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  Sirna targeting rad1 homolog (rad1)Related Patent Categories: Organic Compounds -- Part Of The Class 532-570 Series, Azo Compounds Containing Formaldehyde Reaction Product As The Coupling Component, Carbohydrates Or Derivatives, Nitrogen Containing, Dna Or Rna Fragments Or Modified Forms Thereof (e.g., Genes, Etc.), , Non-coding Sequences Which Control Transcription Or Translation Processes (e.g., Promoters, Operators, Enhancers, Ribosome Binding Sites, Etc.)The Patent Description & Claims data below is from USPTO Patent Application 20070260052. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. Ser. No. 10/714,333, filed Nov. 14, 2003, which claims the benefit of U.S. Provisional Application No. 60/426,137, filed Nov. 14, 2002, and also claims the benefit of U.S. Provisional Application No. 60/502,050, filed Sep. 10, 2003; this application is also a continuation-in-part of U.S. Ser. No. 10/940,892, filed Sep. 14, 2004, which is a continuation of PCT Application No. PCT/US04/14885, international filing date May 12, 2004. The disclosures of the priority applications, including the sequence listings and tables submitted in electronic form in lieu of paper, are incorporated by reference into the instant specification. Sequence Listing [0002] The sequence listing for this application has been submitted in accordance with 37 CFR .sctn. 1.52(e) and 37 CFR .sctn. 1.821 on CD-ROM in lieu of paper on a disk containing the sequence listing file entitled "DHARMA.sub.--2100-US46_CRF.txt" created May 30, 2007, 104 kb. Applicants hereby incorporate by reference the sequence listing provided on CD-ROM in lieu of paper into the instant specification. FIELD OF INVENTION [0003] The present invention relates to RNA interference ("RNAi"). BACKGROUND OF THE INVENTION [0004] Relatively recently, researchers observed that double stranded RNA ("dsRNA") could be used to inhibit protein expression. This ability to silence a gene has broad potential for treating human diseases, and many researchers and commercial entities are currently investing considerable resources in developing therapies based on this technology. [0005] Double stranded RNA induced gene silencing can occur on at least three different levels: (i) transcription inactivation, which refers to RNA guided DNA or histone methylation; (ii) siRNA induced mRNA degradation; and (iii) mRNA induced transcriptional attenuation. [0006] It is generally considered that the major mechanism of RNA induced silencing (RNA interference, or RNAi) in mammalian cells is mRNA degradation. Initial attempts to use RNAi in mammalian cells focused on the use of long strands of dsRNA. However, these attempts to induce RNAi met with limited success, due in part to the induction of the interferon response, which results in a general, as opposed to a target-specific, inhibition of protein synthesis. Thus, long dsRNA is not a viable option for RNAi in mammalian systems. [0007] More recently it has been shown that when short (18-30 bp) RNA duplexes are introduced into mammalian cells in culture, sequence-specific inhibition of target mRNA can be realized without inducing an interferon response. Certain of these short dsRNAs, referred to as small inhibitory RNAs ("siRNAs"), can act catalytically at sub-molar concentrations to cleave greater than 95% of the target mRNA in the cell. A description of the mechanisms for siRNA activity, as well as some of its applications are described in Provost et al. (2002) Ribonuclease Activity and RNA Binding of Recombinant Human Dicer, EMBO J. 21(21): 5864-5874; Tabara et al. (2002) The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell 109(7):861-71; Ketting et al. (2002) Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Developmental Timing in C. elegans; Martinez et al., Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi, Cell 110(5):563; Hutvagner & Zamore (2002) A microRNA in a multiple-turnover RNAi enzyme complex, Science 297:2056. [0008] From a mechanistic perspective, introduction of long double stranded RNA into plants and invertebrate cells is broken down into siRNA by a Type III endonuclease known as Dicer. Sharp, RNA interference--2001, Genes Dev. 2001, 15:485. Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs. Bernstein, Caudy, Hammond, & Hannon (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference, Nature 409:363. The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition. Nykanen, Haley, & Zamore (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway, Cell 107:309. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing. Elbashir, Lendeckel, & Tuschl (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs, Genes Dev. 15:188, FIG. 1. [0009] The interference effect can be long lasting and may be detectable after many cell divisions. Moreover, RNAi exhibits sequence specificity. Kisielow, M. et al. (2002) Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA, J. Biochem. 363:1-5. Thus, the RNAi machinery can specifically knock down one type of transcript, while not affecting closely related mRNA. These properties make siRNA a potentially valuable tool for inhibiting gene expression and studying gene function and drug target validation. Moreover, siRNAs are potentially useful as therapeutic agents against: (1) diseases that are caused by over-expression or misexpression of genes; and (2) diseases brought about by expression of genes that contain mutations. [0010] Successful siRNA-dependent gene silencing depends on a number of factors. One of the most contentious issues in RNAi is the question of the necessity of siRNA design, i.e., considering the sequence of the siRNA used. Early work in C. elegans and plants circumvented the issue of design by introducing long dsRNA (see, for instance, Fire, A. et al. (1998) Nature 391:806-811). In this primitive organism, long dsRNA molecules are cleaved into siRNA by Dicer, thus generating a diverse population of duplexes that can potentially cover the entire transcript. While some fraction of these molecules are non-functional (i.e., induce little or no silencing) one or more have the potential to be highly functional, thereby silencing the gene of interest and alleviating the need for siRNA design. Unfortunately, due to the interferon response, this same approach is unavailable for mammalian systems. While this effect can be circumvented by passing the Dicer cleavage step and directly introducing siRNA, this tactic carries with it the risk that the chosen siRNA sequence may be non-functional or semi-functional. [0011] A number of researches have expressed the view that siRNA design is not a crucial element of RNAi. On the other hand, others in the field have begun to explore the possibility that RNAi can be made more efficient by paying attention to the design of the siRNA. Unfortunately, none of the reported methods have provided a satisfactory scheme for reliably selecting siRNA with acceptable levels of functionality. Accordingly, there is a need to develop rational criteria by which to select siRNA with an acceptable level of functionality, and to identify siRNA that have this improved level of functionality, as well as to identify siRNAs that are hyperfunctional. SUMMARY OF THE INVENTION [0012] The present invention is directed to increasing the efficiency of RNAi, particularly in mammalian systems. Accordingly, the present invention provides kits, siRNAs and methods for increasing siRNA efficacy. [0013] According to a first embodiment, the present invention provides a kit for gene silencing, wherein said kit is comprised of a pool of at least two siRNA duplexes, each of which is comprised of a sequence that is complementary to a portion of the sequence of one or more target messenger RNA, and each of which is selected using non-target specific criteria. [0014] According to a second embodiment, the present invention provides a method for selecting an siRNA, said method comprising applying selection criteria to a set of potential siRNA that comprise 18-30 base pairs, wherein said selection criteria are non-target specific criteria, and said set comprises at least two siRNAs and each of said at least two siRNAs contains a sequence that is at least substantially complementary to a target gene; and determining the relative functionality of the at least two siRNAs. [0015] According to a third embodiment, the present invention also provides a method for selecting an siRNA wherein said selection criteria are embodied in a formula comprising: (-14)*G.sub.13-13*A.sub.1-12*U.sub.7-11*U.sub.2-10*A.sub.1110*U.sub.4-10*- C.sub.3-10*C.sub.5-10*C.sub.6-9*A.sub.10-9*U.sub.9-9*C.sub.18-8*G.sub.10-7- *U.sub.1-7*U.sub.16-7*C.sub.17-7*C.sub.19+7*U.sub.17+8*A.sub.2+8*A.sub.4+8- *A.sub.5+8*C.sub.4+9*G.sub.8+10*A.sub.7+10*U.sub.18+11*A.sub.19+11*C.sub.9- +15*G.sub.1+18*A.sub.3+19*U.sub.10-Tm-3*(GC.sub.total)-6*(GC.sub.15-19)-30- *X; or Formula VIII: (-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(-4)*A9+(-5- )*A10+(-2)*A11+(-5)*A12+(17)*A13+(-3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18- +(30)*A19+(-13)*U1+(-10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8- +(-5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15+(-10) *U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-9)*C5+(-20)*- C6+(-18)*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-5)*C12+(-3)*C13+(-6)*C14+(-2)- *C15+(-5)*C16+(-3)*C17+(-12)*C18+(-18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10)*G4+- (-4) *G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(1)*G11+(9)*G12+(-24) *G13+(18)*G14+(11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+6*(number of A+U in position 15-19)-3*(number of G+C in whole siRNA), Formula X wherein position numbering begins at the 5'-most position of a sense strand, and A.sub.1=1 if A is the base at position 1 of the sense strand, otherwise its value is 0; A.sub.2=1 if A is the base at position 2 of the sense strand, otherwise its value is 0; A.sub.3=1 if A is the base at position 3 of the sense strand, otherwise its value is 0; A.sub.4=1 if A is the base at position 4 of the sense strand, otherwise its value is 0; A.sub.5=1 if A is the base at position 5 of the sense strand, otherwise its value is 0; A.sub.6=1 if A is the base at position 6 of the sense strand, otherwise its value is 0; A.sub.7=1 if A is the base at position 7 of the sense strand, otherwise its value is 0; A.sub.10=1 if A is the base at position 10 of the sense strand, otherwise its value is 0; A.sub.11=1 if A is the base at position 11 of the sense strand, otherwise its value is 0; A.sub.13=1 if A is the base at position 13 of the sense strand, otherwise its value is 0; A.sub.19=1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; C.sub.3=1 if C is the base at position 3 of the sense strand, otherwise its value is 0; C.sub.4=1 if C is the base at position 4 of the sense strand, otherwise its value is 0; C.sub.5=1 if C is the base at position 5 of the sense strand, otherwise its value is 0; C.sub.6=1 if C is the base at position 6 of the sense strand, otherwise its value is 0; C.sub.7=1 if C is the base at position 7 of the sense strand, otherwise its value is 0; C.sub.9=1 if C is the base at position 9 of the sense strand, otherwise its value is 0; C.sub.17=1 if C is the base at position 17 of the sense strand, otherwise its value is 0; C.sub.18=1 if C is the base at position 18 of the sense strand, otherwise its value is 0; C.sub.19=1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; G.sub.1=1 if G is the base at position 1 on the sense strand, otherwise its value is 0; G.sub.2=1 if G is the base at position 2 of the sense strand, otherwise its value is 0; G.sub.8=1 if G is the base at position 8 on the sense strand, otherwise its value is 0; G.sub.10=1 if G is the base at position 10 on the sense strand, otherwise its value is 0; G.sub.13=1 if G is the base at position 13 on the sense strand, otherwise its value is 0; G.sub.19=1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; U.sub.1=1 if U is the base at position 1 on the sense strand, otherwise its value is 0; U.sub.2=1 if U is the base at position 2 on the sense strand, otherwise its value is 0; U.sub.3=1 if U is the base at position 3 on the sense strand, otherwise its value is 0; U.sub.4=1 if U is the base at position 4 on the sense strand, otherwise its value is 0; U.sub.7=1 if U is the base at position 7 on the sense strand, otherwise its value is 0; U.sub.9=1 if U is the base at position 9 on the sense strand, otherwise its value is 0; U.sub.10=1 if U is the base at position 10 on the sense strand, otherwise its value is 0; U.sub.15=1 if U is the base at position 15 on the sense strand, otherwise its value is 0; U.sub.16=1 if U is the base at position 16 on the sense strand, otherwise its value is 0; U.sub.17=1 if U is the base at position 17 on the sense strand, otherwise its value is 0; U.sub.18=1 if U is the base at position 18 on the sense strand, otherwise its value is 0. GC.sub.15-19=the number of G and C bases within positions 15-19 of the sense strand, or within positions 15-18 if the sense strand is only 18 base pairs in length; GC.sub.total=the number of G and C bases in the sense strand; Tm=100 if the siRNA oligo has the internal repeat longer then 4 base pairs, otherwise its value is 0; and X=the number of times that the same nucleotide repeats four or more times in a row. [0016] According to a fourth embodiment, the invention provides a method for developing an algorithm for selecting siRNA, said method comprising: (a) selecting a set of siRNA; (b) measuring gene silencing ability of each siRNA from said set; (c) determining relative functionality of each siRNA; (d) determining improved functionality by the presence or absence of at least one variable selected from the group consisting of the presence or absence of a particular nucleotide at a particular position, the total number of As and Us in positions 15-19, the number of times that the same nucleotide repeats within a given sequence, and the total number of Gs and Cs; and (e) developing an algorithm using the information of step (d). [0017] According to a fifth embodiment, the present invention provides a kit, wherein said kit is comprised of at least two siRNAs, wherein said at least two siRNAs comprise a first optimized siRNA and a second optimized siRNA, wherein said first optimized siRNA and said second optimized siRNA are optimized according a formula comprising Formula X. [0018] The present invention also provides a method for identifying a hyperfunctional siRNA, comprising applying selection criteria to a set of potential siRNA that comprise 18-30 base pairs, wherein said selection criteria are non-target specific criteria, and said set comprises at least two siRNAs and each of said at least two siRNAs contains a sequence that is at least substantially complementary to a target gene; determining the relative functionality of the at least two siRNAs and assigning each of the at least two siRNAs a functionality score; and selecting siRNAs from the at least two siRNAs that have a functionality score that reflects greater than 80 percent silencing at a concentration in the picomolar range, wherein said greater than 80 percent silencing endures for greater than 120 hours. [0019] According to a sixth embodiment, the present invention provides a hyperfunctional siRNA that is capable of silencing Bc12. Continue reading... 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