(12) United States Patent
`Hannon et a].
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 8,153,776 B2
`*Apr. 10, 2012
`
`US008153776B2
`
`METHODS AND COMPOSITIONS FOR RNA
`INTERFERENCE
`
`2004/0229266 A1 11/2004 Tuschlet a1.
`2005/0164210 A1
`7/2005 Mittal et al.
`2005/0197315 A1
`9/2005 Taira et a1.
`
`FOREIGN PATENT DOCUMENTS
`2470903
`7/2003
`CA
`1462525
`9/2004
`EP
`WO-94/01550
`1/1994
`WO
`99/32619
`7/1999
`WO
`WO-99/32619
`7/1999
`WO
`WO-99/49029
`9/1999
`WO
`WO-00/01846
`1/2000
`WO
`WO-00/44895
`8/2000
`WO
`WO-00/44914
`8/2000
`WO
`WO-00/63364
`10/2000
`WO
`WO-01/29058
`4/2001
`WO
`WO-01/36646
`5/2001
`WO
`WO-01/48183
`7/2001
`WO
`WO-01/49844
`7/2001
`WO
`WO-01/68836
`9/2001
`WO
`WO-01/75164
`10/2001
`WO
`WO-02/44321
`6/2002
`WO
`WO-02/059300
`8/2002
`WO
`WO-02/068635
`9/2002
`WO
`WO-03/020931
`3/2003
`WO
`WO WO-2004/029219
`4/2004
`
`OTHER PUBLICATIONS
`
`Caplen et a1. Speci?c inhibition of gene expression by small double
`stranded RNAs in invertebrate and vertebrate systems. PNAS 2001,
`vol. 98, No. 17: 9742-9747.*
`Agrawal, et al., “Antisense therapeutics: is it as simple as comple
`mentary base recognition?,” Molecular Medicine Today, 61:72-81
`(2000).
`Ambros, “Dicing Up RNAs,” Science 293: 811-813 (2001).
`Bass, “Double-Stranded RNA as a Template for Gene Silencing,”
`Cell, 101:235-238 (2000).
`Baulcombe, “Gene silencing: RNA makes RNA makes no protein,”
`Curr. Biol, 9:R599-R601 (1999).
`Baulcombe, “RNA as a target and an initiator of post-transcriptional
`gene silencing in transgenic plants,” Plant Mol. Biol., 32:79-88
`(1996).
`Bernstein, et al., “Dicer is essential for mouse development,” Nat
`Genet., 35(3):215-7 (2003).
`Bernstein, et al., “Role for a bidentate ribonuclease in the initiation
`step of RNA interference,” Nature 409(6818):363-6 (2001).
`Bernstein, et al., “The rest is silence,” RNA 7(11):1509-21 (2001).
`Bohmert, et al., “AGOl de?nes a novel locus ofArabidopsis control
`ling leaf development,” EMBO J., 17: 170-180 (1998).
`(Continued)
`
`Primary Examiner * Kimberly Chong
`(74) Attorney, Agent, or Firm *Wilmer Cutler Pickering
`Hale and Dorr LLP
`
`(57)
`
`ABSTRACT
`
`The present invention provides methods for attenuating gene
`expression in a cell, especially in a mammalian cell, using
`gene-targeted double stranded RNA (dsRNA), such as a hair
`pin RNA. The dsRNA contains a nucleotide sequence that
`hybridiZes under physiologic conditions of the cell to the
`nucleotide sequence of at least a portion of the gene to be
`inhibited (the “target” gene).
`
`10 Claims, 68 Drawing Sheets
`
`(54)
`
`(75)
`
`Inventors: Gregory J. Hannon, Huntington, NY
`(US); Patrick Paddison, Seattle, WA
`(US); Emily Bernstein, New York, NY
`(US); Amy Caudy, LaWrenceville, NJ
`(US); Douglas Conklin, Cold Spring
`Harbor, NY (US); Scott Hammond,
`Cold Spring Harbor, NY (US)
`
`(73)
`
`Assignee: Cold Spring Harbor Laboratory, Cold
`Spring Harbor, NY (US)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is subject to a terminal dis
`claimer.
`
`(21)
`
`Appl. N0.: 11/894,676
`
`(22)
`
`Filed:
`
`Aug. 20, 2007
`
`(65)
`
`Prior Publication Data
`
`US 2008/0213861A1
`
`Sep. 4, 2008
`
`Related US. Application Data
`
`(63)
`
`Continuation of application No. 10/ 997,086, ?led on
`Nov. 23, 2004, and a continuation-in-part of
`application No. 10/055,797, ?led on Jan. 22, 2002.
`
`(51) Int. Cl.
`(2006.01)
`C07H 21/04
`(52) US. Cl. ................. .. 536/245; 536/24.31; 536/241;
`435/6; 435/325; 435/375; 514/44
`(58) Field of Classi?cation Search ...................... .. None
`See application ?le for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`5,246,921 A
`9/1993 Reddy et a1.
`5,624,803 A *
`4/1997 Noonberg et al. .............. .. 435/6
`5,814,500 A
`9/1998 DietZ
`5,998,148 A 12/1999 Bennett et al.
`6,107,027 A
`8/2000 Kay et al.
`6,130,092 A 10/2000 Lieber et a1.
`6,326,193 B1
`12/2001 Liu et al.
`6,506,559 B1
`1/2003 Fire et al.
`6,541,248 B1
`4/2003 Kingsman et al.
`6,573,099 B2
`6/2003 Graham et al.
`6,605,429 B1
`8/2003 Barber et al.
`7,691,995 B2
`4/2010 Zamore et al.
`2002/0086356 A1
`7/2002 Tuschl et al.
`2002/0114784 A1
`8/2002 Li et al.
`2002/0160393 A1* 10/2002 Syrnonds et a1. ............... .. 435/6
`2003/0051263 A1
`3/2003 Fire et al.
`2003/0055020 A1
`3/2003 Fire et al.
`2003/0056235 A1
`3/2003 Fire et al.
`2003/0084471 A1
`5/2003 Beach et al.
`2004/0001811 A1
`1/2004 Kreutzer et al.
`2004/0018999 A1
`1/2004 Beach et al.
`2004/0086884 A1
`5/2004 Beach et al.
`2004/0102408 A1 *
`5/2004 Kreutzer et al. .............. .. 514/44
`
`Benitec - Exhibit 1001 - page 1
`
`

`
`US 8,153,776 B2
`Page 2
`
`OTHER PUBLICATIONS
`Bosher, et al., “RNA Interference Can Target Pre-mRNA: Conse
`quences for Gene Expression in a Caenorhabditis elegans Operon,”
`Genetics, 153:1245-1256 (1999).
`Bosher, et al., “RNA interference: genetic wand and genetic watch
`dog,” Nat. Cell Biol., 2:E31-36 (2000).
`Caplen, N.J., et al., “dsRNA-mediated gene silencing in cultured
`Drosophila cells: a tissue culture model for the analysis of RNA
`interference,” Gene, 252:95-105 (2000).
`Caplen, N.J., et al., “RNAi as a gene therapy approach,” Expert Opin.
`Biol. Ther., 3(4):575-586 (2003).
`Carmell et al., “The Argonaute family: tentacles that reach into
`RNAi, developmental control, stem cell maintenance, and
`tumorigenesis,” Genes Dev., 16(21):2733-42 (2002).
`Carmell MA, et al., “RNase III enzymes and the initiation of gene
`silencing,” Nat Struct Mol Biol., 11(3):214-8 (2004).
`Carmell, et al., “Germline transmission of RNAi in mice,” Nat Struct
`Biol., 10(2):91-2 (2003).
`Catalanotto, et al. “Gene silencing in worms and fungi,” Nature
`404:245 (2000).
`Caudy, et al., “A micrococcal nuclease homologue in RNAi effector
`complexes,” Nature 425(6956):411-4 (2003).
`Caudy, et al., “Fragile X-related protein and VIG associate with the
`RNA interference machinery,” Genes Dev., 16(19):2491-6 (2002).
`Caudy, et al., “Induction and biochemical puri?cation of RNA-in
`duced silencing complex from Drosophila S2 cells,” Methods Mol.
`Biol., 265:59-72 (2004).
`Check, E., “RNA to the rescue? Disease therapies based on a tech
`nique for gene silencing called RNA interference are racing towards
`the clinic. Erika Check investigates molecular medicine’s next big
`thing,” Nature, 425:10-12 (2003).
`Cleary, et al., “Production of complex nucleic acid libraries using
`highly parallel in situ oligonucleotide synthesis,” Nat Methods,
`1(3):241-8 (2004).
`Cogoni, et al., “Gene silencing in Neurospora crassa requires a
`protein homologous to RNA-dependent RNA polymerase,” Nature
`399:166-169 (1999).
`Cogoni, et al., “Posttranscriptional Gene Silencing in Neurospora by
`a RecQ DNA Helicase,” Science, 286:2342-2344 (1999).
`Connelly, et al., “The sbcC and sbcD genes of Escherichia coli
`encode a nuclease involved in palindrome inviability and genetic
`recombination,” Genes Cell 1:285-291 (1996).
`Crooke, “Basic Principles of Antisense Therapeutics,” Antisense
`Research and Application, Chapter 1, Springer-Verlag, New York
`(1998).
`Dalmay, et al., “An RNA-Dependent RNA Polymerase Gene in
`Arabidopsis is Required for Posttranscriptional Gene Silencing
`Mediated by a Transgene but Not by a Virus,” Cell, 101:543-553
`(2000).
`Denli, et al., “Processing of primary microRNAs by the Micropro
`cessor complex,” Nature, 432(70l4):231-5 (2004).
`Denli, et al., “RNAi: an ever-growing puzzle,” Trends Biochem. Sci.,
`28(4):196-201 (2003).
`Di Nocera, et al., “Transient expression of genes introduced into
`cultured cells ofDrosophila,” PNAS, 80:7095-7098 (1983).
`Eck, et al., “Gene-based therapy, Goodman & Gilman’s,” The Phar
`macological Basis of Therapeutics, 9th Edition, 5:77-101 (1996).
`Elbashir, et al., “Functional anatomy of siRNAs for mediating ef?
`cient RNAi in Drosophila melanogaster embryo lysate,” The EMBO
`Journal, 20(23):6877-6888 (2001).
`Fagard, et al., “AG01, QDE-2, and RDE-l are related proteins
`required for post-transcriptional gene silencing in plants, quelling in
`fungi, and RNA interference in animals,” PNAS 97:11650-11654
`(2000).
`Fire, “RNA-triggered gene silencing,” Trends Genet., 15:358-363
`(1999).
`Fire, et al. “Potent and speci?c genetic interference by double
`stranded RNA in Caenorhabditis elegans,” Nature, 391:806-811
`(1998).
`Fortier, “Temperature-Dependent Gene Silencing by an Expressed
`Inverted Repeat in Drosophila,” Genesis 26:240-244 (2000).
`Fraser, “Human Genes Hit the Big Screen,” Nature, 428:375-378
`(2004).
`
`Gillespie, et al., “Homeless is required for RNA localization in
`Drosophila oogenesis and encodes a new member of the DE-H fam
`ily of RNA-dependent ATPases,” Genes Dev. 9:2495-2508 (1995).
`Good et al., “Expression of small, therapeutic RNAs in human cell
`nuclie,” Gene Therapy 4:45- 54 (1997).
`Guo, “par-1, a Gene Required for Establishing Polarity in C. elegans
`Embryos, Encodes a Putative Ser/Thr Kinase that is Asyrnmetrically
`Distributed,” Cell 81 :61 1-620 (1995).
`Gupta, et al., “Inducible, reversible, and stable RNA interference in
`mammalian cells,” Proc Natl Acad Sci USA 101(7): 1927-32 (2004).
`Hamilton, et al., “A Species of Small Antisense RNA in Post
`transcriptional Gene Silencing in Plants,” Science 286:950-952
`(1999).
`Hammond, et al., “An RNA-directed nuclease mediates post-tran
`scriptional gene silencing in Drosophila cells,” Nature 404:293 -296
`(2000).
`Hammond, SM, et al., “Post-transcriptional gene silencing by
`double-stranded RNA,” Nat Rev Genet. 2(2): 1 10-9 (2001).
`Hammond, S., et al., “Argonaute2, a Link Between Genetic and
`Biochemical Analyses RNAi,” Science, 293: 1 146-1 150 (2001).
`Hannon, “RNA interference,” Nature 418(6894):244-51 (2002).
`Hannon, et al., “RNA interference by short hairpin RNAs expressed
`in vertebrate cells,” Methods Mol Biol., 257:255-66 (2004).
`Hannon, et al., “Unlocking the potential of the human genome with
`RNA interference,” Nature, 431(7006):371-8 (2004).
`Hasuwa, H., et al., “Small interfering RNA and gene silencing in
`transgenic mice and rats,” FEBS Letters, 532:227-230 (2002).
`He, et al., “A microRNA polycistron as a potential human oncogene,”
`Nature, 435(7043):828-33 (2005).
`He, et al., “MicroRNAs: small RNAs with a big role in gene regula
`tion,” Nat Rev Genet., 5(7):522-31 (2004).
`Hemann, et al., “An epi-allelic series of p53 hypomorphs created by
`stable RNAi produces distinct tumor phenotypes in vivo,” Nat Genet.
`33(3):396-400 (2003).
`Hunter, “Genetics: A touch of elegance with RNAi,” Curr. Biol.,
`9zR440-R442 (1999).
`Jackson, et al., “Expression pro?ling reveals off-target gene regula
`tion by RNAi”, Nature Biotechnology 21(6), 635-638 (2003).
`Jacobsen, et al., “Disruption of an RNA helicase/RNAse III gene in
`Arabidopsis causes unregulated cell division in ?oral meristems,”
`Development 126:5231-5243 (1999).
`Jen, K.Y., et al., “Suppression of Gene Expression by Targeted Dis
`ruption of Messenger RNA: Available Options and Current Strate
`gies,” Stem Cells, 18:307-319 (2000).
`Jones, et al., “De novo methylation and co-suppression induced by a
`cytoplamically replicating plant RNA virus,” EMBO J. 17:6385
`6393 (1998).
`Jones, et al., “RNA-DNA Interactions and DNA Methylation in P0 st
`Transcriptional Gene Silencing,” Plant Cell, 11:2291-2301 (1999).
`Jorgensen, et al., “An RNA-Based Information Superhighway in
`Plants,” Science, 279: 1486-1487 (1998).
`Kalejta, et al., “An Integral Membrane Green Fluorescent Protein
`Marker, Us9-GFP, is Quantitatively Retained in Cells during
`Propidium Iodide-Based Cell Cycle Analysis by Flow Cytometry,”
`Exp. Cell. Res. 248:322-328 (1999).
`Kennerdell, et al., “Heritable gene silencing in Drosophila using
`double-stranded RNA,” Nat. Biotechnol., 17:896-898 (2000).
`Kennerdell, et al., “Use of dsRNA-Mediated Genetic Interference to
`Demonstrate that frizzled and frizzled 2 Act in the Wingless Path
`way,” Cell 95:1017-1026 (1998).
`Ketting, et al., “mut-7 of C. elegans, Required for Transposon Silenc
`ing and RNA Interference, Is a Homolog of Werner Syndrome
`Helicase and RNaseD,” Cell 99:133-141 (1999).
`Ketting, R. F. et al., “Dicer functions in RNA interference and in
`synthesis of small RNA involved in developmental timing in C.
`elegans”, Genes Dev 15:2654-2659 (2001).
`Kramer, et al., “Activation of the human anaphase-promoting com
`plex by proteins of the CDC20/Fizzy family,” Curr. Biol. 8:1207
`1210 (1998).
`Lam, et al., “Inducible expression of double-stranded RNA directs
`speci?c genetic interference in Drosophila,” Curr. Biol., 10:957-963
`(2000).
`
`Benitec - Exhibit 1001 - page 2
`
`

`
`US 8,153,776 B2
`Page 3
`
`Lee, et al., “Distinct Roles for Drosophila Dicer-l and Dicer-2 in the
`siRNNmiRNA Silencing Pathways”, Cell 117:69-81 (2004).
`Lingel, et al., “Nucleic acid 3‘-end recognition by the Argonaute2
`PAZ domain,” Nature Structural & Molecular Biology, 11(6):576
`577 (2004).
`Lipardi, et al., “RNAi as Raondon Degradative PCR: siRNA Primers
`Convert mRNA into dsRNAs that are Degraded to Generate New
`siRNAs,” Cell , 107:297-307 (2001).
`Liu J, et al., MicroRNA-dependent localization of targeted mRNAs
`to mammalian P-bodies, Nat Cell Biol. 7(7):719-23 (2005); Epub
`Jun. 5, 2005.
`Liu, et al., “Argonaute2 is the catalytic engine of mammalian RNAi,”
`Science, 305(5689):1437- 41 (2004).
`Lohmann, et al., “Silencing of Developmental Genes in Hydra,” Dev.
`Biol., 214: 211-214 (1999).
`Lund, et al., “Nuclear Export of MicroRNA Precursors,” Science
`303:95-98 (2004).
`Manche, et al., “Interactions between Double-Stranded RNA Regu
`lators and the Protein Kinase DAI,” Molecular and Cellular Biology,
`12(11):5238-5248 (1992).
`Marshall, “Gene therapy’s growing pains,” Science, 269:1050-1055
`(1995).
`Matsuda, et al., “Molecular cloning and characterization of a novel
`human gene (HERNA) which encodes a putative RNA-helicase,”
`Biochim. Biophys., Acta 1490:163-169 (2000).
`McCaffrey, et al., “RNA interference in adult mice,” Nature
`418(6893):38-9 (2002).
`Mette, et al., “Transcriptional silencing and promoter methylation
`triggered by double stranded RNA,” The EMBO Journal,
`19(19):5194-5201 (2000).
`Misquitta, et al., “Targeted disruption of gene function in Drosophila
`by RNA interference (RNA-i): A role for nautilus in embryonic
`somatic muscle formation,” PNAS 96: 1451-1456 (1999).
`Montgomery, et al., “Double-stranded RNA as a mediator in
`sequence-speci?c genetic silencing and co-suppression,” Trends
`Genet, 14:255-258 (1998).
`Montgomery, M.K. et al., “RNA as a target of double-stranded RNA
`mediated genetic interference in Caenorhabditis elegans,” PNAS
`95:15502-15507 (1998).
`Moss, Eric G., “RNA interference: It’s a small RNA world,” Current
`Biology, 11(19):R772-R775 (2001).
`Mourrain, et al., “Arabidopsis SGS2 and SGS3 Genes are Required
`for Posttranscriptional Gene Silencing and Natural Virus Resis
`tance,” Cell 101:533-542 (2000).
`Murchison, et al ., “miRNAs on the move: miRNA bio genesis and the
`RNAi machinery,” Curr Opin Cell Biol. 16(3):223 -9 (2004).
`Ngo, et al., “Double-stranded RNA induces mRNA degradation
`inTrypanosoma brucei,” PNAS 95:14687-14692 (1998).
`Novina, et al., “The RNAi Revolution,” Nature 430: 161-164 (2004).
`Opalinska, et al., “Nucleic acid based therapeutics: basic principals
`and recent applications,” Nature Reviews: Drug Discovery, 1:503
`514 (2002).
`Paddison, et al., “A resource for large-scale RNA-interference-based
`screens in mammals,” Nature, 428(6981):427-31 (2004).
`Paddison, et al., “Cloning of short hairpin RNAs for gene knockdown
`in mammalian cells,” Nature Meth., 1(2):163-167 (2004).
`Paddison, et al., “RNA interference: the new somatic cell genetics?”
`Cancer Cell, 2(1):17-23 (2002).
`Paddison, et al., “Short hairpin activated gene silencing in mamma
`lian cells,” Methods Mol Biol., 265:85-100 (2004).
`Paddison, et al., “Short hairpin RNAs (shRNAs) induce sequence
`speci?c silencing in mammalian cells,” Genes & Development,
`16:948-958 (2002).
`Paddison, et al., “siRNAs and shRNAs: skeleton keys to the human
`genome,” Curr Opin Mol Ther., 5(3):217-24 (2003).
`Paddison, et al., “Stable suppression of gene expression by RNAi in
`mammalian cells,” 99(3): 1443-1448 (2002).
`Paroo, et al., “Challenges for RNAi in vivo,” TRENDS in Biotechnol
`ogy 22:390-394 (2004).
`Pham, et al., “A Dicer-2-Dependent 80S Complex Cleaves Targeted
`mRNAs during RNAi in Drosophila,” Cell 117:83-94 (2004).
`
`Piccin, et al., “Ef?cient and heritable functional knock-out of an adult
`phenotype in Drosophilia using a GAL4 -driven hairpin RNA incor
`porating a heterologous spacer,” Nucleic Acids Research,
`29(12)e55:1-5 (2001).
`Qi, et al., “Biochemical Specialization within Arabidopsis RNA
`Silencing Pathways,” Mol Cell. 19(3):421-8 (2005).
`Ratcliff, et al., “A Similarity BetweenViral Defense and Gene Silenc
`ing in Plants,” Science 276:1558-1560 (1997).
`Rivas, et al., “Puri?ed Argonaute2 and an siRNA form recombinant
`human RISC,” Nat Struct Mol Biol., 12(4):340-9 (2005).
`Sanchez, “Double-stranded RNA speci?cally disrupts gene expres
`sion during planarian regeneration,” PNAS 96:5049-5054 (1999).
`Schneider, “Cell lines derived from late embryonic stages of
`Drosophila melanogaster,” J. Embryol. Exp. Morpho., 27:353-365
`(1972).
`Schramke, et al., "RNA-interference-directed chromatin modi?ca
`tion coupled to RNA polymerase II transcription,” Nature,
`435(7046):1275-9 (2005).
`Sharp, “RNAi and double-strand RNA,” Genes Dev., 13:139-141
`(1999).
`Shi, et al. “Genetic interference in Typanosoma brucei by heritable
`and inducible double-stranded RNA,” RNA, 6: 1069-1076 (2000).
`Shuttleworth, et al., “Antisense oligonucleotide-directed cleavage of
`mRNA in Xenopus oocytes and eggs,” EMBO J., 7:427-434 (1988).
`Sij en, “Post-transcriptional gene-silencing: RNAs on the attack or on
`the defense?” Bioessays, 22:520-531 (2000).
`Silva, et al., “Free energy lights the path toward more effective
`RNAi,” Nat Genet. 35(4):303-5 (2003).
`Silva, et al., “RNA interference microarrays: High-throughput loss
`of-function genetics in mammalian cells,” Proceedings of the
`National Academy of Sciences ofUSA, 101(17):6548-6552 (2004).
`Silva, et al., “RNA interference: a promising approach to antiviral
`therapy.” Trends Mol Med. 8(11):505-8 (2002).
`Silva, et al., “RNA-interference-based functional genomics in mam
`malian cells: reverse genetics coming of age,” Oncogene,
`23(51):8401-9 (2004).
`Silva, et a1 ., “Second-generation shRNA libraries covering the mouse
`and human genomes,” Nature Genetics, 37(1 1): 1281-1288 (2005).
`Singh, et al., “Inverted-repeat DNA: a new gene-silencing tool for
`seed lipid modi?cation,” Biochemical Society, 28(6):925-927
`(2000).
`Siolas, et al., “Synthetic shRNAs as potent RNAi triggers,” Nature
`Biotechnology, 23(2):227-231 (2005).
`Smardon, et a1 ., “EGO-1 is related to RNA-directed RNA polymerase
`and functions in germ-line development and RNA interference in C.
`elegans,” Curr. Biol. 10: 169-178 (2000).
`Smith, et al., “Total silencing by intron-spliced hairpin RNAs,”
`Nature, 407:319-320 (2000).
`Song, et al., “Crystal structure of Argonaute and its implications for
`RISC slicer activity,” Science, 305(5689):1434-7 (2004).
`Song, et al., “The crystal structure of the Argonaute2 PAZ domain
`reveals an RNA binding motif in RNAi effector complexes,” Nat.
`Struct. Biol. 10(12):1026-32 (2003).
`Svoboda, et al., “RNAi and expression of retrotranspo sons MuERV-L
`and IAP in preimplantation mouse embryos,” Dev. Biol., 269(1):276
`85 (2004).
`Tabara, et al., “RNAi in C. elegans: Soaking in the Genome
`Sequence,” Science, 282:430-432 (1998).
`Tabara, et al., “The dsRNA Binding Protein RDE-4 Interacts with
`RDE-l, DCR-1, and a DExH-Box Helicase to Direct RNAi in C.
`elegans,” Cell, 109:861-871. (2002).
`Tabara, et al., “The rde-l Gene, RNA Interference, and Transposon
`Silencing in C. elegans,” Cell, 99:123-132 (1999).
`Tavernarakis, et al., “Heritable and inducible genetic interference by
`double-stranded RNA encoded by transgenes,” Nat. Genet., 24: 180
`183 (2000).
`Timmons, et al., “Speci?c interference by ingested dsRNA,” Nature,
`395:854 (1998).
`Tomari, et al., “RISC Assembly Defects in the Drosophila RNAi
`Mutant armitage”, Cell 116:831-841 (2004).
`Tuschl, et al. “Targeted mRNA degradation by double- stranded RNA
`in vitro,” Genes Dev., 13:3191-3197 (1999).
`
`Benitec - Exhibit 1001 - page 3
`
`

`
`US 8,153,776 B2
`Page 4
`
`Ui-Tei, et al., “Sensitive Assay of RNA Interference in Drosophila
`and Chinese Hamster Cultured Cells Using Fire?y Luciferase Gene
`as Target,” FEBS Letters, 479179-82 (2000).
`Vaucheret, et al., “Transgene-induced gene silencing in plants,” Plant
`J. 161651-659 (1998).
`Wadhwa, et al., “Know-how of RNA interference and its applications
`in research and therapy,” Mutation Research, 567171-84 (2004).
`Wassenegger, “A model for RNA-mediated gene silencing in higher
`plants,” Plant Mol. Biol. 371349-362 (1998).
`Waterhouse, et al., “Virus resistance and gene silencing in plants can
`be induced by simultaneous expression of sense and antisense RNA,”
`PNAS 95113959-13964 (1998).
`Wianny, “Speci?c interference With gene function by double
`stranded RNA in early mouse development,” Nature Cell Biol., 2170
`75 (2000).
`Wolf, et al., “Cell cycle: Oiling the gears of anaphase,” Curr. Biol.
`81R636-R639 (1998).
`Zamore, et al., “RNAi: Double-Stranded RNA Directs the ATP
`Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals” Cell
`101125-33 (2000).
`Zhang, et al., “Human Dicer preferentially cleaves dsRNAs at their
`termini Without a requirement for ATP,” The Embo Journal, 2115875 -
`5885. (2002).
`Zhang, et al., “Single Processing Center Models for Human Dicer
`and Bacterial RNase III,” Cell, 118157-68 (2004).
`Zhang, et al., “Targeted gene silencing by small interfering RNA
`based knock down technology,” Curr. Pharma. Biotech., 511-7
`(2004).
`European Search Report for European PAtent Application No.
`058570086, mailed May 8, 2008.
`Bosher et al., “RNA interference can target pre-mRNA: conse
`quences for gene expression in a Caenorhabditis elegans operon,”
`Genetics, vol. 153, No. 3, p. 1245-1256 (Nov. 1999).
`European Search report for European Patent application No.
`037320520, mailed May 23, 2008.
`HasuWa et al., “Small interfering RNA and gene silencing in
`transgenic mice and rats,” FEBS Letters, Elsevier, Amsterdam, NL,
`vol. 532, pp. 227-230 (Dec. 2002).
`Manche et al., “Interactions between double-stranded RNA regula
`tors and the proteinkinase Dai,” Molecular and cellular Biology,
`Amercian Society for Microbiology, Washington, US, vol. 12, pp.
`5238-5248 (Nov. 1992).
`Marked-up U.S. Appl. No. 09/866,557, ?led May 24, 2001.
`Marked-up U.S. Appl. No. 60/243,097, ?led Oct. 24, 2000.
`Declaration of Dr. Vladimir DroZdoff (executed Aug. 5, 2008).
`Declaration of Mr. John Maroney (executed Aug. 5, 2008).
`Declaration of Professor Gregory Hannon (executed Aug. 5, 2008).
`Letter ofApr. 22, 2008 from Douglass N. Ellis, Jr. of Ropes & Gray
`LLP to John Maroney, Esq. of Cold Spring Harbor Laboratory.
`Letter of Apr. 28, 2008 from John Maroney of Cold Spring Harbor
`Laboratory to Douglass N. Ellis, Jr. of Robes & Gray LLP.
`Letter of Apr. 29,2008 from Douglass N. Ellis, Jr. from Robes & Gray
`LLP to John Maroney, Esq. of Cold Spring Harbor Laboratory.
`Letter of May 9, 2008 to Eric R. Hubbard, Esq. of Robes & Gray LLP
`from John Maroney, Esq. of Cold Spring Harbor Laboratory.
`Letter of Jun. 4, 2008 from Eric R. Hubbard of Robes & Gray LLP to
`John Maroney, Esq. of Cold Spring Harbor Laboratory.
`Letter of Jun. 13, 2008 from John Maroney, Esq. of Cold Spring
`Harbor Laboratory to James Haley, Esq. of Robes & Gray LLP
`BuchholZ et al., “EnZymatically prepared RNAi libraries,” Nature
`Methods, vol. 3, No. 9, pp. 696-700 (Sep. 2006).
`Caplen et al., “Rescue of polyglutamine-mediated cytotoxicity by
`double-stranded RNA-mediated RNA interference,” Human
`Molecular Genetics, vol. 11, pp. 175-184 (2002).
`Chang et al., “Lessons from Nature: microRNA-based ShRNA librar
`ies,” Nature Methods, vol. 3, No. 9, pp. 707-714 (Sep. 2006).
`
`Cullen, “Enhancing and con?rming the speci?city of RNAi experi
`ments,” Nature Methods, vol. 3, pp. 677-681 (Sep. 2006).
`Elbashir et al., “Duplexes of 21-nucleotide RNA’s mediate RNA
`interference in cultured mammalian cells,” Nature, vol. 411, pp.
`494-498 (May 2001).
`Elbashir et al., “RNA interference is mediated by 21- and
`22-nucleotide RNA,s,” Gene and Development, vol. 15, pp. 188-200
`(2001).
`Gil et al., “Induction of apoptosis by the DsRNA-dependent protein
`Kinase(PKR)1 mechanism of Action,” Apoptosis, vol. 5, pp. 107-114
`(2000).
`Hutvagner et al., ’A Cellular Function for the RNA-Interference
`Enzyme Dicer i the maturation of the let-7 Small Temporal RNA,
`Science, vol. 293, pp. 834-838 (Aug. 2001).
`McManus et al., “Gene Silencing in mammals by small interfering
`RNA’s,” Nature Reviews, vol. 3, pp. 737-747 (Oct. 2002).
`Pei et al., “On the art of identifying effective and speci?c siRNAs,”
`Nature Methods, vol. 3, No. 9, pp. 670-676 (Sep. 2006).
`Sen et al., “A brief history of RNAi: the silence of the genes,” FASEB
`J., vol. 20, pp. 1293-1299 (2006).
`Snove Jr et al., “Expressing short Hairpin RNAs in vivo,” Nature
`Methods, vol. 3 No. 9, pp. 689-695 (Sep. 2006).
`Svoboda et al., “RNAI in mouse Oocytes and Preimplantation
`Embryos: effectiveness of Hairpin dsRNA,” Biochem. Biophys. Res.
`Commum. vol. 287, pp. 1099-1104 (2001).
`Vermeulen et al., “the contributions of DsRNA structure to Dicer
`speci?city and ef?ciency,” RNA, vol. 11, pp. 674-682 (2005).
`Brummelkamp et al., “A system for stable expression of short inter
`fering RNAs in mammalian cells,” Science, vol. 296, pp. 550-553
`(Apr. 2002).
`European Search Result mailed on Feb. 17, 2010, for European
`Application No. EP 03732052 ?led Jan. 22, 2003.
`European Search Result mailed on Sep. 22, 2009 for European Appli
`cation No. EP 03732052 ?led Jan. 22, 2003.
`Miller et al., “Improved retroviral vectors for gene transfer and
`expression,” Biotechniques, vol. 7(9), pp. 980-990 (1989).
`Non ?nal of?ce action mailed on Feb. 9, 2005 for US. Appl. No.
`10/055,797, ?led Jan. 22,2002.
`Non ?nal of?ce action mailed on Nov. 8, 2005 for US. Appl. No.
`10/055,797, ?led Jan. 22,2002.
`Non ?nal of?ce action mailed on Jun. 23, 2010, for US. Appl. No.
`12/152,837, ?led Jan. 22,2002.
`Final of?ce action mailed on Apr. 17, 2007, for US. Appl. No.
`10/055,797, ?led Jan. 22,2002.
`Non ?nal of?ce action mailed on Jul. 26, 2006, for US. Appl. No.
`10/055,797, ?led Jan. 22,2002.
`Final Of?ce Action mailed on May 12, 2009, for US. Appl. No.
`10/997,086, ?led Nov. 23, 2004.
`Final Of?ce Action mailed on Jul. 2, 2010, for US. Appl. No.
`10/997,086, ?led Nov. 23, 2004.
`Non Final Of?ce Action mailed on Aug. 26, 2009, for US. Appl. No.
`10/997,086, ?led Nov. 23, 2004.
`Non Final Of?ce Action mailed on Feb. 12, 2007, for US. Appl. No.
`10/997,086, ?led Nov. 23, 2004.
`Brummelkamp et a1 ., “Stable suppression of tumorigenicity by virus
`mediated RNA interference,” Cancer cell, vol. 2, pp. 243-247 (2002).
`Final Of?ce Action mailed on Mar. 18, 2011 for US. Appl. No.
`12/152,837, ?led May 16, 2008.
`McManus et al., “Gene silencing using micro-RNA designed hair
`pins,” RNA, vol. 8, pp. 842-850 (2002).
`Sorensen et al., “Gene Silencing by systemic delivery of Synthetic
`siRNAs in adult Mice,” J. Mol. Biol., vol. 327, pp. 761-766 (2003).
`US. Appl. No. 60/305,185 ?led Jul. 12,2001.
`
`* cited by examiner
`
`Benitec - Exhibit 1001 - page 4
`
`

`
`Benitec - Exhibit 1001 - page 5
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 2 0f 68
`
`US 8,153,776 B2
`
`cyc?n E wtracrt
`
`{ac-Z extract
`
`Fig. 2A
`
`Benitec - Exhibit 1001 - page 6
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 3 0f 68
`
`US 8,153,776 B2
`
`Benitec - Exhibit 1001 - page 7
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 4 0f 68
`
`US 8,153,776 B2
`
`cE+<hOm+oU
`
`<POM+UU
`
`Substrate
`
`cyc/in E
`
`lacZ
`
`~25 nt
`
`iqw Wu
`
`cyclin F
`Northern
`
`Fig. 4A
`
`FigT4B
`
`Benitec - Exhibit 1001 - page 8
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 5 0f 68
`
`US 8,153,776 B2
`
`S2 CELLS EMBRYO
`
`r
`
`510
`
`S100
`
`510
`
`S100
`
`nu
`
`:.
`
`Iuc
`
`Fig. 5C
`
`Benitec - Exhibit 1001 - page 9
`
`

`
`Benitec - Exhibit 1001 - page 10
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 7 0f 68
`
`US 8,153,776 B2
`
`r
`
`f
`
`‘i 13
`£ 5
`
`u u
`
`
`
`k. \ L’J-Hbi: 10ml
`
`f
`
`DicerlP
`
`73E
`Q 8 g
`
`u E \
`
`"
`Fig. 6D
`
`ii, (a \___?____J
`
`Fig. 6E
`
`Fig. 6F
`
`Benitec - Exhibit 1001 - page 11
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 8 0f 68
`
`US 8,153,776 B2
`
`<52. 38g
`
`Fig. 7A Fig. 7B
`
`V
`
`D Luc ds + dicer ds
`Z GFP ds + control ds
`5] GFP ds + dicer ds
`
`Exp. 2
`
`Exp. 3
`
`Fig. 7C
`
`Benitec - Exhibit 1001 - page 12
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 9 0f 68
`
`US 8,153,776 B2
`
`helicase
`
`helicose
`
`_
`
`ZAP
`
`7 RI! 0
`
`helico
`
`ZAP
`
`Ifu
`
`RIbd_srm
`
`b.
`
`Benitec - Exhibit 1001 - page 13
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 10 0f 68
`
`US 8,153,776 B2
`
`CELL FREE ExTRAcT
`
`l 200K SPIN
`
`RIBosoIvIE PELLET
`i HIGH SALT EXTRACTION
`
`RIBOSOME AssocIATEo PROTEINS
`
`LOW SALT PRECIPITATION
`
`.
`
`PELLET
`RNAi ACTIVITY
`
`SUPERNATANT
`
`RESOLUBIUZE HIGH SALTl
`
`CHROMATOGRAPHY
`s'uPERosE 6
`
`MONO s
`
`MONO Q
`
`HYDROXYAPATITE
`
`Fig. 9
`
`Benitec - Exhibit 1001 - page 14
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 11 0f 68
`
`US 8,153,776 B2
`
`.m 1 a A a
`
`mm: wmw 0% $0 9. 6% $6
`
`
`
`
`
`m" w w w w .m Qmd Q E520: :EEog w :56
`
`2N 11'.
`
`amp lit.
`
`Silt.
`
`2 .9“.
`
`
`
`630528. NémZn
`
`Benitec - Exhibit 1001 - page 15
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 12 0f 68
`
`US 8,153,776 B2
`
`2w lit.
`
`0Q llY
`
`a"
`
`: .mE
`
`ww
`
`m
`
`c269»
`
`
`
`Ecumco: $2263
`
`Benitec - Exhibit 1001 - page 16
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 13 0f 68
`
`US 8,153,776 B2
`
`‘.,..?wawémwwwwwH
`
`
`
`5596: 39203
`
`'
`
`Q .5
`
`
`
`630588” @0940
`
`m
`
`C260;
`
`Benitec - Exhibit 1001 - page 17
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 14 0f 68
`
`US 8,153,776 B2
`
`
`
`BEQCDEEM @6966
`
`Benitec - Exhibit 1001 - page 18
`
`

`
`Benitec - Exhibit 1001 - page 19
`
`

`
`US. Patent
`
`Apr. 10, 2012
`
`Sheet 16 0f 68
`
`US 8,153,776 B2
`
`=3 :9; - o3 .
`
`
`
`=8 >>2 - om< EEK? ,.
`
`Fig. 15
`
`Benitec - Exhibit 1001 - page 20
`
`

`
`Benitec - Exhibit 1001 - page 21
`
`

`
`Benitec - Exhibit 1001 - page 22
`
`

`
`Benitec - Exhibit 1001 - page 23
`
`

`
`Benitec - Exhibit 1001 - page 24
`
`

`
`Benitec - Exhibit 1001 - page 25
`
`

`
`Benitec - Exhibit 1001 - page 26
`
`

`
`Benitec - Exhibit 1001 - page 27
`
`

`
`Benitec - Exhibit 1001 - page 28
`
`

`
`Benitec - Exhibit 1001 - page 29
`
`

`
`Benitec - Exhibit 1001 - page 30
`
`

`
`Benitec - Exhibit 1001 - page 31
`
`

`
`Benitec - Exhibit 1001 - page 32
`
`

`
`Benitec - Exhibit 1001 - page 33
`
`

`
`Benitec - Exhibit 1001 - page 34
`
`

`
`Benitec - Exhibit 1001 - page 35
`
`

`
`Benitec - Exhibit 1001 - page 36
`
`

`
`Benitec - Exhibit 1001 - page 37
`
`

`
`Benitec - Exhibit 1001 - page 38
`
`

`
`Benitec - Exhibit 1001 - page 39
`
`

`
`Benitec - Exhibit 1001 - page 40
`
`

`
`Benitec - Exhibit 1001 - page 41
`
`

`
`Benitec - Exhibit 1001 - page 42
`
`

`
`Benitec - Exhibit 1001 - page 43
`
`

`
`Benitec - Exhibit 1001 - page 44
`
`

`
`Benitec - Exhibit 1001 - page 45
`
`

`
`Benitec - Exhibit 1001 - page 46
`
`

`
`Benitec - Exhibit 1001 - page 47
`
`

`
`Benitec - Exhibit 1001 - page 48
`
`

`
`Benitec - Exhibit 1001 - page 49
`
`

`
`Benitec - Exhibit 1001 - page 50
`
`

`
`Benitec - Exhibit 1001 - page 51
`
`

`
`Benitec - Exhibit 1001 - page 52
`
`

`
`Benitec - Exhibit 1001 - page 53
`
`

`
`Benitec - Exhibit 1001 - page 54
`
`

`
`Benitec - Exhibit 1001 - page 55
`
`

`
`Benitec - Exhibit 1001 - page 56
`
`

`
`Benitec - Exhibit 1001 - page 57
`
`

`
`Benitec - Exhibit 1001 - page 58
`
`

`
`Benitec - Exhibit 1001 - page 59
`
`

`
`Benitec - Exhibit 1001 - page 60
`
`

`
`Benitec - Exhibit 1001 - page 61
`
`

`
`Benitec - Exhibit 1001 - page 62
`
`

`
`Benitec - Exhibit 1001 - page 63
`
`

`
`Benitec - Exhibit 1001 - page 64
`
`

`
`Benitec - Exhibit 1001 - page 65
`
`

`
`Benitec - Exhibit 1001 - page 66
`
`

`
`Benitec - Exhibit 1001 - page 67
`
`

`
`Benitec - Exhibit 1001 - page 68
`
`

`
`Benitec - Exhibit 1001 - page 69
`
`

`
`Benitec - Exhibit 1001 - page 70
`
`

`
`Benitec - Exhibit 1001 - page 71
`
`

`
`Benitec - Exhibit 1001 - page 72
`
`

`
`Benitec - Exhibit 1001 - page 73
`
`

`
`Benitec - Exhibit 1001 - page 74
`
`

`
`Benitec - Exhibit 1001 - page 75
`
`

`
`Benitec - Exhibit 1001 - page 76
`
`

`
`Benitec - Exhibit 1001 - page 77
`
`

`
`Benitec - Exhibit 1001 - page 78
`
`

`
`Benitec - Exhibit 1001 - page 79
`
`

`
`Benitec - Exhibit 1001 - page 80
`
`

`
`Benitec - Exhibit 1001 - page 81
`
`

`
`Benitec - Exhibit 1001 - page 82
`
`

`
`Benitec - Exhibit 1001 - page 83
`
`

`
`Benitec - Exhibit 1001 - page 84
`
`

`
`Benitec - Exhibit 1001 - page 85
`
`

`
`Benitec - Exhibit 1001 - page 86
`
`

`
`Benitec - Exhibit 1001 - page 87
`
`

`
`Benitec - Exhibit 1001 - page 88
`
`

`
`Benitec - Exhibit 1001 - page 89
`
`

`
`Benitec - Exhibit 1001 - page 90