and it can be hypothesized that only higher or lower values at every position) but retain the relative those siRNA that exhibit heightened affinity for RISC (i.e., shape and position of the profile along the X-axis can be siRNA that exhibit an average internal stability profile similar foreseen as being equally effective as the model profile to those observed in strong siRNA) would survive in an intra described here. Moreover, it is likely that siRNA that have cellular environment. This hypothesis was tested using GFP strong or even stronger gene-specific silencing effects might specific siRNA isolated from N. benthamiana. Llave et al. have exaggerated AG values (either higher or lower) at key (2002) Endogenous and Silencing-Associated Small RNAs positions. Thus, for instance, it is possible that the 5'-most in Plants. The Plant Cell 14, 1605-1619, introduced long position of the sense strand (position 19) could have AG double-stranded GFP-encoding RNA into plants and subse values of 7.4 kcal/mol or lower and still be a strong siRNA if, quently re-isolated GFP-specific siRNA from the tissues. The for instance, a G-CYG-T/U mismatch were substituted at AISP of fifty-nine of these GFP-siRNA were determined, position 19 and altered duplex stability. Similarly, position 12 averaged, and Subsequently plotted alongside the AISP pro and position 7 could have values above 8.3 kcal/mol and file obtained from the cyclophilin B/DBI/luciferase siRNA below 7.7 kcal/mole, respectively, without abating the silenc having >90% silencing properties (FIG. 6b). Comparison of ing effectiveness of the molecule. Thus, for instance, at posi the two groups show that profiles are nearly identical. This tion 12, a stabilizing chemical modification (e.g., a chemical finding validates the information provided by the internal modification of the 2' position of the sugar backbone) could stability profiles and demonstrates that: (1) the profile iden be added that increases the average internal stability at that tified by analysis of the cyclophilin B/DBI/luciferase siRNAs position. Similarly, at position 7, mismatches similar to those are not gene specific; and (2) AISP values can be used to described previously could be introduced that would lower search for strong siRNAS in a variety of species. the AG values at that position. 0357 Both chemical modifications and base-pair mis 0354 Lastly, it is important to note that while functional matches can be incorporated into siRNA to alter the duplex's and non-functional siRNA were originally defined as those AISP and functionality. For instance, introduction of mis molecules having specific silencing properties, both broader matches at positions 1 or 2 of the sense strand destabilized the or more limiting parameters can be used to define these mol 5' end of the sense strand and increases the functionality of the ecules. As used herein, unless otherwise specified, “non molecule (see Luc, FIG. 7). Similarly, addition of 2'-O-me functional siRNA are defined as those siRNA that induce thyl groups to positions 1 and 2 of the sense strand can also less than 50% (<50%) target silencing, “semi-functional alter the AISP and (as a result) increase both the functionality siRNA’ induce 50-79% target silencing, “functional siRNA'. of the molecule and eliminate off-target effects that results are molecules that induce 80-95% gene silencing, and from sense strand homology with the unrelated targets (FIG. “highly-functional siRNA are molecules that induce great 8). than 95% gene silencing. These definitions are not intended to be rigid and can vary depending upon the design and needs of Rationale for Criteria in a Biological Context the application. For instance, it is possible that a researcher 0358. The fate of siRNA in the RNAi pathway may be attempting to map a gene to a chromosome using a functional described in 5 major steps: (1) duplex recognition and pre assay, may identify an siRNA that reduces gene activity by RISC complex formation; (2) ATP-dependent duplex only 30%. While this level of gene silencing may be “non unwinding/strand selection and RISC activation; (3) mRNA functional for, e.g., therapeutic needs, it is Sufficient for gene target identification; (4) mRNA cleavage, and (5) product mapping purposes and is, under these uses and conditions, release (FIG. 1). Given the level of nucleic acid-protein inter “functional.” For these reasons, functional siRNA can be actions at each step, siRNA functionality is likely influenced defined as those molecules having greater than 10%. 20%, by specific biophysical and molecular properties that promote 30%, 40%, 50%, 60%, 70%, 80%, or 90% silencing capabili efficient interactions within the context of the multi-compo ties at 100 nM transfection conditions. Similarly, depending nent complexes. Indeed, the systematic analysis of the siRNA upon the needs of the study and/or application, non-func test set identified multiple factors that correlate well with tional and semi-functional siRNA can be defined as having functionality. When combined into a single algorithm, they different parameters. For instance, semi-functional siRNA proved to be very effective in selecting active siRNAs. US 2009/0088,563 A1 Apr. 2, 2009 20

0359. The factors described here may also be predictive of identified by any of the formulas can be filtered to remove any key functional associations important for each step in RNAi. and all sequences that induce toxicity or cellular stress. Intro For example, the potential formation of internal hairpin struc duction of an siRNA containing a toxic motif into a cell can 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 0360 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 0364. 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 0361. 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 0365. 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 0362 Among the criteria associated with base preferences 0366 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. 0367 By increasing the number of siRNAs directed to a Post Algorithm Filters particular target using a pool or kit, one is able both to 0363 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 US 2009/0088,563 A1 Apr. 2, 2009

levels of functionality alone, if combined, they may satisfac calculation of the number of base pairs. The two nucleotide 3' torily promote degradation of the target messenger RNA and overhangs mimic natural siRNAS and are commonly used but successfully inhibit translation. By including multiple siR are not essential. Preferably, the overhangs should consist of 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. NAR 31: 2401-21407). proprietary ACER) technology. However, other methods for 0368. 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. 0369 More preferably, the kit is comprised ofat least three siRNAs, wherein one strand of each siRNA comprises a 0373 The siRNA duplexes within the aforementioned sequence that is Substantially similar to a sequence of the pools of siRNAS may correspond to overlapping sequences target mRNA and the other Strand comprises a sequence that within a particular mRNA, or non-overlapping sequences of is substantially complementary to the region of the target the mRNA. However, preferably they correspond to non mRNA. As with the kit that comprises at least two siRNAs, overlapping sequences. Further, each siRNA may be selected more preferably one strand will comprise a sequence that is randomly, or one or more of the siRNA may be selected identical to a sequence that is contained in the mRNA and according to the criteria discussed above for maximizing the another strand that is 100% complementary to a sequence that effectiveness of siRNA. is contained in the mRNA. During experiments, when three 0374. Included in the definition of siRNAs are siRNAs siRNAs were combined together, 60% of the pools induced that contain substituted and/or labeled nucleotides that may, more than 95% gene silencing and 92% of the pools induced for example, be labeled by radioactivity, fluorescence or more than 80% gene silencing. mass. The most common Substitutions are at the 2' position of 0370 Further, even more preferably, the kit is comprised the ribose Sugar, where moieties such as H (hydrogen) F. NH of at least four siRNAs, wherein one strand of each siRNA OCH and other O-alkyl, alkenyl, alkynyl, and orthoesters, comprises a sequence that is Substantially similar to a region may be substituted, or in the phosphorous backbone, where of the sequence of the target mRNA, and the other strand sulfur, amines or hydrocarbons may be substituted for the comprises a sequence that is Substantially complementary to bridging of non-bridging atoms in the phosphodiester bond. the region of the target mRNA. As with the kit or pool that Examples of modified siRNAs are explained more fully in comprises at least two siRNAs, more preferably one strand of commonly assigned U.S. patent application Ser. No. 10/613, each of the siRNA duplexes will comprise a sequence that is 077, filed Jul. 1, 2003. identical to a sequence that is contained in the mRNA, and 0375 Additionally, as noted above, the cell type into another strand that is 100% complementary to a sequence that which the siRNA is introduced may affect the ability of the is contained in the mRNA. siRNA to enter the cell; however, it does not appear to affect 0371. Additionally, kits and pools with at least five, at least the ability of the siRNA to function once it enters the cell. six, and at least seven siRNAs may also be useful with the Methods for introducing double-stranded RNA into various present invention. For example, pools of five siRNA induced cell types are well known to persons skilled in the art. 95% gene silencing with 77% probability and 80% silencing 0376. As persons skilled in the art are aware, in certain with 98.8% probability. Thus, pooling of siRNAs together species, the presence of proteins such as RdRP, the RNA can result in the creation of a target-specific silencing reagent dependent RNA polymerase, may catalytically enhance the with almost a 99% probability of being functional. The fact activity of the siRNA. For example, RdRP propagates the that such high levels of Success are achievable using Such RNAi effect in C. elegans and other non-mammalian organ pools of siRNA, enables one to dispense with costly and isms. In fact, in organisms that contain these proteins, the time-consuming target-specific validation procedures. siRNA may be inherited. Two other proteins that are well 0372 For this embodiment, as well as the other aforemen studied and known to be a part of the machinery are members tioned embodiments, each of the siRNAs within a pool will of the Argonaute family and Dicer, as well as their homo preferably comprise 18-30 base pairs, more preferably 18-25 logues. There is also initial evidence that the RISC complex base pairs, and most preferably 19 base pairs. Within each might be associated with the ribosome so the more efficiently siRNA, preferably at least 18 contiguous bases of the anti translated mRNAs will be more susceptible to silencing than sense strand will be 100% complementary to the target others. mRNA. More preferably, at least 19 contiguous bases of the 0377 Another very important factor in the efficacy of antisense strand will be 100% complementary to the target siRNA is mRNA localization. In general, only cytoplasmic mRNA. Additionally, there may be overhangs on either the mRNAs are considered to be accessible to RNAi to any appre sense Strand or the antisense Strand, and these overhangs may ciable degree. However, appropriately designed siRNAs, for beat either the 5' end or the 3' end of either of the strands, for example, siRNAs modified with internucleotide linkages or example there may be one or more overhangs of 1-6 bases. 2'-O-methyl groups, may be able to cause silencing by acting When overhangs are present, they are not included in the in the nucleus. Examples of these types of modifications are US 2009/0088,563 A1 Apr. 2, 2009 22 described in commonly assigned U.S. patent application Ser. expression vectors. Any vector that has an adequate siRNA Nos. 10/431,027 and 10/613,077. expression and procession module may be used. Further 0378. As described above, even when one selects at least more, certain chemical modifications to siRNAS, including two siRNAs at random, the effectiveness of the two may be but not limited to conjugations to other molecules, may be 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 0384. 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 0379 Within the kits of the present invention, preferably 0385. In addition to developing kits that contain multiple each siRNA will be present in a concentration of between siRNA directed against a single gene, another embodiment 0.001 and 200 uM, more preferably between 0.01 and 200 includes the use of multiple siRNA targeting multiple genes. nM, and most preferably between 0.1 and 10 nM. Multiple genes may be targeted through the use of high- or 0380. In addition to preferably comprising at least four or hyper-functional siRNA. High- or hyper-functional siRNA five siRNAs, the kits of the present invention will also pref that exhibit increased potency, require lower concentrations erably comprise a buffer to keep the siRNA duplex stable. to induce desired phenotypic (and thus therapeutic) effects. Persons skilled in the art are aware of buffers suitable for This circumvents RISC Saturation. It therefore reasons that if keeping siRNA stable. For example, the buffer may be com lower concentrations of a single siRNA are needed for knock prised of 100 mM KC1, 30 mM HEPES-pH 7.5, and 1 mM out or knockdown expression of one gene, then the remaining MgCl2. Alternatively, kits might contain complementary (uncomplexed) RISC will be free and available to interact Strands that contain any one of a number of chemical modi with siRNA directed against two, three, four, or more, genes. fications (e.g., a 2'-O-ACE) that protect the agents from deg Thus in this embodiment, the authors describe the use of radation by nucleases. In this instance, the user may (or may highly functional or hyper-functional siRNA to knock out not) remove the modifying protective group (e.g., deprotect) three separate genes. More preferably, Such reagents could be before annealing the two complementary Strands together. combined to knockout four distinct genes. Even more prefer 0381. By way of example, the kits may be organized such ably, highly functional or hyperfunctional siRNA could be that pools of siRNA duplexes are provided on an array or used to knockout five distinct genes. Most preferably, siRNA microarray of wells or drops for a particular gene set or for of this type could be used to knockout or knockdown the unrelated genes. The array may, for example, be in 96 wells, expression of six or more genes. 384 wells or 1284 wells arrayed in a plastic plate or on a glass Hyperfunctional siRNA slide using techniques now known or that come to be known (0386 The term hyperfunctional siRNA (hf-siRNA) to persons skilled in the art. Within an array, preferably there describes a subset of the siRNA population that induces RNAi will be controls such as functional anti-lamin A/C, cyclophi in cells at low- or Sub-nanomolar concentrations for extended lin and two siRNA duplexes that are not specific to the gene of periods of time. These traits, heightened potency and interest. extended longevity of the RNAi phenotype, are highly attrac 0382. In order to ensure stability of the siRNA pools prior tive from a therapeutic standpoint. Agents having higher to usage, they may be retained in lyophilized form at minus potency require lesser amounts of the molecule to achieve the twenty degrees (-20°C.) until they are ready for use. Prior to desired physiological response, thus reducing the probability usage, they should be resuspended; however, even once resus of side effects due to “off-target' interference. In addition to pended, for example, in the aforementioned buffer, they the potential therapeutic benefits associated with hyperfunc should be kept at minus twenty degrees, (-20°C.) until used. tional siRNA, hf-siRNA are also desirable from an economic The aforementioned buffer, prior to use, may be stored at perspective. Hyperfunctional siRNA may cost less on a per approximately 4°C. or room temperature. Effective tempera treatment basis, thus reducing overall expenditures to both tures at which to conduct transfections are well known to the manufacturer and the consumer. persons skilled in the art and include for example, room 0387 Identification of hyperfunctional siRNA involves temperature. multiple steps that are designed to examine an individual 0383. The kits may be applied either in vivo or in vitro. siRNA agent's concentration- and/or longevity-profiles. In Preferably, the siRNA of the pools or kits is applied to a cell one non-limiting example, a population of siRNA directed through transfection, employing standard transfection proto against a single gene are first analyzed using the previously cols. These methods are well known to persons skilled in the described algorithm (Formula VIII). Individual siRNA are art and include the use of lipid-based carriers, electropora then introduced into a test cell line and assessed for the ability tion, cationic carriers, and microinjection. Further, one could to degrade the target mRNA. It is important to note that when apply the present invention by synthesizing equivalent DNA performing this step it is not necessary to testall of the siRNA. sequences (either as two separate, complementary strands, or Instead, it is sufficient to test only those siRNA having the as hairpin molecules) instead of siRNA sequences and intro highest SMARTSCORESTM, or siRNA ranking (i.e., ducing them into cells through vectors. Once in the cells, the SMARTSCORESTM, or siRNA ranking >-10). Subse cloned DNA could be transcribed, thereby forcing the cells to quently, the gene silencing data is plotted against the generate the siRNA. Examples of vectors suitable for use with SMARTSCORESTM, or siRNA rankings (see FIG.9), siRNA the present application include but are not limited to the that (1) induce a high degree of gene silencing (i.e., they standard transient expression vectors, adenoviruses, retrovi induce greater than 80% gene knockdown) and (2) have Supe ruses, lentivirus-based vectors, as well as other traditional rior SMARTSCORESTM (i.e., a SMARTSCORETM, or US 2009/0088,563 A1 Apr. 2, 2009

siRNA ranking, of>-10, Suggesting a desirable average inter determine would be useful in connection with the present nal stability profile) are selected for further investigations invention in enabling siRNA to cross the cellular membrane. 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 0391 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 0392 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 0388 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 0389. 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% silencing for a study, all sub-F50 siRNAs were considered non-functional. period of 10 days. Yet identical studies of a second gene (gene 0393 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 1 (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 0394 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 0390 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 US 2009/0088,563 A1 Apr. 2, 2009 24 cose. Proc. Natl. Acad. Sci. USA 1997, 94:4360.) are used according to manufacturer instructions. To measure TABLE III - continued luciferase activity, media is removed from HEK293 cells 24 hrs post-transfection, and 50 ul of Steady-GLO reagent (Promega) is added. After 5 minutes, plates are analyzed on a Cyclo 3 O SEQ. ID OO61 ACAAAAACAGCAAAUUCCA plate reader. Cyclo 31 SEQ. ID OO62 AAAAACAGCAAAUUCCAUC Example I Cyclo 32 SEQ. ID OO63 AAACACCAAAUUCCAUCGU SEQUENCES USED TO DEVELOPE THE ALGORITHM Cyclo 33 SEQ. ID OO64 ACAGCAAAUUCCAUCGUGU 0395 Anti-Firefly and anti-Cyclophilin siRNAs panels Cyclo 34 SEO. ID OO65 AGCAAAUUCCAUCGUCUAA (FIG.5a, b) sorted according to using Formula VIII predicted Cyclo 35 SEO. ID OO66 CAAAUUCCAUCGUGUAAUC values. All siRNAs scoring more than 0 (formula VIII) and more then 20 (formula IX) are fully functional. All ninety Cyclo 36 SEO. ID OO67 AAUUCCAUCGUGUAAUCAA sequences for each gene (and DBI) appear below in Table III. Cyclo 37 SEO. ID OO68 UUCCAUCGUGUAAUCAAGG

TABLE III Cyclo 38 SEQ. ID OO69 CCAUCGUGUAAUCAAGGAC

Cyclo 1 SEQ. ID OO32 GUUCCAAAAACAGUGGAUA Cyclo 39 SEO. ID OO 70 AUCGUGUAAUCAAGGACUU Cyclo 2 SEQ. ID OO33 UCCAAAAACAGUGGAUAAU Gyclo 4 O SEQ. ID OO71 CGUGUAAUCAAGGACUUCA

Cyclo 3 SEQ. ID OO34 CAAAAACAGUGGAUAAUUU Cyclo 41 SEQ. ID OO72 UGUAAUCAAGGACUUCAUG Cyclo 4 SEQ. ID 0035 AAAACAGUGGAUAAUUUUG Cyclo 42 SEQ. ID OO73 UAAUCAAGGACUUCAUGAU

Cyclo 5 SEQ. ID OO3 6 AACAGUGGAUAAUUUUGUG Cyclo 43 SEO. ID OO74 AUCAAGCACUUCAUGAUCC Cyclo 6 SEO. ID OO37 CAGUGGAUAAUUUUGUGGC Cyclo 44 SEQ. ID OO75 CAAGGACUUCAUGAUCCAG

Cyclo 7 SEO. ID OO38 GUGGAUAAUUUUGUGGCCU Cyclo 45 SEQ. ID OO76 AGGACUUCAUGAUCCAGGG

Cyclo 8 SEO. ID OO39 GGAUAAUUUUGUGGCCUUA Cyclo 46 SEO. ID OO77 GACUUCAUGAUCCAGGGCG

Cyclo 9 SEO. ID OO4. O AUAAUUUUGUGGCCUUAGC Cyclo 47 SEO. ID OO78 CUUCAUGAUCCAGGGCGGA Cyclo O SEO. ID OO41 AAUUUUGUGGCCUUAGCUA Cyclo 48 SEQ. ID OO79 UCAUGAUCCAGGGCGGAGA

Cyclo 1 SEO. ID OO42 UUUUGUGGCCUUAGCUACA Cyclo 49 SEQ. ID OO80 AUGAUCCAGGGCGGAGACU Cyclo 2 SEO. ID OO43 UUGUGGCCUUAGCUACAGG Cyclo 50 SEO. ID OO81 GAUCCAGGGCGGAGACUUC

Cyclo 3 SEQ. ID OO44 GUGGCCUUAGCUACAGGAG Cyclo 51 SEO. ID OO82 UCCAGGGCGGAGACUUCAC Cyclo 4 SEQ. ID OO45 GGCCUUAGCUACAGGAGAG Cyclo 52 SEO. ID 0083 CAGGGCGGAGACUUCACCA

Cyclo 5 SEQ. ID OO46 CCUUAGCUACAGGAGAGAA Cyclo 53 SEO. ID OO84 GGGCGGAGACUUCACCAGG Cyclo 6 SEQ. ID OO47 UUAGCUACAGGAGAGAAAG Cyclo 54 SEO. ID OO85 GCGGAGACUUCACCAGGGG

Cyclo 7 SEQ. ID OO48 AGCUACAGGAGAGAAAGGA Cyclo 55 SEO. ID OO86 GGAGACUUCACCAGGGGAG Cyclo 8 SEQ. ID OO49 CUACAGGAGAGAAAGGAUU Cyclo 56 SEO. ID 0087 AGACUUCACCAGCGGAGAU

Cyclo 9 SEQ. ID OO5 O ACAGGAGAGAAAGGAUUUG Cyclo 57 SEO. ID OO88 ACUUCACCAGGGGAGAUGG Cyclo 2O SEQ. ID OO51 AGGAGAGAAAGGAUUUGGC Cyclo 58 SEO. ID OO89 UUCACCAGGGGAGAUGGCA

Cyclo 21 SEQ. ID OO52 GAGAGAAAGGAUUUGGCUA. Cyclo 59 SEO. ID OO90 CACCAGGGGAGAUGGCACA Cyclo 22 SEQ. ID OO53 GAGAAAGGAUUUGGCUACA Cyclo 6 O SEO. ID OO91 CCAGGGGAGAUGGCACAGG

Cyclo 23 SEQ. ID OO54 GAAAGGAUUUGGCUACAAA Cyclo 61 SEO. ID OO92 AGGGGAGAUGGCACAGGAG

Cyclo 24 SEQ. ID OO55 AAGGAUUUGGCUACAAAAA Cyclo 62 SEO. ID OO93 GGGAGAUGGCACAGGAGGA Cyclo 25 SEQ. ID OO56 GGAUUUGGCUACAAAAACA Cyclo 63 SEO. ID OO94 GAGAUGGCACAGGAGGAAA

Cyclo 26 SEQ. ID OO57 AUUUGGCUACAAAAACAGC Cyclo 64 SEO. ID OO95 GAUGGCACAGGAGGAAAGA Cyclo 27 SEQ. ID OO58 UUGGCUACAAAAACAGCAA Cyclo 65 SEO. ID OO96 UGGCACAGGAGGAAAGAGC

Cyclo 28 SEQ. ID OO59 GGCUACAAAAACAGCAAAU Cyclo 66 SEO. ID OO97 GCACAGGAGGAAAGAGCAU Cyclo 29 SEQ. ID OO6O CUACAAAAACAGCAAAUUC Cyclo 67 SEO. ID OO98 ACAGGAGGAAAGAGCAUCU

US 2009/0088,563 A1 Apr. 2, 2009 27

TABL H III - continued TABLE III - continued

Lull 4 O SEQ. D O251 UUGCGCGGAGGAGUUGUGU Lull 78 SEO. ID O289 UUUGGAGCACGGAAAGACG

Lull 41 SEQ. D O252 AGUUGCGCGGAGGAGUUGU Lull 79 SEO. ID O29O GUUUUGGAGCACGGAAAGA

Lull 42 SEQ. D O253 AAAGUUGCGCGGAGGAGUU Lull 8 O SEO. ID O291 UUGUUUUGGAGCACGGAAA

Lull 43 SEQ. D O254 AAAAAGUUGCGCGGAGGAG Lull 81 SEO. ID O292 UGUUGUUUUGGAGCACGGA

Lull 44 SEQ. D O255 CGAAAAAGUUGCGCGGAGG Lull 82 SEO. ID O293 GUUGUUGUUUUGGAGCACG

Lull 45 SEQ. D O256 CGCGAAAAAGUUGCGCGGA Lull 83 SEO. ID O294 CCGUUGUUGUUUUGGAGCA

Lull 46 SEQ. D O257 ACCGCGAAAAAGUUGCGCG Lull 84 SEO. ID O295 CGCCGUUGUUGUUUUGGAG

Lull 47 SEQ. D O258 CAACCGCGAAAAAGUUGCG Lull 85 SEO. ID O296 GCCGCCGUUGUUGUUUUGG

Lull 48 SEQ. D O259 AACAACCGCGAAAAAGUUG. Lull 86 SEO. ID O297 CCGCCGCCGUUGUUGUUUU

Lull 49 SEQ. D O26 O GUAACAACCGCGAAAAAGU Lull 87 SEO. ID O298 UCCCGCCGCCGUUGUUGUU

Lull SO SEQ. D O261 AAGUAACAACCGCGAAAAA Lull 88 SEO. ID O299 CUUCCCGCCGCCGUUGUUG

Lull 51 SEQ. D O262 UCAAGUAACAACCGCGAAA Lull 89 SEO. ID O3OO AACUUCCCGCCGCCGUUGU

Lull 52 SEQ. D O263 AGUCAAGUAACAACCGCGA Lull 9 O SEO. ID O301 UGAACUUCCCGCCGCCGUU

Lull 53 SEQ. D O264 CCAGUCAAGUAACAACCGC

Lull 54 SEQ. D O265 CGCCAGUCAAGUAACAACC Example II

Lull 55 D O266 GUCGCCAGUCAAGUAACAA VALIDATION OF THE ALGORITHMUSING DBI, SEQ. LUCIFERASE, PLK, EGFR, AND SEAP Lull 56 SEQ. D O267 ACGUCGCCAGUCAAGUAAC 0396 The algorithm (Formula VIII) identified siRNAs for Lull f SEQ. D O268 UUACGUCGCCAGUCAAGUA five genes, human DBI, firefly luciferase (fLuc), renilla luciferase (ruc), human PLK, and human secreted alkaline Lull 58 SEQ. D O269 GAUUACGUCGCCAGUCAAG phosphatase (SEAP). Four individual siRNAs were selected on the basis of their SMARTSCORESTM derived by analysis Lull 59 SEQ. D O27 O UGGAUUACGUCGCCAGUCA of their sequence using Formula VIII (all of the siRNAs Lull 6 O SEQ. D O271 CGUGGAUUACGUCGCCAGU would be selected with Formula IX as well) and analyzed for their ability to silence their targets expression. In addition to Lull 61 SEQ. D O272 AUCGUGGAUUACGUCGCCA the scoring, a BLAST search was conducted for each siRNA. Lull 62 SEQ. D O273 AGAUCCUGGAUUACGUCGC To minimize the potential for off-target silencing effects, only those target sequences with more than three mismatches Lull 63 SEQ. D O274 AGAGAUCGUGGAUUACGUC against un-related sequences were selected. Semizarov, et al. (2003) Specificity of short interfering RNA determined Lull 64 SEQ. D O275 AAAGAGAUCGUGGAUUACG through gene expression signatures, Proc. Natl. Acad. Sci. Lull 65 SEQ. D O276 AAAAAGAGAUCGUGGAUUA. USA, 100:6347. These duplexes were analyzed individually and in pools of 4 and compared with several siRNAs that were Lull 66 SEQ. D O277 GGAAAAAGAGAUCGUGGAU randomly selected. The functionality was measured as a per Lull 67 SEQ. D O278 ACGGAAAAAGAGAUCGUGG centage of targeted gene knockdown as compared to controls. All siRNAs were transfected as described by the methods Lull 68 SEQ. D O279 UGACGGAAAAAGAGAUCGU above at 100 nM concentration into HEK293 using Lipo fectamine 2000. The level of the targeted gene expression was Lull 69 SEQ. GAUGACGGAAAAAGAGAUC evaluated by B-DNA as described above and normalized to Lull 70 SEQ. D O281 ACGAUGACGGAAAAAGAGA the non-specific control. FIG. 10 shows that the siRNAs selected by the algorithm disclosed herein were significantly Lull 71. SEQ. D O282 AGACGAUGACGGAAAAAGA more potent than randomly selected siRNAs. The algorithm Lull 72 SEQ. D O283 AAAGACGAUGACGGAAAAA increased the chances of identifying an F50 siRNA from 48% to 91%, and an F80 siRNA from 13% to 57%. In addition, Lull 73 SEQ. D O284 GGAAAGACGAUGACGGAAA pools of SMART siRNA silence the selected target better than randomly selected pools (see FIG. 10F). Lull 74 SEQ. D O285 ACGGAAAGACGAUGACGGA Example III Lull SEQ. D O286 GCACGGAAAGACGAUGACG VALIDATION OF THE ALGORITHMUSING GENES Lull 76 SEQ. D O287 GAGCACGGAAAGACGAUGA INVOLVED IN CLATHRIN-DEPENDENT ENDOCYTOSIS Lull 77 SEQ. D 0288 UGGAGCACGGAAAGACGAU 0397) Components of clathrin-mediated endocytosis path way are key to modulating intracellular signaling and play US 2009/0088,563 A1 Apr. 2, 2009 28 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 0402. 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, Colo., USA); mono 0398 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 (U45976), 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 0399. 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 0403 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 0400. 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 1xsiRNA Universal buffer (Dharmacon, Inc.) 04.04. 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 04.01 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) 04.05 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 2009/0088,563 A1 Apr. 2, 2009 29

Endocytic-coated Pits, Interactions with Clathrin, and the In Silico Identification of Functional siRNA Impact of Overexpression on Clathrin-mediated Traffic, Mol. 0410. To identify functional and hyperfunctional siRNA Biol. Cell 10:2687). against the Bcl2 gene, the sequence for Bcl-2 was down loaded from the NCBI Unigene database and analyzed using Example IV the Formula VIII algorithm. As a result of these procedures, both the sequence and SMARTSCORESTM, or siRNA rank VALIDATION OF THE ALGORITHMUSINGEG-5, ings of the Bcl2 siRNA were obtained and ranked according GADPH, ATE1, MEK2, MEK1, QB, LAMINA/C, to their functionality. Subsequently, these sequences were C-MYC, HUMANCYCLOPHILIN, AND MOUSE CYCLOPHI BLASTed (database) to insure that the selected sequences LIN were specific and contained minimal overlap with unrelated 0406 A number of genes have been identified as playing genes. The SMARTSCORESTM, or siRNA rankings for the potentially important roles in disease etiology. Expression top 10 Bcl-2 siRNA are identified in FIG. 13. profiles of normal and diseased kidneys has implicated Edg5 in immunoglobulin A neuropathy, a common renal glomeru In Vivo Testing of Bcl-2 SiRNA lar disease. Myc1, MEK1/2 and other related kinases have been associated with one or more cancers, while lamins have 0411 Bcl-2 siRNAs having the top ten SMART been implicated in muscular dystrophy and other diseases. SCORESTM, or siRNA rankings were selected and tested in a For these reasons, siRNA directed against the genes encoding functional assay to determine silencing efficiency. To accom these classes of molecules would be important research and plish this, each of the ten duplexes were synthesized using therapeutic tools. 2'-O-ACE chemistry and transfected at 100 nM concentra 04.07 FIG. 12 illustrates four siRNAs targeting 10 differ tions into cells. Twenty-four hours later assays were per ent genes (Table V for sequence and accession number infor formed on cell extracts to assess the degree of target silencing. mation) that were selected according to the Formula VIII and Controls used in these experiments included mock trans assayed as individuals and pools in HEK293 cells. The level fected cells, and cells that were transfected with a non-spe of siRNA induced silencing was measured using the B-DNA cific siRNA duplex. assay. These studies demonstrated that thirty-six out of the 0412. The results of these experiments are presented forty individual SMART-selected siRNA tested are func below (and in FIG. 14) and show that all ten of the selected tional (90%) and all 10 pools are fully functional. 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 ALGORITHMUSING BCL2 therapeutic environments. 0408. Bcl-2 is a ~25kD, 205-239 amino acid, anti-apop totic protein that contains considerable homology with other siRNA 1 GGGAGAUAGUGAUGAAGUA SEO. ID NO. 3O2 members of the BCL family including BCLX, MCL1, BAX, siRNA 2 GAAGUACAUCCAUUAUAAG SEO. ID NO. 303 BAD, and BIK. The protein exists in at least two forms (Bcl2a, which has a hydrophobic tail for membrane anchor siRNA 3 GUAGCACAACCGGGAGAUA SEO. ID NO. 304 age, and Bcl2b, which lacks the hydrophobic tail) and is predominantly localized to the mitochondrial membrane. siRNA 4 AGAUAGUGAUGAAGUACAU SEO. ID NO. 305 While Bcl2 expression is widely distributed, particular inter siRNA 5 UGAAGACUCUGCUCAGUUU SEO. ID NO. 306 est has focused on the expression of this molecule in Band T cells. Bcl2 expression is down-regulated in normal germinal siRNA 6 GCAUGCGGCCUCUGUUUGA SEO. ID NO. 3 O7 center B cells yet in a high percentage of follicular lympho siRNA 7 UGCGGCCUCUGUUUGAUUU SEO. ID NO. 3O8 mas, Bcl2 expression has been observed to be elevated. Cyto logical studies have identified a common translocation (14: siRNA 8 GAGAUAGUGAUGAAGUACA SEO. ID NO. 309 18)(q32;q32)) amongst a high percentage (>70%) of these lymphomas. This genetic lesion places the Bcl2 gene injux siRNA 9 GGAGAUAGUGAUGAAGUAC SEO. ID NO. 310 taposition to immunoglobulin heavy chain gene (IgEI) encod siRNA 1 O GAAGACUCUGCUCAGUUUG. SEO. ID NO. 311 ing sequences and is believed to enforce inappropriate levels of gene expression, and resistance to programmed cell death 0413 Bel2 siRNA: Sense Strand, 5'-->3' in the follicle center B cells. In other cases, hypomethylation of the Bcl2 promoter leads to enhanced expression and again, Example VI inhibition of apoptosis. In addition to cancer, dysregulated expression of Bcl-2 has been correlated with multiple sclero SEQUENCES SELECTED BY THE ALGORITHM sis and various neurological diseases. 04.09. The correlation between Bcl-2 translocation and 0414 Sequences of the siRNAs selected using Formulas cancer makes this gene an attractive target for RNAi. Identi (Algorithms) VIII and IX with their corresponding ranking, fication of siRNA directed against the bc12 transcript (or which have been evaluated for the silencing activity in vivo in Bcl2-Igh fusions) would further our understanding Bcl2 the present study (Formula VIII and IX, respectively) are gene function and possibly provide a future therapeutic agent shown in Table V. It should be noted that the “t' residues in to battle diseases that result from altered expression or func Table V, and elsewhere, when referring to siRNA, should be tion of this gene. replaced by “u' residues.

US 2009/0088,563 A1 Apr. 2, 2009 32

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 GUGCCGAGCUUUAGAAUUU 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

0415 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 0420 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 0416 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: 0421 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 0417 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 0422 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 0418 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 HUMANCYCLOPHILIN B which 100% of pools of oligonucleotides distanced by 2, 10 0423 Table III above lists the siRNA sequences for the and 20 base pairs were functional). human cyclophilin B protein. A particularly functional 0419. 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 2009/0088,563 A1 Apr. 2, 2009

0424. Alternatively, one could pool 2, 3, 4, 5 or more of were incubated with 'I-EGF (1 ng/ml) or 'I-transferrin (1 these sequences to create a kit for silencing a gene. Preferably, ug/ml) in binding medium (DMEM, 0.1% bovine serum albu within the kit there would be at least one sequence that has a min) at 37° C., and the ratio of internalized and surface relatively high predicted functionality when any of Formulas radioactivity was determined during 5-min time course to I-VII is applied. calculate specific internalization rate constant k as described previously (Jiang, X et al.). The measurements of the uptakes Example XI of radiolabeled transferrin and EGF were performed using short time-course assays to avoid influence of the recycling SAMPLE POOLS OF SIRNAS AND THEIRAPPLICATION on the uptake kinetics, and using low ligand concentration to TO HUMAN DISEASE avoid saturation of the clathrin-dependent pathway (for EGF 0425 The genetic basis behind human disease is well Lund, K. A., Opresko, L. K., Strarbuck, C. Walsh, B. J. & documented and siRNA may be used as both research or Wiley, H. S. (1990).J. Biol. Chem. 265, 15713-13723). diagnostic tools and therapeutic agents, either individually or 0428 The effects of knocking down Rab5a, 5b, 5c, Eps, or in pools. Genes involved in signal transduction, the immune Eps 15R (individually) are shown in FIG.22 and demonstrate response, apoptosis, DNA repair, cell cycle control, and a that disruption of single genes has little or no effect on EGF or variety of other physiological functions have clinical rel Tfn internalization. In contrast, simultaneous knock down of evance and therapeutic agents that can modulate expression Rab5a, 5b, and 5c, or Eps and Eps 15R, leads to a distinct of these genes may alleviate Some or all of the associated phenotype (note: total concentration of siRNA in these symptoms. In some instances, these genes can be described as experiments remained constant with that in experiments in a member of a family or class of genes and siRNA (randomly, which a single siRNA was introduced, see FIG. 23). These conventionally, or rationally designed) can be directed experiments demonstrate the effectiveness of using rationally against one or multiple members of the family to induce a designed siRNA to knockdown multiple genes and validates desired result. the utility of these reagents to override genetic redundancy. 0426 To identify rationally designed siRNA to each gene, the sequence was analyzed using Formula VIII or Formula X Example XIII to identify rationally designed siRNA. To confirm the activity VALIDATION MULTIGENETARGETING USING G6PD, of these sequences, the siRNA are introduced into a cell type GAPDH, PLK, AND UQC of choice (e.g., HeLa cells, HEK293 cells) and the levels of the appropriate message are analyzed using one of several art 0429 Further demonstration of the ability to knock down proven techniques. siRNA having heightened levels of expression of multiple genes using rationally designed potency can be identified by testing each of the before men siRNA was performed using pools of siRNA directed against tioned duplexes at increasingly limiting concentrations. four separate genes. To achieve this, siRNA were transfected Similarly, siRNA having increased levels of longevity can be into cells (total siRNA concentration of 100 nM) and assayed identified by introducing each duplex into cells and testing twenty-four hours later by B-DNA. Results shown in FIG. 24 functionality at 24, 48, 72, 96, 120, 144, 168, and 192 hours show that pools of rationally designed molecules are capable after transfection. Agents that induce >95% silencing at sub of simultaneously silencing four different genes. nanomolar concentrations and/or induce functional levels of silencing for >96 hours are considered hyperfunctional. Example XIV VALIDATION OF MULTIGENE KNOCKOUTS AS DEMON Example XII STRATED BY GENE EXPRESSION PROFILING, A PRO VALIDATION OF MULTIGENE KNOCKOUT USINGRAB5 PHETIC EXAMPLE AND EPS 0430. To further demonstrate the ability to concomitantly 0427 Two or more genes having similar, overlapping knockdown the expression of multiple gene targets, single functions often leads to genetic redundancy. Mutations that siRNA or siRNA pools directed against a collection of genes knockout only one of, e.g., a pair of Such genes (also referred (e.g., 4, 8, 16, or 23 different targets) are simultaneously to as homologs) results in little or no phenotype due to the fact transfected into cells and cultured for twenty-four hours. Sub that the remaining intact gene is capable of fulfilling the role sequently, mRNA is harvested from treated (and untreated) of the disrupted counterpart. To fully understand the function cells and labeled with one of two fluorescent probes dyes of Such genes in cellular physiology, it is often necessary to (e.g., a red fluorescent probe for the treated cells, a green knockout or knockdown both homologs simultaneously. fluorescent probe for the control cells.). Equivalent amounts Unfortunately, concomitant knockdown of two or more genes of labeled RNA from each sample is then mixed together and is frequently difficult to achieve in higher organisms (e.g., hybridized to sequences that have been linked to a solid mice) thus it is necessary to introduce new technologies dis support (e.g., a slide, “DNA CHIP). Following hybridiza sect gene function. One Such approach to knocking down tion, the slides are washed and analyzed to assess changes in multiple genes simultaneously is by using siRNA. For the levels of target genes induced by siRNA. example, FIG. 11 showed that rationally designed siRNA directed against a number of genes involved in the clathrin Example XV mediated endocytosis pathway resulted in significant levels IDENTIFYING.HYPERFUNCTIONAL SIRNA of protein reduction (e.g., >80%). To determine the effects of gene knockdown on clathrin-related endocytosis, internaliza 0431) Identification of Hyperfunctional Bcl-2 siRNA tion assays were performed using epidermal growth factor 0432. The ten rationally designed Bcl2 siRNA (identified and transferrin. Specifically, mouse receptor-grade EGF in FIG. 13, 14) were tested to identify hyperpotent reagents. (Collaborative Research Inc.) and iron-saturated human To accomplish this, each of the ten Bcl-2 siRNA were indi transferrin (Sigma) were iodinated as described previously vidually transfected into cells at a 300 pM (0.3 nM) concen (Jiang, X., Huang, F., Marusyk, A. & Sorkin, A. (2003) Mol trations. Twenty-four hours later, transcript levels were Biol Cell 14, 858-70). HeLa cells grown in 12-well dishes assessed by B-DNA assays and compared with relevant con US 2009/0088,563 A1 Apr. 2, 2009 34 trols. As shown in FIG. 25, while the majority of Bcl-2 siRNA failed to induce functional levels of silencing at this concen TABLE XI-continued tration, siRNA 1 and 8 induced >80% silencing, and siRNA 6 exhibited greater than 90% silencing at this subnanomolar 96-WELL 24-WELL concentration. By way of prophetic examples, similar assays could be per MIXTURE 4 (MEDIA-SIRNATRANSFECTION formed with any of the groups of rationally designed genes REAGENT MIXTURE) described in the Examples. Thus for instance, rationally Mixture 3 20 ul 100 ul designed siRNA sequences directed against a gene of interest Complete media 80 ul 400 ul could be introduced into cells at increasingly limiting con MIXTURE 4 100 ul 500 ul centrations to determine whether any of the duplexes are FINAL VOLUME hyperfunctional. Incubate 48 hours at 37°C. Example XVI 0438 Transfection. Create a Mixture 1 by combining the GENESILENCING specified amounts of OPTI-MEM serum free media and PROPHETIC EXAMPLE transfection reagent in a sterile polystyrene tube. Create a Mixture 2 by combining specified amounts of each siRNA 0433 Below is an example of how one might transfect a with OPTI-MEM media in Sterile 1 ml tubes. Create a Mix cell. ture 3 by combining specified amounts of Mixture I and 0434 Select a cell line. The selection of a cell line is usually determined by the desired application. The most Mixture 2. Mix gently (do not vortex) and incubate at room important feature to RNAi is the level of expression of the temperature for 20 minutes. Create a Mixture 4 by combining gene of interest. It is highly recommended to use cell lines for specified amounts of Mixture 3 to complete media. Add which siRNA transfection conditions have been specified and appropriate volume to each cell culture well. Incubate cells validated. with transfection reagent mixture for 24-72 hours at 37° C. 0435 Plate the cells. Approximately 24 hours prior to This incubation time is flexible. The ratio of silencing will transfection, plate the cells at the appropriate density so that remain consistent at any point in the time period. Assay for they will be approximately 70-90% confluent, or approxi gene silencing using an appropriate detection method such as mately 1x10 cells/ml at the time of transfection. Cell densi RT-PCR, Western blot analysis, immunohistochemistry, phe ties that are too low may lead to toxicity due to excess expo notypic analysis, mass spectrometry, fluorescence, radioac Sure and uptake of transfection reagent-siRNA complexes. tive decay, or any other method that is now known or that Cell densities that are too high may lead to low transfection comes to be known to persons skilled in the art and that from efficiencies and little or no silencing. Incubate the cells over reading this disclosure would useful with the present inven night. Standard incubation conditions for mammalian cells tion. The optimal window for observing a knockdown phe are 37°C. in 5% CO. Other cell types, such as insect cells, notype is related to the mRNA turnover of the gene of interest, require different temperatures and CO concentrations that although 24-72 hours is standard. Final Volume reflects are readily ascertainable by persons skilled in the art. Use amount needed in each well for the desired cell culture for conditions appropriate for the cell type of interest. mat. When adjusting volumes for a Stock Mix, an additional 0436 siRNA re-suspension. Add 20 ul siRNA universal 10% should be used to accommodate variability in pipetting, buffer to each siRNA to generate a final concentration of 50 etc. Duplicate or triplicate assays should be carried out when uM. possible. 0437 siRNA-lipid complex formation. Use RNase-free solutions and tubes. Using the following table, Table XI: Example XVII TABLE XI SIRNAS THAT TARGET HOMO SAPIENSTRANSDUC 96-WELL 24-WELL TION (BETA)-Like 3 MIXTURE 1 (TRANSIT-TKO-PLASMID DILUTION MIXTURE) 0439 siRNAs that target nucleotide sequences for homo sapiens transducin (beta)-like 3 (NCBI accession number Opti-MEM 9.3 46.5 NM 006453) and having sequences generated in silico by TransIT-TKO (1 gul) O.S 2.5 the algorithms herein, are provided. In various embodiments, MIXTURE 1 1O.O SO.O the siRNAs are rationally designed. In various embodiments. FINAL VOLUME the siRNAs are functional or hyperfunctional. These siRNA MIXTURE 2 (SIRNA DILUTION MIXTURE) that have been generated by the algorithms of the present Opti-MEM 9.O 45.O invention include: siRNA (1 M) 1.O S.O

MIXTURE 2 1O.O SO.O FINAL VOLUME siRNA. Sense Sequence Sequence ID Number MIXTURE 3 (SIRNA-TRANSFECTION REAGENT MIXTURE) AAAUUGAGCCUUUCUACAA 438 Mixture 1 10 50 Mixture 2 10 50 AAUUGAGCCUUUCUACAAG 439

MIXTURE3 2O 100 ACAACCUGCUGCAUGAGAA 44 O FINAL VOLUME Incubate 20 minutes at room temperature ACACUGAGCGGCACUUUCA 441 US 2009/0088,563 A1 Apr. 2, 2009 35

- Continued - Continued siRNA. Sense Sequence Sequence ID Number siRNA. Sense Sequence Sequence ID Number

ACAGUUCGCUGGCUACAGU 442 GCUUCUGCGUCACGUGGAA 479

ACUAUGCUGUGGAGCGCAA 443 GGACAUCACUGCCUUUGAC 48O

ACUCAGCGCUGCCAUGAUA 444 GGACCGUGCCUGUGUUUGA 481

AGAAACAGUUCGCUGGCUA. 445 GGACUUCAGCUGUCUCAAG 482

AGCAAGAGCUGGACAACCU 446 GGAGGACAUCACUGCCUUU 483

AGCCAGGACUGCACUGUGA 447 GGAGGCUGCUGUGCUGUUG. 484

AGGAGGACCAGGAGGACAU 4 48 GGCACCGUCUGCUGCUCUA 485

CAAUAGCCCCUGCCUAAAA 449 GGCCGUGACAAGAUAUGUA. 486

CAUCAACAGCGUGGCUAUU 450 GGCGUUCUGUGAAGAGUUC 487

CAUCAAGAACAACGAGUGU 451 GGGCAUUGCUGCUGGCUCA 488

CCAAAGCCUUGCUGUCCAA 452 GGUUUUGGAUGUCCGGUUU 489

CCACUCAGCGCUGCCAUGA 453 GUAUCUGGAGAAUGAACAA 490

CCAGGGCUGUACUUUCUGA 454 GUCCGUAUCUGGAGAAUGA 491

CCGGGACCCUAGCAGGUUU 45.5 GUGAAGAGUUCGGUGCUAA 492

CGAGGACCGUGCCUGUGUU 456 GUGAAGCUCUGGACCAUCA 493

CGGAUGGCCUCGUGAAGCU 457 GUGAGGAGGUUUUGGAUGU 494

CUACACGCCUGCUGCUCUU 458 GUGCCAAGGAUCAGAGCGU 495

CUACGGGACACACCACUUC 459 UCAAGAACAACGAGUGUGU 496

CUUCAGAGCUGCCAGGCCA 460 UCACCUCACUGGCCUUCAG 497

GAACACAGCCCCAGACAAC 461 UCUGACAGCUGGCGACCAA 498

GACAACCUGCUGCAUGAGA 462 UCUGGACCAUCAAGAACAA 499

GACAGCCAGUCGGGCAUUG. 463 UGGACAACCUGCUGCAUGA SOO

GACCGUGCCUGUGUUUGAG 464 UGGCAAGAGGGCAGCGUUA. 5O1

GACGUCAGCCUGCCAGAUC 465 UGUGAAGAGUUCGGUGCUA. 5O2

GACUUCAGCUGUCUCAAGA 466 0440 Thus, consistent with Example XVII, the present GAGGACAUCACUGCCUUUG. 467 invention provides an siRNA that targets a nucleotide sequence for TBL3, wherein the siRNA is selected from the GAGGACCGUGCCUGUGUUU 468 group consisting of SEQ. ID NOS. 438-502. GAGUCAACAUUCUGGAAGU 469 0441. In another embodiment, an siRNA is provided, said siRNA comprising a sense region and an antisense region, GAGUGUGGCGUUCUGUGAA 47 O wherein said sense region and said antisense region are at least 90% complementary, said sense region and said anti GAUCAGAGCGUCCGUAUCU 471 sense region togetherform a duplex region comprising 18-30 base pairs, and said sense region comprises a sequence that is GCACCAGAGUCAACAUUCU 472 at least 90% similar to a sequence selected from the group GCACCUCUUCUGCGUCUGU 473 consisting of: SEQ. ID NOS 438-502. 0442. In another embodiment, an siRNA is provided GCCGUGACAAGAUAUGUAU 474 wherein the siRNA comprises a sense region and an antisense GCGAGAAGCUGGAAGCCAC 47s region, wherein said sense region and said antisense region are at least 90% complementary, said sense region and said GCUCAGGGUUCCGGUCACA 476 antisense region together form a duplex region comprising 18-30 base pairs, and said sense region comprises a sequence GCUCUAGGCUGAAGGAGUC 477 that is identical to a contiguous stretch of at least 18 bases of GCUGCUGGGUGUCUUCUCA 478 a sequence selected from the group consisting of: SEQ. ID NOS 438-5O2. US 2009/0088,563 A1 Apr. 2, 2009 36

0443) In another embodiment, an siRNA is provided pairs and comprises a second sense region that comprises a wherein the siRNA comprises a sense region and an antisense sequence that is at least 90% similar to a sequence selected region, wherein said sense region and said antisense region from the group consisting of: SEQ. ID NOS 438-502. are at least 90% complementary, said sense region and said 0447. In another embodiment, a pool of at least two siR antisense region together form a duplex region comprising NAs is provided, wherein said pool comprises a first siRNA 19-30 base pairs, and said sense region comprises a sequence and a second siRNA, said first siRNA comprises a duplex region of length 19-30 base pairs and has a first sense region that is identical to a contiguous stretch of at least 18 bases of comprising a sequence that is identical to at least 18 bases of a sequence selected from the group consisting of SEQ. ID a sequence selected the group consisting of: SEQ. ID NOS NOS 438-5O2. 438-502 and said duplex of said second siRNA is 19-30 base 0444. In another embodiment, a pool of at least two siR pairs and comprises a second sense region comprising a NAs is provided, wherein said pool comprises a first siRNA sequence that is identical to a sequence selected from the and a second siRNA, said first siRNA comprises a duplex group consisting of: SEQ. ID NOS 438-502. region of length 18-30 base pairs that has a first sense region 0448. In each of the aforementioned embodiments, pref that is at least 90% similar to 18 bases of a first sequence erably the antisense region is at least 90% complementary to selected from the group consisting of: SEQ. ID NOS 438-502 a contiguous stretch of bases of one of the NCBI sequences and said second siRNA comprises a duplex region of length identified in Example XVII; each of the recited NCBI 18-30 base pairs that has a second sense region that is at least sequences is incorporated by reference as if set forth fully 90% similar to 18 bases of a second sequence selected from herein. In some embodiments, the antisense region is 100% the group consisting of: SEQ. ID NOS 438-502 and wherein complementary to a contiguous stretch of bases of one of the said first sense region and said second sense region are not NCBI sequences identified in Example XVII. identical. 0449 Further, in some embodiments that are directed to 0445. In another embodiment, a pool of at least two siR siRNA duplexes in which the antisense region is 20-30 bases NAs is provided, wherein said pool comprises a first siRNA in length, preferably there is a stretch of 19 bases that is at and a second siRNA, said first siRNA comprises a duplex least 90%, more preferably 100% complementary to the region of length 18-30 base pairs that has a first sense region recited sequence id number and the entire antisense region is that is identical to at least 18 bases of a sequence selected at least 90% and more preferably 100% complementary to a from the group consisting of: SEQ. ID NOS 438-502 and contiguous stretch of bases of one of the NCBI sequences wherein the second siRNA comprises a second sense region identified in Example XVII. that comprises a sequence that is identical to at least 18 bases 0450 While the invention has been described in connec of a sequence selected from the group consisting of: SEQ. ID tion with specific embodiments thereof, it will be understood NOS 438-5O2. that it is capable of further modifications and this application 0446. In another embodiment, a pool of at least two siR is intended to cover any variations, uses, or adaptations of the NAs is provided, wherein said pool comprises a first siRNA invention following, in general, the principles of the invention and a second siRNA, said first siRNA comprises a duplex and including such departure from the present disclosure as region of length 19-30 base pairs and has a first sense region come within known or customary practice within the art to comprising a sequence that is at least 90% similar to a which the invention pertains and as may be applied to the sequence selected from the group consisting of SEQ. ID NOS essential features hereinbefore set forth and as follows in the 438-502, and said duplex of said second siRNA is 19-30 base Scope of the appended claims.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 5O2

<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 &212> TYPE RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic US 2009/0088,563 A1 Apr. 2, 2009 37

- Continued

FEATURE: NAMEAKEY: misc feature LOCATION: 1-2, 4, 6-9, 12, 15-18 OTHER INFORMATION: n is any nucleotide SEQUENCE: 2 nnanannnnu gnaannnna 19

SEQ ID NO 3 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: 3

ala Laala 19

SEQ ID NO 4 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: 4

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 US 2009/0088,563 A1 Apr. 2, 2009 38

- Continued

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 OTHER INFORMATION: n is any nucleotide SEQUENCE: 9

ala Lala 19

SEQ ID NO 10 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: 1.O

accl. Claala 19

SEQ ID NO 11 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: 11 nnancinnnnu gnaannnna 19 US 2009/0088,563 A1 Apr. 2, 2009 39

- Continued

SEQ ID NO 12 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: 12

ac. Laala 19

SEQ ID NO 13 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: 13

lac Coala 19

SEQ ID NO 14 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: 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 US 2009/0088,563 A1 Apr. 2, 2009 40

- Continued

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 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 US 2009/0088,563 A1 Apr. 2, 2009 41

- Continued

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 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 US 2009/0088,563 A1 Apr. 2, 2009 42

- Continued

&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: 26 nnangnnnnu gnuannnna 19

<210 SEQ ID NO 27 <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: 27 nnangnnnnu unuannnna 19

<210 SEQ ID NO 28 <211 LENGTH: 22 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 4-5, 7, 9-12, 15, 18-21 <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 NAMEAKEY: misc feature <222> LOCATION: (1) . . . (2OO) <223> OTHER INFORMATION: firefly luciferase fragment <4 OO SEQUENCE: 30 US 2009/0088,563 A1 Apr. 2, 2009 43

- Continued 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 <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 US 2009/0088,563 A1 Apr. 2, 2009 44

- Continued

&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: <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 US 2009/0088,563 A1 Apr. 2, 2009 45

- Continued

<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 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 US 2009/0088,563 A1 Apr. 2, 2009 46

- Continued

<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 <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 US 2009/0088,563 A1 Apr. 2, 2009 47

- Continued

&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: <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 US 2009/0088,563 A1 Apr. 2, 2009 48

- Continued

<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 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 US 2009/0088,563 A1 Apr. 2, 2009 49

- Continued

<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 <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 ulaaucaagga cullcalugau. 19

<210 SEQ ID NO 74 <211 LENGTH: 19 US 2009/0088,563 A1 Apr. 2, 2009 50

- Continued

&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: <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 US 2009/0088,563 A1 Apr. 2, 2009 51

- Continued

<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

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 US 2009/0088,563 A1 Apr. 2, 2009 52

- Continued

<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 <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 US 2009/0088,563 A1 Apr. 2, 2009 53

- Continued

&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: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 96 luggcacagga gaaagagc 19

<210 SEQ ID NO 97 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 97 gcacaggagg aaagagcau, 19

<210 SEQ ID NO 98 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 98 acaggaggaa agagcaucu. 19

<210 SEQ ID NO 99 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic US 2009/0088,563 A1 Apr. 2, 2009 54

- Continued

<4 OO SEQUENCE: 99 aggaggaaag agcaucuac 19

<210 SEQ ID NO 100 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 1.OO gaggaaagag caucuacgg 19

<210 SEQ ID NO 101 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 101 ggaaagagca ulculacggug 19

<210 SEQ ID NO 102 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 102 aaagagcauc ulacggugag 19

<210 SEQ ID NO 103 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 103 agagcaucua C9gugagcg 19

<210 SEQ ID NO 104 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 104 agcaucuacg glugagcgcul 19

<210 SEQ ID NO 105 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 105 caucuacggu gag cqcuuc 19 US 2009/0088,563 A1 Apr. 2, 2009 55

- Continued

<210 SEQ ID NO 106 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 106 ulculacgguga gcgcuuccC 19

<210 SEQ ID NO 107 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 107 ulacggugagc gculuccc.cg 19

<210 SEQ ID NO 108 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 108 cggugagcgc lulu.ccc.cgau. 19

<210 SEQ ID NO 109 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 109 gugagcgcuu ccc.cgauga 19

<210 SEQ ID NO 110 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 110 gagggculu.cc ccgaugaga 19

<210 SEQ ID NO 111 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 111 gcgcuuccCC gaugagaac 19

<210 SEQ ID NO 112 <211 LENGTH: 19 US 2009/0088,563 A1 Apr. 2, 2009 56

- Continued

&212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 112 gculu.ccc.cga ugagaacuu. 19

<210 SEQ ID NO 113 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 113 uuccc.cgalug agaaculuca 19

<210 SEQ ID NO 114 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 114

CCCC gaugag aacuucaaa 19

<210 SEQ ID NO 115 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 115 cc.galugagaa cullcaaacul 19

<210 SEQ ID NO 116 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 116 gaugagaacul ulcaaaculga 19

<210 SEQ ID NO 117 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 117 ulgagaacuuc aaaculgaag 19

<210 SEQ ID NO 118 <211 LENGTH: 19 &212> TYPE : RNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic