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  Functional and hyperfunctional sirna directed against bcl-2USPTO Application #: 20080108803Title: Functional and hyperfunctional sirna directed against bcl-2 Abstract: Efficient sequence specific gene silencing is possible through the use of siRNA technology. By selecting particular siRNAs by rationale design, one can maximize the generation of an effective gene silencing reagent, as well as methods for silencing Bcl-2. (end of abstract) Agent: Kalow & Springut LLP - New York, NY, US Inventors: Anastasia Khvorova, Angela Reynolds, Devin Leake, William Marshall, Stephen Scaringe USPTO Applicaton #: 20080108803 - Class: 536024500 (USPTO) 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.), , Nucleic Acid Expression Inhibitors The Patent Description & Claims data below is from USPTO Patent Application 20080108803. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of the filing dates of U.S. patent application Ser. No. 11/083,784, filed Mar. 18, 2005 entitled, "Functional and Hyperfunctional siRNA Directed Against Bcl-2," U.S. patent application Ser. No. 10/714,333, filed Nov. 14, 2003 entitled, "Functional and Hyperfunctional siRNA," U.S. Provisional Application Ser. No. 60/426,137, filed Nov. 14, 2002, entitled "Combinatorial Pooling Approach for siRNA Induced Gene Silencing and Methods for Selecting siRNA," and U.S. Provisional Application Ser. No. 60/502,050, filed Sep. 10, 2003, entitled "Methods for Selecting siRNA," the entire disclosures of which are hereby incorporated by reference into the present disclosure. 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.--0100-US28_CRF.txt" created Oct. 17, 2007, 80 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., Ribonuclease Activity and RNA Binding of Recombinant Human Dicer, E.M.B.O. J., 2002 Nov. 1; 21(21): 5864-5874; Tabara et al., The ds RNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell 2002, June 28; 109(7):861-71; Ketting et al., 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 2002, Sep. 6; 110(5):563; Hutvagner & Zamore, A microRNA in a multiple-turnover RNAi enzyme complex, Science 2002, 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, Role for a bidentate ribonulclease in the initiation step of RNA interference, Nature 2001, 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, ATP requirements and small interfering RNA structure in the RNA interference pathway, Cell 2001, 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, RNA interference is mediated by 21- and 22-nucleotide RNAs, Genes Dev 2001, 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. of Biochemistry 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 bypassing 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 one 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. [0014] According to a second embodiment, the present invention provides a method for optimizing RNA interference by using one or more siRNAs that are optimized according to a formula (or algorithm) selected from: Relative functionality of siRNA=-(GC/3)+(AU.sub.15-19)-(Tm.sub.20.degree. C.)*3-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.sub.3)+(U.sub.10)+(A.sub.14- )-(U.sub.5)-(A.sub.11) Formula I Relative functionality of siRNA=-(GC/3)-(AU.sub.15-19)*3-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.su- b.3) Formula II Relative functionality of siRNA=-(GC/3)+(AU.sub.15-19)-(Tm.sub.20.degree. C.)*3 Formula III Relative functionality of siRNA=-GC/2+(AU.sub.15-19)/2-(Tm.sub.20.degree. C.)*2-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.sub.3)+(U.sub.10)+(A.sub.14- )-(U.sub.5)-(A.sub.11) Formula IV Relative functionality of siRNA=-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.sub.3)+(U.sub.10)+(A.sub.1- 4)-(U.sub.5)-(A.sub.11) Formula V Relative functionality of siRNA=-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.sub.3) Formula VI Relative functionality of siRNA=-(GC/2)+(AU.sub.15-19)/2-(Tm.sub.20.degree. C.)*1-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*3+(A.sub.3)*3+(U.sub.10)/2+(A.su- b.14)/2-(U.sub.5)/2-(A.sub.11)/2 Formula VII wherein in Formulas I-VII: [0015] Tm.sub.20.degree. C.=1 if the Tm is greater than 20.degree. C.; [0016] A.sub.19=1 if A is the base at position 19 on the sense strand, otherwise its value is 0; [0017] AU.sub.15-19=0-5 depending on the number of A or U bases on the sense strand at [0018] positions 15-19; [0019] G.sub.13=1 if G is the base at position 13 on the sense strand, otherwise its value is 0; [0020] C.sub.19=1 if C is the base at position 19 of the sense strand, otherwise its value is 0; Continue reading... Full patent description for Functional and hyperfunctional sirna directed against bcl-2 Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Functional and hyperfunctional sirna directed against bcl-2 patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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