I 1111111111111111 11111 111111111111111 IIIII IIIII IIIII IIIII 111111111111111111
`US007452987B2
`
`c12) United States Patent
`Giese et al.
`
`(IO) Patent No.:
`(45) Date of Patent:
`
`US 7,452,987 B2
`Nov. 18, 2008
`
`(54)
`
`INTERFERING RNA MOLECULES
`
`(75)
`
`Inventors: Klaus Giese, Berlin (DE); Jiirg
`Kaufmann, Berlin (DE); Anke
`Klippel-Giese, Berlin (DE)
`
`(73) Assignee: Silence Therapeutics
`Aktiengesellschaft (AG), Berlin (DE)
`
`( *) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 107 days.
`
`(21) Appl. No.: 10/633,630
`
`(22) Filed:
`
`Aug. 5, 2003
`
`(65)
`
`Prior Publication Data
`
`US 2004/0180351 Al
`
`Sep. 16, 2004
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/402,541, filed on Aug.
`12, 2002.
`
`(30)
`
`Foreign Application Priority Data
`
`Aug. 5, 2002
`Apr. 10, 2003
`
`(EP)
`(EP)
`
`.................................. 02017601
`.................................. 03008383
`
`(51)
`
`Int. Cl.
`C07H 21104
`(2006.01)
`(52) U.S. Cl. .................... 536/24.5; 536/23.1; 536/24.1;
`536/24.31; 536/24.32; 536/24.33; 536/24.3
`(58) Field of Classification Search ................ 536/24.5,
`536/24.3, 24.31, 24.33
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,217,294 A
`5,466,786 A
`5,514,546 A *
`5,589,389 A
`5,670,633 A
`5,792,847 A
`5,872,232 A
`5,898,031 A *
`5,914,396 A
`5,955,589 A *
`6,005,087 A
`6,107,094 A *
`6,129,912 A
`6,153,737 A
`6,239,272 Bl
`6,284,267 Bl
`6,476,205 Bl
`6,506,559 Bl
`6,531,584 Bl
`6,617,438 Bl*
`6,812,204 Bl
`7,056,704 B2
`7,074,863 B2
`7,078,196 B2
`7,138,517 B2
`7,148,342 B2
`
`................ 536/23.1
`
`8/1980 Petty
`11/1995 Buhr et al.
`5/1996 Kool ............................. 435/6
`12/1996 Pages et al.
`9/1997 Cook et al.
`8/1998 Buhr et al.
`2/1999 Cook et al.
`4/1999 Crooke ...................... 435/91.3
`6/1999 Cook et al.
`9/1999 Cook et al.
`12/1999 Cook et al.
`8/2000 Crooke ....................... 435/455
`10/2000 Hortin et al.
`11/2000 Manoharan et al.
`5/2001 Beigelman et al.
`9/2001 Aneja
`11/2002 Buhr et al.
`1/2003 Fire et al.
`3/2003 Cook et al.
`9/2003 Beigelman et al. ......... 536/23.1
`11/2004 McHale et al.
`6/2006 Tuschl et al.
`7/2006 Ekholm et al.
`7/2006 Tuschl et al.
`11/2006 Cook et al.
`12/2006 Tolentino et al.
`
`3/2007 Heidenreich et al.
`7,196,184 B2
`3/2008 John et al.
`7,348,314 B2
`2/2003 Zernicka-Goetz et al.
`2003/0027783 Al
`2003/0108923 Al
`6/2003 Tuschl et al.
`7/2003 Klippel-Giese et al.
`2003/0135033 Al
`2003/0190635 Al* 10/2003 McSwiggen ................... 435/6
`2004/0053875 Al
`3/2004 Kreutzer et al.
`2004/0072779 Al
`4/2004 Kreutzer et al.
`2004/0102408 Al
`5/2004 Kreutzer et al.
`2004/0106569 Al
`6/2004 Klippel-Giese et al.
`2004/0175703 Al
`9/2004 Kreutzer et al.
`2004/0180351 Al
`9/2004 Giese et al.
`2004/0229266 Al * 11/2004 Tuschl et al ....................
`2005/0043263 Al
`2/2005 Giese et al.
`2005/0100907 Al
`5/2005 Kreutzer et al.
`2005/0142535 Al*
`6/2005 Darnha et al ...................
`2006/0094678 Al
`5/2006 Vornlocher et al.
`2006/0142226 Al
`6/2006 Po Ii sky et al.
`2006/0217329 Al
`9/2006 Feinstein
`2006/0240022 Al
`10/2006 Klippel-Giese et al.
`2007/0027097 Al
`2/2007 Lewis
`
`435/6
`
`435/5
`
`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`
`02018572.4
`
`8/2002
`
`(Continued)
`
`OTHER PUBLICATIONS
`
`Holen et al. Positional effects of short interfering RN As targeting the
`human coagulation trigger tissue factor. Nucleic Acids Research,
`2002, vol. 30, No. 8: 1757-1766. Oxford University Press.*
`
`(Continued)
`
`Primary Examiner-Sean McGarry
`Assistant Examiner-Kimberly Chong
`(74) Attorney, Agent, or Firm-Saliwanchik, Lloyd &
`Saliwanchik
`
`(57)
`
`ABSTRACT
`
`The present invention is related to a ribonucleic acid com(cid:173)
`prising a double stranded structure whereby the double(cid:173)
`stranded structure comprises a first strand and a second
`strand, whereby the first strand comprises a first stretch of
`contiguous nucleotides and whereby said first stretch is at
`least partially complementary to a target nucleic acid, and the
`second strand comprises a second stretch of contiguous
`nucleotides whereby said second stretch is at least partially
`identical to a target nucleic acid, and whereby the double
`stranded structure is blunt ended.
`
`7 Claims, 29 Drawing Sheets
`
`Alnylam Exh. 1020
`
`i
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`

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`US 7,452,987 B2
`Page 2
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`U.S. PATENT DOCUMENTS
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`2007 /0093445 Al
`2007/0135372 Al
`2007 /0265220 Al
`
`4/2007 Tuschl et al.
`6/2007 MacLachlan et al.
`11/2007 Rossi et al.
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`EP
`GB
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
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`1 230 375 Bl
`1 527 176 Bl
`1 407 044 Bl
`2417727 B
`WO 92/07065
`WO 92/19732
`WO 94/01550
`WO 96/18736
`WO 97/46570
`WO 98/05770
`WO 98/53083
`WO 99/15682
`WO 99/32619
`WO 99/49029
`WO 99/53050
`WO 99/55857
`WO 99/61631
`WO 00/01846
`WO 00/44495
`WO 00/44895
`WO 00/44914
`WO 00/63364
`WO 01/36646
`WO 01/75164 A2
`WO 02/16620
`WO 02/44321
`WO 02/055693 A2
`WO 02/097114
`WO 03/064441 A2
`WO 03/070895
`WO 03/070918
`WO 2004/015107 A3
`WO 2004/019973 Al
`WO 2004/035615 A2
`WO 2005/000320 A2
`WO 2005/105152
`WO 2006/023544 A2
`WO 2006/069782 A2
`WO 2007 /048244 A2
`WO 2007 /084684 A2
`
`7/2005
`1/2007
`9/2007
`1/2008
`4/1992
`11/1992
`* 1/1994
`6/1996
`12/1997
`2/1998
`11/1998
`4/1999
`7 /1999
`9/1999
`10/1999
`11/1999
`12/1999
`1/2000
`8/2000
`8/2000
`8/2000
`10/2000
`5/2001
`10/2001
`2/2002
`* 6/2002
`7/2002
`12/2002
`8/2003
`8/2003
`8/2003
`2/2004
`3/2004
`4/2004
`1/2005
`11/2005
`3/2006
`7/2006
`5/2007
`7/2007
`
`OTHER PUBLICATIONS
`
`The Merriam Webster Dictionary, pp. 37 and 540. 1997 Merriam(cid:173)
`Webster, Inc.*
`Holen et al. Positional effects of short intefering RN As targeting the
`human coagulation trigger Tissue Factor. Nucleic Acids Research
`(2002)vol. 30, No. 8: 1757-1766. Oxford University Press.*
`Mahato et al. Modulation of gene expression by antisense and
`antigene oligodeoxynucleotides and small interfering RNA. Expert.
`Opin. Deliv. 2005, vol. 2(1): 3-28.*
`Scherer et al. Approaches for the Sequence-specific knockdown of
`mRNA (Nat. Biotechnol., 2003, 21(12), pp. 1457-1465).*
`Zhang et al. Targeted Gene Silencing by Small Interfering RNA
`based Knock-down TEchnology. Current
`Pharmaceutical
`Biotechnology 2004, vol. 5, pp. 1-7.*
`Robert Barstead, "Genome-wide RN Ai", Current Opinion in Chemi(cid:173)
`cal Biology, 2001, pp. 63-66, vol. 5.
`David C. Baulcombe, "Fast forward genetics based on virus-induced
`gene silencing", Current Opinion in Plant Biology, 1999, pp. 109-
`113, vol. 2.
`Emily Bernstein et al., "Role for a bidentate ribonuclease in the
`initiation step of RNA interference", Nature, Jan. 18, 2001, p. 363-
`366, vol. 409.
`Eric Billy et al., "Specific Interference with Gene Expression
`Induced by Long, Double-Stranded RNA in Mouse Embryonal
`
`Teratocarcinoma Cell Lines", Proceedings of the National Academy
`of Sciences of the United States of America, Dec. 4, 2001, pp.
`14428-14433, vol. 98, No. 25.
`Christian Cazenave et al., "Rate of degradation of [a]- and [f:l](cid:173)
`oligodeoxynucleotides in Xenopus oocytes. Implications for anti(cid:173)
`messenger strategies", Nucleic Acids Research, 1987, pp. 10507-
`10521, vol. 15, No. 24.
`Sayda M. Elbashir et al., "Duplexes of21-nucleotide RNAs mediate
`RNA interference in cultured mammalian cells", Nature, May 24,
`2001, pp. 494-498, vol. 411.
`Andrew Fire, "RNA-triggered gene silencing", TIG, Sep. 1999, pp.
`358-363, vol. 15, No. 9.
`Jason R. Kennerdell et al., "Heritable gene silencing in Drosophila
`using double-standard RNA", Nature Biotechnology, Jul. 2000, pp.
`896-898, vol. 17.
`Jason R. Kennerdell et al., "Use of dsRNA-Mediated Genetic Inter(cid:173)
`ference to Demonstrate that frizzled and frizzled 2 Act in the Wing(cid:173)
`less Pathway", Cell, Dec. 23, 1998, pp. 1017-1026, vol. 95.
`Stuart K. Kim, "Functional genomics: The worm scores a knockout",
`Current Biology, 2001, pp. R85-R87, vol. 11.
`Ikuma Maeda et al., "Large-scale analysis of gene function in
`Caenorhabditis elegans by high-throughput RNAi", Current Biol(cid:173)
`ogy, 2001, pp. 171-176, vol. 11.
`Mary K. Montgomerry et al., "RNA as a Target of Double-Stranded
`RNA-Mediated Genetic Interference in Caenorhabditis elegans",
`Proceedings of the National Academy of Science of the United States
`of America, Dec. 22, 1998, pp. 15502-15507, vol. 95, No. 26.
`Antti Nykanen et al., "ATP Requirements and Small Interfering RNA
`Structure in the RNA Interference Pathway", Cell, No. 2, 2001, pp.
`309-321, vol. 107.
`Phillip A. Sharp, "RNA interference-200 l", Genes & Development,
`2001, pp. 485-490, vol. 15.
`Maria Sternberger et al., "GeneBlocs Are Powerful Tools to Study
`and Delineate Signal Transduction Process That Regulate Cell
`Growth and Transformation", Anti sense & Nucleic Acid Drug Devel(cid:173)
`opment, 2002, pp. 131-143, vol. 12.
`Thomas Tuschl et al., "Targeted mRNA degradation by double(cid:173)
`stranded RNA in vitro", Genes & Development, 1999, pp. 3191-
`3197, vol. 13.
`Peter M. Waterhouse et al., "Gene silencing as an adaptive defense
`against viruses", Nature, Jun. 14, 2001, pp. 834-842, vol. 411.
`Florence Wianny et al., "Specific interference with gene function by
`double-stranded RNA in early mouse development", Nature Cell
`Biology, Feb. 2000, pp. 70-75, vol. 2.
`Eric Wickstrom, "Oligodeoxynucleotide stability in subcellular
`extracts and culture media", Journal of Biochemical and Biophysical
`Methods, 1986, pp. 97-102, vol. 13.
`U.S. Appl. No. 60/352,873, filed Feb. 1, 2002, Darnha et al.
`Andrew J. Hamilton and David C. Balcombe, Species of Small
`Antisense RNA In Posttrancriptional Gene Silencing in Plants, Sci(cid:173)
`ence, vol. 286, Oct. 29, pp. 950-951.
`Andrew Fire, et al., Potent and specific genetic interference by
`double-stranded RNA in Caenorhabditis elegans, Nature O Macmil(cid:173)
`ian Publishers Ltd., 1993, vol. 391/191, pp. 806-811.
`Peter M. Waterhouse, et al., Virus resistance and gene silencing in
`plants can be induced by simultaneous expression of sense and
`antisense RNA, Communicated by W. James Peacock, Common(cid:173)
`wealth Scientific and Industrial Research Organization, Canberra,
`Australia, Aug. 17, 1998, vol. 95, pp. 13959-13964.
`Mohammad B. Baluamian, et al., Transcriptional and Post(cid:173)
`transcriptional Silencing of Rodent al(I) collagen by a Homologous
`Transcriptionally Self-Silenced Transgene, Molecular and Cellular
`Biology, Jan. 1999, vol. 19, No. 1. pp. 274-283.
`Phillip A. Sharp, RN Ai and Double-strand RNA, Genes & Develop(cid:173)
`ment, vol. 13, pp. 139-141, Cold Spring Harbor Laboratory Press,
`1999.
`Thomas Tusch, et al., Targeted mRNA Degradation by double(cid:173)
`stranded RNA in vitro, Genes & Development, vol. 13, pp. 3191-
`3197; Cold Spring Harbor Laboratory, 1999.
`News of the Week, Science, Canidate Gene Silencers Found, Fetal
`Cells Help Parkinson's Patients, Science, vol. 286, pp. 886, Oct. 29,
`1999.
`
`ii
`
`

`

`US 7,452,987 B2
`Page 3
`
`Rueyling Lin and Leon Avery, Policing Rogue Genes, Nature, vol.
`402, pp. 128-129, Nov. 11, 1999.
`Michael T. McManus, et al., Gene Silencing in Mannnals by Small
`Interfering RN As, Center for Cancer Research Massachussets, vol. 3,
`pp. 737-750,Oct. 2002.
`Mary K. Montgomery, et al., Double-stranded RNA As a Mediator in
`Sequence-Specific Genetic Silencing and Co-Suppression, TIG, vol.
`14, No. 7, pp. 255-256 and 258, Jul. 1999.
`John Wiley & Sons, Current Protocols in Molecular Biology, vol. 1,
`1999.
`Michael Y. X. Ma, et al., Design and Synthesis of RNA Miniduplexes
`via a Synthetic Linker Approach, Biochemistry, vol. 32, No. 7, 1993,
`pp. 1751-1758.
`Richard H. Griffey, et al., 2'-O-Aminoprophyl Ribonucleotides; A
`Zwitterionic Modification that Enhances the Exonuclease Resistance
`and Biological Activity of Antisense Oligonucleotides, J. Med.
`Chem, 1996, vol. 39, pp. 5100-5109.
`Jane A. Grasby, et al., Purine Functional Groups in Essential Resi(cid:173)
`dues of the Hairpin Ribozyme Required for Catalytic Cleavage of
`RNA, Biochemistry 1995, vol. 34, pp. 4068-4706.
`Thomas Horn, et al., Chemical Synthesis and Characterization of
`Branched Oligodeoxyribonucleotides (bDNA) For Use As Signal
`Amplifiers in Nucleic Acid Quantification Assays, Nucleic Acids
`Research, 1997, vol. 25, No. 23, pp. 4842-4849.
`Eugene Skripkin, et al., Psoralen Crosslinking Between Human
`Immunodeficiency Virus Type 1 RNA and Primer tRNA3Lys,
`Nucleic Acids Research, 1996, vol. 24, No. 3, pp. 509-514.
`Sergei M. Gryaznov, et al., Template Controlled Coupling and
`Recombination
`of Oligonucleotide
`Blocks
`Containing
`Thiophosphoryl Groups, Nucleic Acids Research, 1993, vol. 21, No.
`6, pp. 1403-1408.
`Ravinderjit S. Braich, et al., Regiospecific Solid-Phase Synthesis of
`Branched Oligonucleotides. Effect ofVicinal 2',5'-(or 2',3'-) and 3',
`5'-Phosphodiester Linkages on the Formation of Hairepin DNA,
`Bioconjugate Chem., 1997, vol. 8, pp. 370-377.
`Alokes Majumdar, et al., Targeted Gene Knockout Medicated by
`Triple Helix Forming Oligonucleotides, Nature Genetics, vol. 20,
`Oct. 1998, pp. 212-214.
`Dinesh A. Barawkar, et al., Synthesis, Biophysical Properties, and
`Nuclease
`Resistance
`Properties
`of Mixed Backbone
`Oligodeoxynucleotides Containing Cationic
`Internucleoside
`Guanidinium Linkages: Deoxynucleic Guanidine/DNA Chimeras,
`Proc. Natl. Acad. Sci. USA, vol. 95, pp. 11047-11052, Sep. 1998
`Chemistry, Biochemistry.
`Mark D. Pegram, et al., Phase II Study of Receptor-Enhanced
`Chemosensitivity Using Recombinant Humanized Anti-pl85her2/
`Neu Monoclonal Antibody Plus Cisplatin in Patients With HER2/
`neu-Overexpressing Metastatic Breast Cancer Refractory to Chemo(cid:173)
`therapy Treatment, Journal of Clinical Oncology, vol. 16, No. 8
`(Aug.), 1998: pp. 2659-2671.
`Sayda M. Elbashir, et al., Functional Anatomy of siRNAs for Medi(cid:173)
`ating Efficient RNAi in Drosophila melanogaster embryo Lysate,
`The EMBO Journal vol. 20, No. 23, pp. 6877-6888, 2001.
`Frank Czauderna, et al., Structural Variations and Stabilising Modi(cid:173)
`fications of Synthetic siRNAs in Mannnalian Cells, Nucleic Acids
`Research, 2003, vol. 31, No. 11, pp. 2705-2716.
`Dianne S. Schwarz, Evidence that siRNAs Function as Guides, Not
`Primers, in the Droshphila and Human RNAi Pathways, Molecular
`Cell, vol. 10, pp. 537-548, Sep. 2002.
`John G. Doench, et al., siRNAs Can Function as miRNAs, Genes &
`Development, vol. 17, pp. 438-442, 2003.
`Rosalind C. Lee, et al., The C. Elegans Heterochronic Gene Lin-4
`Encodes Small RNAs with Antisense Complementarity to Lin-14,
`Cell, vol. 75, pp. 843-854, Dec. 3, 1993.
`Eric G. Moss, et al., The Cold Shock Domain Protein Lin-28 Controls
`Developmental Timing in C. Elegans and is Regulated by the Lin-4
`RNA, Cell, vol. 88, pp. 637-646, Mar. 7, 1997.
`Yang Shi, et al., A CBP/p300 Homolog Specifies Multiple Differen(cid:173)
`tiation Patheways in Caenorhabditis elegans, Genes & Development,
`vol. 12, No. 7, pp. 943-955, Apr. 1, 1998.
`Jason R. Kennerdeil, et al., Use of dsRNA-Mediated Genetic
`Inberference to Demonstrate that Frizzled and Frizzled 2 Act in the
`Wingless Pathway, Cell, vol. 95, pp. 1017-1026, Dec. 23, 1998.
`
`Timmons L. Fire, A., Specific Interference by Ingested dsRNA.,
`Nature, Oct. 29, 1998, pp. 395.
`Wargelius A., Ellingsen S. Fjose A., Double-stranded RNA Induces
`Specific Development Defects in Zebrafish Embryos, Biochem
`Biophys Res Commun., Sep. 16, 1999, vol. 1, pp. 156-161.
`Philip D. Zamore, et al., RNAi: Double-Stranded RNA Directs the
`ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals,
`Cell, vol. 101, pp. 25-33, Mar. 31, 2000.
`Eugen Uhlmann, et al., Antisense Oligonucleotides: A New Thera(cid:173)
`peutic Principle, vol. 90, No. 4, pp. 553-584, Jun. 1990.
`Mary K. Montgomery, et al., RNA as a Target of Double-Stranded
`RNA-Mediated Genetic Interference in Caenorhabditis elegans,
`Proc. Nat'! Acad. Sci. USl, vol. 95, pp. 15502-15507, Dec. 1998.
`Florence Wianny, et al, Specific Interference with Gene Function by
`Double-Stranded RNA in Early Mouse Development, Nature Cell
`Biology, vol. 2, Feb. 2000, pp. 70-75.
`Borecky L, et al., Therapeutic Use of Double-Stranded RN As in man,
`Tex Rep Biol Med 1981-1982, vol. 41, pp. 575-581.
`Madhur Kumar, et al., Antisense RNA: Function and Fate of Duplex
`RNA in Cells of Higher Eukaryotes, Microbiology and Molecular
`Biology Reviews, Dec. 1998, pp. 1415-1434.
`R. Kreutzer, et al., Specific Inhibition of Viral Gene Expression by
`Double-Stranded RNA in vitro, Nature 391, pp. 806, 1998.
`Sudhir Agrawal, et al., Self-Stabilized Oligonucleotides as Novel
`Antisense Agents, Nucleic Acids, Res., vol. 21, 1993.
`Patrick J. Paddison, et al., Short Hairpin RNAs (shRNAs) Induce
`Sequence-Specific Silencing in Mannnalian Cells, Genes & Devel(cid:173)
`opment vol. 16, pp. 948-958, 2002.
`Sayda M. Elbashir, et al., Analysis of Gene Function in Somatic
`Mannnalian Cells Using Small Interfering RNAs, Methods, vol. 26,
`2002, pp. 199-213.
`Pierre G. Milhaud, et al., Free and Liposome-encapsulated Double(cid:173)
`Stranded RN As as Inducers of Interferon, Interleukin6, and Cellular
`Toxicity, Journal oflnterferon Research vol. 11, pp. 261-265, 1991.
`Jennifer Couzin, Breakthrough, Small RNAs Make Big Splash, Sci(cid:173)
`ence, vol. 298, pp. 2296-2297, Dec. 2002.
`Antonio Regalado, Turning Off Genes Sheds New Light On How
`They Work, The Wall Street Journal, Aug. 2002.
`Wess et al., Managing Complexity, Early Days for RN Ai, Biocentury,
`vol. 11, No. 12, pp. 123, Mar. 2003.
`Billy E, et al., Specific Interference with Gene Expression Induced by
`Long, Double-Stranded RNA in Mouse Embryonal Teratocarcinoma
`Cell Lines, PNAS, vol. 98, No. 25, Dec. 2001.
`Elbashir SM, et al., Duplexes of 21-Nucleotide RN As Mediate RNA
`Interference in Cultured Mannnalian Cells, Nature, vol. 411, pp.
`494-498, May 2001.
`Lipinski et al., Experimental and Computational Approaches to Esti(cid:173)
`mate Solubility and Permeability in Drug Discovery and Develop(cid:173)
`ment Settings, Adv. Drug Delivery Reviews vol. 23, pp. 3-25, 1997.
`Bhan et al., Nucleic Acid Research, vol. 25, 1997, pp. 3310.
`Li et al., Dev. Biology, vol. 210, 1999, pp. 238, Abstr. 346.
`Ngo et al., PNAS, vol. 95, Dec. 1998, p. 14687.
`Voinnet, 0., and Baulcombe, DC., Nature (1997) vol. 398, pp. 553.
`McCaffrey, AP et al., Nature (2000), vol. 418, pp. 38-39.
`Barber, GN et al., Mo!. and Cell. Biol. (1995), vol. 15, No. 6, p.
`3138-46.
`S. Blair Hedges, Nature Reviews, Genetics (2002), vol. 3, pp. 838-
`849.
`Hornung et al., Nature Medicine, vol. 11, No. 3, pp. 263-270, Mar.
`2005.
`Judge et al, Nature Biotechnology, vol. 23, No. 4, pp. 457-462, Apr.
`2005.
`Applicant's Response to the Written Opinion in the Examination
`proceedings Mar. 28, 2001.
`Brennicke et al., FEMS Microbiol. Rev., vol. 23, p. 297-316 1999.
`Perler, Nucl. Acids Res., vol. 30, No. I, pp. 383-384 2002.
`Zhao, et al., Developmental Biol., vol. 229, pp. 215-223, 2001.
`Hu-Lieskovan, et al, Cancer Res., vol. 65, No. 19, pp. 8984-8992,
`Oct. 1, 2005.
`Caplen, et al, PNAS, vol. 98, No. 17, pp. 9742-9747, Aug. 14, 2001.
`Hunter, et al., JBC, vol. 250, No. 2, pp. 409-417, Jan. 25, 1975.
`Manche et al., Mo!. Cell, Biol., vol. 12, No. 11, p. 5238-48, Nov.
`1992.
`
`iii
`
`

`

`US 7,452,987 B2
`Page 4
`
`Zeng, et al., RNA, vol. 8, pp. 855-860, 2002.
`Zheng, et al., RNA, vol. 10, pp. 1934-1945, 2004.
`Martinez et al., Cell, vol. 110, pp. 563-574, Jun. 9, 2002.
`Chien, et al., Cancer Gene Therapy, 1-8, 2004.
`Soutschek, et al., Nature, vol. 432, 1-8, 2004.
`Karberg, Mach <loch ma! das Gen aus, Die Ziet, No. 41, May 10,
`2006.
`Napoli, et al., Introduction of a Chimeric Chalcone Synthase Gene
`into Petunia Results in Reversible Co-Suppression of Homologous
`Genes in trans, The Plant Cell, vol. 2, pp. 279-289, Apr. 1990.
`Caplen et al; "Specific inhibition of gene expression by small double(cid:173)
`stranded RNAs in invertebrate and vertebrate systems", Proc Natl
`Acad Sci US A. Aug. 14, 2001;98(17):9742-7.
`Kraynack et al., "Small interfering RNAs containing full 2'-O(cid:173)
`methylribonucleotide-modified sense strands display Argonaute2/
`eIF2Cs-dependent activity." RNA. Jan. 2006;12(1):163-76.
`Chiu et al. "siRNA function in RN Ai: a chemical modification analy(cid:173)
`sis."RNA. 2003 ep;9(9): 1034-48.
`Griinweller et al. "Comparison of different antisense strategies in
`mammalian cells using locked nucleic acids, 2'-O-methyl RNA,
`phosphorothioates and small interfering RNA."Nucleic Acids Res.
`Jun. 15, 2003;31(12):3185-93.
`Morrissey et al"Activity of stabilized short interfering RNA in a
`mouse model of hepatitis B virus replication." Hepatology. Jun.
`2005;41(6): 1349-56.
`Choung et al "Chemical modification of siRNAs to improve serum
`stability without loss of efficacy."Biochem Biophys Res Commun.
`Apr. 14, 2006;342(3):919-27.
`Conrad et al. "Ribonuclease III: new sense from nuisance." Int J
`Biochem Cell Biol. Feb. 2002;34(2): 116-29.
`Zamore et al. "RNAi: double-stranded RNA directs the ATP-depen(cid:173)
`dent cleavage ofmRNA at 21 to 23 nucleotide intervals."Cell. Mar.
`31, 2000;101(1):25-33.
`Kalota et al. "2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F(cid:173)
`ANA) modified oligonucleotides (ON) effect highly efficient, and
`persistent,
`gene
`silencing."Nucleic Acids Res.
`Jan.
`18,
`2006;34(2):451-61. Print 2006.
`Williams,
`"Dicing with
`siRNA." Nat Biotechnol. Feb.
`2005;23(2): 181-2.
`Priority Document of9927444.l, Cancer Res. Camp. Technol. Ltd.
`Inhibiting Gene Expression, Pub!. Nov. 19, 1999, Issued Nov. 22,
`2000.
`Priority Document of U.S. Appl. No. 60/130,377, Method and Com(cid:173)
`positions for Inhibiting the Function of Polynucleotide, Issued Jun. 6,
`2000.
`Priority Document of U.S. Appl. No. 60/117,635, Double-Stranded
`RNA Blocks Specific Gene Expression in Multicellular Setting in in
`Vivo and in Vitro, Mar. 14, 2000.
`International Preliminary Examination Report, (English Translation)
`DE 0000244 (IPER), Jul. 7, 2003.
`P. Dan Cook, "Medicinal Chemistry of Antisense Oligoucleotides";
`(ed. Crroke, S.T.), pp. 29-56 Marcel Dekker, NY 2001.
`Parrish et al., "Functional Anatomy of a dsRNA Trigger:" (2000)
`Molecular Cell 6, 1077-1087.
`Bevilacqua and Cech; "Minor-Groove Recognition of Double(cid:173)
`Stranded RNA by the Double-Stranded RNA-Binding;" Biochemis(cid:173)
`try 1996, 35, 9983-9994.
`Wu, et al.,"Identification and Partial Purification of Human Double
`Strand RNase Activity"; The Journal of Biological Chem; vol. 273,
`No. 6, 2532-2542 (1998).
`in antisense
`M. Monoharan;"2'-Carbohydrate modifications
`oligonucleotide therapy ... ;" Biochimica et BiophysicaActa; ( 1999)
`117-130.
`Inoue et al., "Synthesis and hybridization studies on two complemen(cid:173)
`tary nona (2'O-methyl)ribonucleotides";Nucleic Acids Res. 1987,
`15:6131-48.
`U.S. Appl. No. 10/205,309; (priority document No. 1, filed Jul. 25,
`2002.
`Paddison, et al.,"Stable suppression of gene expression by RNAi in
`mammalian cells;"PNAS, 99(3); 99 1443-1448; (2002).
`Park, et al., "Prevention ofHIV-1 infection in human peripheral blood
`mononuclear cells by specific RNA interference;" Nucleic Acids
`Res., vol. 30, No. 22, pp. 4830-4835; (2002).
`
`Harborth, et al., Identification of essential genes in cultured mamma(cid:173)
`lian cells using small interfering RNAs; J Cell Science; 114: pp.
`4557-4565; (2001).
`Kurreck, "Improvement through novel chemical modifications;" Eur.
`J. Biochem, 270, 1628-1644 (2003).
`Rose, et al., "Functional polarity is introduced by Dicer processing of
`short substrate RNAs;"Nucleic Acids Research, 2005, vol. 33, No.
`13, pp. 4140-4156.
`U.S. Appl. No. 60/352,873, filed Feb. 1, 2002, Darnha, M. J. et al.
`U.S. Appl. No. 60/358,580, filed Feb. 20, 2002, Beigelman, L. et al.
`U.S. Appl. No. 60/363,124, filed Mar. 11, 2002, Beigelman, L. et al.
`U.S. Appl. No. 60/386,782, filed Jun. 6, 2002, Biegelman, L. et al.
`Amarzguioui, M. et al. "Tolerance for mutations and chemical modi(cid:173)
`fications in a siRNA", Nucleic Acids Research, 2003, pp. 589-595,
`vol. 31, No. 2.
`Bertrand, J. et al. "Comparison of antisense oligonucleotides and
`siRNAs in cell culture and in vivo", Biochemical and Biophysical
`Research Communications, 2002, pp. 1000-1004, vol. 296.
`Billy, E. et al. "Specific interference with gene expression induced by
`long, double-stranded RNA in mouse embryonal teratocarcinoma
`cell lines", PNAS, Dec. 4, 2001, pp. 14428-14433, vol. 98, No. 25.
`Boutla, A. et al. "Short 5'-phosphorylated doubled-stranded RNAs
`induce RNA interference in Drosophila", Current Biology, Nov. 13,
`2001, pp. 1776-1780, vol. 11.
`Braasch, D.A. et al. "RNA Interference in Mammalian Cells by
`Chemically-Modified RNA", Biochemistry, 2003, pp. 7967-7975,
`vol. 42.
`Brown, K. M. et al. "Target accessibility dictates the potency of
`human RISC", Nature Structural & Molecular Biology, May 2005,
`pp. 469-470, vol. 12, No. 5.
`Chiu, Y. et al. "RNAi in Human Cells: Basic Structural and Func(cid:173)
`tional Features of Small Interfering RNA", Molecular Cell, Sep.
`2002, pp. 549-561, vol. 10.
`Clayton, J. "The Silent Treatment", Nature, Sep. 30, 2004, pp. 599-
`605, vol. 431.
`Clemens, M. J. "PKR- A Protein Kinase Regulated by Double(cid:173)
`stranded RNA", Int. J Biochem. Cell Biol., 1997, pp. 945-949, vol.
`29, No. 7.
`Clemens, J.C. et al. "Use of double-stranded RNA interference in
`Drosophilia cell lines to dissect signal transduction pathways",
`PNAS, Jun. 6, 2000, pp. 6499-6503, vol. 97, No. 12.
`Crooke, R. M. et al. "Metabolism of Antisense Oligonucleotides in
`Rat Liver Homogenates", The Journal of Pharmacology and Experi(cid:173)
`mental Therapeutics, 2000, pp. 140-149, vol. 292.
`Czauderna, F. et al. "Functional Studies of the PI(3)-kinase signaling
`pathway employing synthetic and expressed siRNA", Nucleic Acids
`Research, 2003, pp. 670-682, vol. 31, No. 2.
`Dorsett, Y. et al. "siRNAs: Applications in Functional Genomics and
`Potential as Therapeutics", Drug Discovery, Apr. 2004, pp. 318-329,
`vol. 3.
`Gary, D. J. et al. "Polymer-based siRNA delivery: Perspectives on the
`fundamental and phenomenological distinctions from polymer(cid:173)
`based DNA delivery", Journal of Controlled Release, 2007, pp.
`64-73, vol. 121.
`Hall,
`I. M. et al "Establishment and Maintenance of a
`Heterochrornatin Domain", Science, Sep. 27, 2002, pp. 2232-2237,
`vol. 297.
`Hammond, S. M. et al. "Argonaute2, a Link Between Genetic and
`Biochemical Analyses ofRNAi", Science, Aug. 10, 2001, pp. 1146-
`1150, vol. 293.
`Hammond, S.M. et al. "An RNA-directed nuclease mediates post(cid:173)
`transcriptional gene silencing in Drosophila cells", Nature, Mar. 16,
`2000, pp. 293-296, vol. 404.
`Hannon, G. J. "RNA interference", Nature, Jul. 11, 2002, pp. 244-
`251, vol. 418.
`Harborth, J. et al. "Sequence, Chemical, and Structural Variation of
`Small Interfering RNAs and Short Hairpin RNAs and the Effect on
`Mammalian Gene Silencing", Antisense and Nucleic Acid Drug
`Development, 2003, pp. 83-105, vol. 13.
`Hoerter, J. A. et al. "Chemical Modification Resolves the Asymmetry
`of siRNA Strand Degradation in Human Blood Serum", RNA, 2007,
`pp. 1-7, vol. 13.
`
`iv
`
`

`

`US 7,452,987 B2
`Page 5
`
`Hu, X. et al. "Relative gene-silencing efficiencies of small interfering
`RN As targeting sense and antisense transcripts from the same genetic
`locus", Nucleic Acids Research, 2004, pp. 4609-4617, vol. 32, No.
`15.
`Inou, A. et al. "Molecular Design and Delivery of siRNA", Journal of
`Drug Targeting, 2006, pp. 448-455, vol. 14, No. 7.
`Jackson, A. L. et al. "Position-specific chemical modification of
`siRNAs reduces "off-target" transcript silencing", RNA, 2006, pp.
`1197-1205, vol. 12.
`Klippel, A. et al. "The Interaction of Small Domains between the
`Subunits of Phosphatidylinositol 3-Kinase Determines Enzyme
`Activity", Molecular and Cellular Biology, Apr. 1994, pp. 2675-
`2685, vol. 14, No. 4.
`Koller, E. et al. "Competition for RISC binding predicts in vitro
`potency of siRNA", Nucleic Acids Research, 2006, pp. 4467-4476,
`vol. 34, No. 16.
`Layzer, J. M. et al. "In vivo activity of nuclease-resistant siRNAs",
`RNA,2004,pp. 766-771,vol. 10.
`Leenders, F. et al. "PKN3 is required for malignant prostate cell
`growth downstream of activated PI 3-kinase", The EMBO Journal,
`2004, pp. 3303-3313, vol. 23, No. 16.
`Lima, W. F. et al. "Structural Requirements at the Catalytic Site of the
`Heteroduplex Substrate of Human RNase Hl Catalysis", The Journal
`of Biological Chemistry, Aug. 27, 2004, pp. 36317-36326, vol. 279,
`No. 35.
`Lubini, P. et al. "Stabilizing effects of the RNA 2'-substituent: crystal
`structure of an oligodeoxynucleotide duplex containing 2'-O(cid:173)
`methylated adenosines", Chemistry &Biology, Sep. 1994, pp. 39-45,
`vol. 1.
`Manoharan, M. "RNA interference and chemically modified small
`interfering RNAs", Current Opinion in Chemical Biology, 2004, pp.
`570-579, vol. 8.
`Martinez, J. et al. "RISC is a 5' phosphomonoester-producing RNA
`endonuclease", Genes & Development, 2004, pp. 975-980, vol. 18.
`Martinez, J. et al. "Single-Stranded Antisense siRNAs Guide Target
`RNA Cleavage in RNAi", Cell, Sep. 6, 2002, pp. 563-574, vol. 110.
`Morrissey, D. V. et al. "Chemical Modification of Synthetic siRNA",
`Pharmaceutical Discovery, May 13, 2005.
`Oliveira, S. et al. "Targeted Delivery of siRNA",Journal of Biomedi(cid:173)
`cine and Biotechnology, 2006, pp. 1-9, vol. 2006.
`Paddison, P. J. et al. "Stable suppression of gene expression by RN Ai
`in mammalian cells", PNAS, Feb. 5, 2002, pp. 1443-1448, vol. 99,
`No.3.
`Pancoska, P. et al. "Efficient RNA interference depends on global
`context of the target sequence: quantitative analysis of silencing
`efficiency using Eulerian graph representation of siRNA", Nucleic
`Acids Research, 2004, pp. 1469-1479, vol. 32, No. 4.
`Parrish, S. et al. "Functional Anatomy of a dsRNA Trigger: Differ(cid:173)
`ential Requirement for the Two Trigger Strands in RNA Interfer(cid:173)
`ence", Molecular Cell, Nov. 2000, pp. 1077-1087, vol. 6.
`
`Patel, D. J. et al. "Structural Biology of RNA Silencing and Its
`Functional Implications", Cold Spring Harbor Symposia on Quan(cid:173)
`titative Biology, 2006, pp. 81-93, vol. LXXI.
`Peek, A. S. et al. "Design of Active Small Interfering RN As", Current
`Opinion in Molecular Therapeutics, 2007, pp. 110-118, vol. 9, No. 2.
`Pei, Y. et al. "On the Art of Identifying Effective and Specific
`siRNAs", Nature Methods, Sep. 2006, pp. 670-676, vol. 3, No. 9.
`Potera, C. "Antisense- down, but not out", Nature Biotechnology,
`May 2007, pp. 497-499, vol. 25, No. 5.
`Rana, T. M. "Illuminating the silence: understanding the structure
`and function of small RN As", Molecular Cell Biology, Jan. 2007, pp.
`23-36, vol. 8.
`Rand, T. A. et al. "Argonaute2 Cleaves the Anti-Guide Strand of
`siRNA during RISC Activation", Cell, Nov. 18, 2005, pp. 621-629,
`vol. 123.
`Rossi, J. J. "A Cholesterol connection in RNAi", Nature, Nov. 11,
`2004, pp. 155-156, vol. 432.
`Santel, A. "Get the balance right: Mitofusins roles in health and
`disease", Biochimica et Biophysica Acta, 2006, pp. 490-499, vol.
`1763.
`Santel, A. et al. "RNA interference in the mouse vascular
`endothelium by systemic administration of siRNA-lipoplexes for
`cancer therapy", Gene Therapy, 2006, pp. 1360-1370, vol. 13.
`Santel, A. et al. "A novel siRNA-lipoplex technology for RNA inter(cid:173)
`ference in the mouse vascular endothelium", Gene Therapy, 2006,
`pp. 1222-1234, vol. 13.
`Scherer, L. J. et al. "Approaches for the sequence-specific knock(cid:173)
`down ofmRNA", Nature Biotechnology, Dec. 2003, pp. 1457-1465,
`vol. 21, No. 12.
`Schwarzer, R. et al. "TRB3 is a PI 3-kinase dependent indicator for
`nutrient starvation", Cellular Signalling, 2006, pp. 899-909, vol. 18.
`Snove, 0. et al. "Chemical Modifications Rescue Off-Target Effects
`ofRNAi", ACS Chemical Biology, 2006, pp. 274-276, vol. 1, No. 5.
`Turner, J. J. et al. "MALDI-TOF mass spectral analysis of siRNA
`degradation in serum confirms an RNAse A-like activity", Molecular
`BioSystems, 2007, pp. 43-50, vol. 3.
`Tuschl, T. "Expanding small RNA interference", Nature Biotechnol(cid:173)
`ogy, May 2002, pp. 446-448, vol. 20.
`Tuschl, T. et al. "Small Interfering RNAs: A Revolutionary Tool for
`the Analysis of Gene Function and Gene Therapy", Molecular Inter(cid:173)
`ventions, Jun. 2002, pp. 158-167, vol. 2, Issue 3.
`Volpe, T. A. et al. "Regulation of Heterochromatic Silencing and
`Histone H3 Lysine-9 Methylation by RN Ai", Science, Sep. 13, 2002,
`pp. 1833-1837, vol. 297.
`Wilson, C. et al. "Building oligonucleotide therapeutics using non(cid:17