(12) United States Patent
`Giese et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 9,695.423 B2
`*Jul. 4, 2017
`
`USOO9695423B2
`
`(54) INTERFERING RNA MOLECULES
`(71) Applicant: SILENCE THERAPEUTICS GMBH,
`Berlin (DE)
`(72) Inventors: Klaus Giese, Berlin (DE); Jörg
`Kaufmann, Berlin (DE); Anke
`Klippel-Giese, Berlin (DE)
`
`(*) Notice:
`
`(73) Assignee: SILENCE THERAPEUTICS GMBH,
`Berlin (DE)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 14 days.
`This patent is Subject to a terminal dis
`claimer.
`
`(21) Appl. No.: 14/977,710
`
`(22) Filed:
`
`Dec. 22, 2015
`
`(65)
`
`Prior Publication Data
`US 2016/O 130587 A1
`May 12, 2016
`
`Related U.S. Application Data
`(63) Continuation of application No. 14/578.636, filed on
`Dec. 22, 2014, now Pat. No. 9,222,092, which is a
`continuation of application No. 13/692,178, filed on
`Dec. 3, 2012, now Pat. No. 8,933,215, which is a
`continuation of application No. 12/986,389, filed on
`Jan. 7, 2011, now Pat. No. 8,324,370, which is a
`continuation of application No. 12/200,296, filed on
`Aug. 28, 2008, now Pat. No. 7,893,245, which is a
`continuation of application No. 10/633,630, filed on
`Aug. 5, 2003, now Pat. No. 7,452,987.
`(60) Provisional application No. 60/402,541, filed on Aug.
`12, 2002.
`Foreign Application Priority Data
`
`(30)
`
`Aug. 5, 2002 (EP) ..................................... O2O176O1
`Apr. 10, 2003 (EP) ..................................... O3OO8383
`
`(2006.01)
`(2010.01)
`(2006.01)
`(2006.01)
`
`(51) Int. Cl.
`C7H 2L/04
`CI2N IS/IT3
`CI2N IS/II
`A61 K 38/00
`(52) U.S. Cl.
`CPC ........ CI2N 15/1137 (2013.01); C12N 15/III
`(2013.01): CI2N 15/113 (2013.01); A61 K
`38/00 (2013.01); C12N 23 10/14 (2013.01);
`CI2N 23.10/3183 (2013.01); C12N 23.10/32
`(2013.01); C12N 23.10/321 (2013.01); C12N
`2310/322 (2013.01); C12N 23.10/343
`(2013.01); C12N 23 10/346 (2013.01); C12N
`2310/53 (2013.01); C12N 2310/531 (2013.01);
`CI2N 2320/51 (2013.01)
`(58) Field of Classification Search
`CPC .................................................... CO7H 21AO74
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`8/1980 Petty
`4,217,294 A
`5,466,786 A 11/1995 Buhr et al.
`5,514,546 A
`5, 1996 Kool
`5,589,389 A 12/1996 Pages et al.
`5,670,633. A
`9, 1997 Cook et al.
`5,792,847 A
`8, 1998 Buhr et al.
`5,872,232 A
`2f1999 Cook et al.
`5,898,031 A
`4/1999 Crooke
`5,914,396 A
`6/1999 Cook et al.
`5,955,589 A
`9, 1999 Cook et al.
`6,005,087 A 12/1999 Cook et al.
`6,107,094. A
`8, 2000 Crooke
`6,129,912 A 10/2000 Hortin et al.
`6,153,737 A 1 1/2000 Manoharan et al.
`6,165,772 A 12/2000 Bertling
`6,239,272 B1
`5/2001 Beigelman et al.
`6,284,267 B1
`9/2001 Aneja
`6,476,205 B1
`1 1/2002 Buhr et al.
`6,506,559 B1
`1/2003 Fire et al.
`6,531,584 B1
`3/2003 Cook et al.
`6,617.438 B1
`9/2003 Matulic-Adamic et al.
`6,812.204 B1
`1 1/2004 McHale et al.
`7,056,704 B2
`6/2006 Tuschl et al.
`7,074,863 B2
`7/2006 Ekholm et al.
`7,078,196 B2
`7/2006 Tuschl et al.
`7,138,517 B2 11/2006 Cook et al.
`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`
`CA
`DE
`
`8, 2003
`2476112
`11, 1997
`196 18 797
`(Continued)
`
`OTHER PUBLICATIONS
`
`U.S. Appl. No. 60/352,873, filed Feb. 1, 2002, Damha, M. J. et al.
`U.S. Appl. No. 60/358,580, filed Feb. 20, 2002, Beigelman, Let 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, Beigelman, L. et al.
`Amarzguioui. M. et al. “Tolerance for mutations and chemical
`modifications 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.
`(Continued)
`Primary Examiner — Kimberly Chong
`(74) Attorney, Agent, or Firm — Morrison & Foerster
`LLP
`
`ABSTRACT
`(57)
`The present invention is related to a ribonucleic acid com
`prising a double stranded structure whereby the double
`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.
`
`25 Claims, 29 Drawing Sheets
`
`Alnylam Exh. 1015
`
`i
`
`

`

`US 9,695.423 B2
`Page 2
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`7,148,342 B2 12/2006 Tolentino et al.
`7,196, 184 B2
`3/2007 Heidenreich et al.
`7,348,314 B2
`3/2008 John et al.
`2003/0027783 A1
`2/2003 Zernicka-Goetz et al.
`2003. O108923 A1
`6, 2003 TuSchl et al.
`2003/0135033 A1
`7/2003 Klippel-Giese et al.
`2003/0190635 A1 10/2003 McSwiggen
`2004.0053875 A1
`3, 2004 Kreutzer et al.
`2004/OO72779 A1
`4/2004 Kreutzer et al.
`2004.0102408 A1
`5, 2004 Kreutzer et al.
`2004/0106569 A1
`6/2004 Klippel-Giese et al.
`2004/0175703 A1
`9, 2004 Kreutzer et al.
`2004/O180351 A1
`9, 2004 Giese et al.
`2004/0229266 Al
`11/2004 Tuschl et al.
`58.85: A.
`23.9 R. al al
`2005. O142535 A1
`6, 2005 Damha et al.
`2006/0094678 A1
`5, 2006 Vornlocher et al.
`2006, O142226 A1
`6/2006 Polisky et al.
`2006/0217329 A1
`9, 2006 Feinstein
`2006/024.0022 A1 10/2006 Klippel-Giese et al.
`2007/0027097 A1
`2/2007 Lewis
`2007/0093445 A1
`4/2007 Tuschl et al.
`2007/O135372 A1
`6/2007 MacLachlan et al.
`2007,0265220 A1 11, 2007 Rossi et al.
`2014/0220676 A1
`8/2014 Kreutzer et al.
`
`FOREIGN PATENT DOCUMENTS
`
`RE
`EP
`E.
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`GB
`W.
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`W.
`W.
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`W
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`A1 s28
`:
`1 144 623 B1 38
`9: 29s
`1214 945
`6, 2002
`O2O185724
`8, 2002
`1 230 375 B1
`7/2005
`1550719 A1
`7/2005
`1527, 176 B1
`1, 2007
`1798.285 A1
`6, 2007
`1 407 044 B1
`9, 2007
`992744. A
`5, 1965
`WO 576. B
`A.
`WO92, 19732
`11, 1992
`WO 92,208.23
`11, 1992
`WO 93/13121
`7, 1993
`WO 94/O1550
`1, 1994
`WO95/13834
`5, 1995
`WO-96, 18376 A1
`6, 1996
`WO96, 18736
`6, 1996
`WO 97/41141
`11, 1997
`WO 97/4657O
`12/1997
`W. 3.36.
`i8
`W. 23S
`2.3%
`WO99/49029
`9, 1999
`WO 99.53050
`10, 1999
`WO 99,55857
`11, 1999
`WO 99,61631
`12/1999
`WOOOO1846
`1, 2000
`WOOOf 44495
`8, 2000
`WOOOf 44895
`8, 2000
`WOOOf 44914
`8, 2000
`WOOOf 63364
`10, 2000
`W 9.3. A2 1 3.398-
`WO O2, 1662O
`2, 2002
`WO O2/44321
`6, 2002
`WO O2/O55693 A2
`T 2002
`WO O2/O97114
`12/2002
`WO 03/064441 A2
`8, 2003
`WO 03/070895
`8, 2003
`WO 03/070912
`8, 2003
`
`WO O3,070918
`WO
`WO WO 2004/O15107 A3
`WO WO 2004/O19973 A1
`WO WO 2004/035615 A2
`WO WO 2005.000320 A2
`WO WO 2005,105,152
`WO WO 2006/023544 A2
`WO WO 2006/069782 A2
`WO WO 2007/048244 A2
`WO WO 2007/084684 A2
`
`8, 2003
`2, 2004
`3, 2004
`4/2004
`1/2005
`11/2005
`3, 2006
`7, 2006
`5, 2007
`7, 2007
`
`& 8 OTHER PUBLICATIONS
`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 double-stranded RNAs
`induce RNA interference in Drosophila', Current Biology, Nov. 13,
`R pp. 1776-1780, vol. 1,
`raasch, 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
`Functional 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
`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
`Experimental Therapeutics, 2000, pp. 140-149, vol. 292.
`Czauderna, F. et al. "Functional Studies of the PI(3)-kinase signal
`ing pathway employing synthetic and expressed siRNA'. Nucleic
`Acids Research, 2003, pp. 670-682, vol. 31, No. 2.
`Dorsett, Y. etal. “siRNAs. Applications in Functional Genomics and
`Potential as Therapeutics'. Drug Discovery, Apr. 2004, pp. 318
`& vol. 3.
`is rary r.
`ary, D. J. et al. “Polymer-based siRNA delivery: Perspectives on
`the fundamental and phenomenological distinctions from polymer
`based DNA delivery”, Journal of Controlled Release, 2007, pp.
`64-73, vol. 121.
`Hall, I. M. et al. “Establishment and Maintenance of a
`Heterochromatin Domain', Science, Sep. 27, 2002, pp. 2232-2237.
`vol. 297.
`Hammond, S. M. et al. "Argonaute2, a Link Between Genetic and
`Biochemical Analyses of RNAi'. Science, Aug. 10, 2001, pp.
`1146-1150, vol. 293.
`Hammond, S.M. et al "An RNA-directed nuclease mediates post
`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 Asym
`metry of siRNA Strand Degradation in Human Blood Serum', RNA,
`g pp. 17 vol. 13.
`Hu, X. et al. Relative gene-silencing efficiencies of Small interfer
`ing RNAS targeting sense and antisense transcripts from the same
`genetic locus'. Nucleic Acids Research, 2004, pp. 4609-4617, vol.
`32, No. 15.
`Inoue, A. et al. “Molecular Design and Delivery of siRNA'. Journal
`of Drug Targeting, 2006, pp. 448-455, vol. 14, No. 7.
`
`ii
`
`

`

`US 9,695.423 B2
`Page 3
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`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 EMBOJournal,
`2004, pp. 3303-3313, vol. 23, No. 16.
`Lima, W. F. et al. “Structural Requirements at the Catalytic Site of
`the Heteroduplex Substrate for Human RNaseH1 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-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
`Biomedicine and Biotechnology, 2006, pp. 1-9, vol. 2006.
`Paddison, P. J. et al. "Stable suppression of gene expression by
`RNAi 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
`ential Requirement for the Two Trigger Strands in RNA Interfer
`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
`titative Biology, 2006, pp. 81-93, vol. LXXI.
`Peek, A. S. et al. “Design of Active Small Interfering RNAs'.
`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 RNAs'. 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
`interference in the mouse vascular endothelium”. Gene Therapy,
`2006, pp. 1222-1234, vol. 13.
`Scherer, L. J. et al. “Approaches for the sequence-specific knock
`down of mRNA'. 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, O. et al. “Chemical Modifications Rescue Off-Target Effects
`of RNAi’, 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. Molecu
`lar BioSystems, 2007, pp. 43-50, vol. 3.
`Tuschl, T. “Expanding small RNA interference”. Nature Biotech
`nology, 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
`Interventions, 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 RNAi'. Science, Sep. 13,
`2002, pp. 1833-1837, vol. 297.
`Wilson, C. et al. “Building oligonucleotide therapeutics using
`non-natural chemistries'. Current Opinion in Chemical Biology,
`2006, pp. 607-614, vol. 10.
`Delivery Note, 2 pages, Dec. 20, 2001.
`Holen, T. et al. "Positional Effects of Short Interfering RNAs
`targeting the human coagulation trigger Tissue Factor'. Nucleic
`Acids Research, 2006, pp. 1757-1766, vol. 30, No. 8.
`The Merriam Webster Dictionary, pp. 37 and 540, 1997, Merriam
`Webster, Inc.
`P. Dan Cook, “Medicinal Chemistry of Antisense Oligoucleotides';
`(ed. Croke, S.T.), pp. 29-56 Marcel Dekker, NY 2001.
`Bevilacqua and Cech; "Minor-Groove Recognition of Double
`Stranded RNA by the Double-Stranded RNA-Binding.” Biochem
`istry 1996, 35, pp. 9983-9994.
`Wu, et al., “Identification and Partial Purification of Human Double
`Strand RNase Activity': The Journal of Biological Chem; vol. 273,
`No. 6, pp. 2532-2542 (1998).
`M. Monoharan;"2"-Carbohydrate modifications in antisense oligo
`nucleotide therapy . . . .” Biochimica et Biophysica Acta; 1999, pp.
`117-130.
`Inoue et al., “Synthesis and hybridization studies on two comple
`mentary nonac2"O-methyl)ribonucleotides': Nucleic Acids Res.,
`1987, pp. 6131-6148, vol. 15.
`U.S. Appl. No. 10/205.309; (priority document for No. 1, filed Jul.
`25, 2002.
`Fire."RNA-triggered gene Silencing.” TIG, pp. 358-363, vol. 15.
`No. 9, Sep. 1999.
`Paddison, et al...Stable Suppression of gene expression by RNAi in
`mammalian cells; PNAS, 99(3), pp. 1443-1448; (2002).
`Park, et al., “Prevention of HIV-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 mam
`malian cells using small interfering RNAs;” JCell Science; 114; pp.
`4557-4565; (2001).
`Kurreck, “Improvement through novel chemical modifications;”
`Eur, J. Biochem, 270, 1628-1644 (2003).
`Jennifer Couzin, Breakthrough, Small RNAS Make Big Splash,
`Science, 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 RNAi.
`Biocentury, vol. 11, No. 12, p. 123, Mar. 2003.
`Kitabwalla, M. et al. “RNA-Interference—A New Weapon Against
`HIV and Beyond”. N. Engl. J. Med... vol. 347, No. 17, pp. 1364
`1367, Oct. 24, 2002.
`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, pp.
`14428-14433.
`
`iii
`
`

`

`US 9,695,423 B2
`
`Page 4
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`Williams, "Dicing with siRNA." Nat Biotechnol. Feb.
`
`
`
`
`
`
`
`
`
`International Preliminary Examination Report, (English Transla­
`
`
`
`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
`
`
`
`Elbashir SM, et al., Duplexes of21-Nucleotide RN As Mediate RNA
`
`
`
`
`
`
`
`
`stability without loss of efficacy." Biochem Biophys Res Commun.,
`
`
`
`
`
`Interference in Cultured Manunalian Cells, Nature, vol. 411, pp.
`Apr. 14, 2006;342(3):919-27.
`
`494-498, May 2001.
`Conrad et al. "Ribonuclease III: new sense from nuisance." Int J
`
`
`
`Biochem Cell Biol. Feb. 2002; 34(2): 116-29.
`Lipinski et al., Experimental and Computational Approaches to
`
`
`
`
`
`
`Zamore et al. "RNAi: double-stranded RNA directs the ATP­
`
`
`
`
`Estimate Solubility and Permeability in Drug Discovery and Devel­
`
`
`
`
`
`dependent cleavage ofmRNAat 21 to 23 nucleotide intervals." Cell.
`
`
`
`vol. 23, pp. 3-25, opment Settings, Adv. Drug Delivery Reviews
`Mar. 31, 2000; 101(1):25-33.
`1997.
`Kalota et al. "2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F­
`
`Bhan et al., Nucleic Acid Research, vol. 25, 1997, p. 3310.
`
`
`
`
`
`
`
`ANA) modified oligonucleotides (ON) effect highly efficient, and
`
`
`Li et al., Dev. Biology, vol. 210, 1999, p. 238, Abstr. 346.
`
`
`
`persistent, gene silencing." Nucleic Acids Res. Jan. 18, 2006;
`
`Ngo et al., PNAS, vol. 95, Dec. 1998, p. 14687.
`
`34(2):451-61. Print 2006.
`
`
`Voinnet, 0., and Baulcombe, DC., Nature (1997) vol. 398, p. 553.
`
`
`
`
`
`McAffrey, AP et al., Nature (2000), vol. 418, pp. 38-39.
`
`2005;23(2): 181-2.
`
`
`Barber, GN et al., Mo!. and Cell. Biol. (1995), vol. 15, No. 6, p.
`Priority Document of9927444.l, Cancer Res. Camp. Technol. Ltd.
`
`
`
`
`3138-46.
`
`
`
`Inhibiting Gene Expression, Pub!. Nov. 19, 1999, Issued Nov. 22,
`
`
`Byrom, MW., et al., TechNotes 10(1), Ambion, http://www.ambion.
`2000.
`com, Mar. 2003.
`Priority Document of U.S. Appl. No. 60/130,377, Methods and
`
`
`
`
`
`S.Blair Hedges, Nature Reviews, Genetics (2002), vol. 3, pp.
`
`
`
`
`Compositions for Inhibiting the Function of Polynucleotide, Issued
`838-849.
`Jun. 6, 2000.
`Sledz, CA., et al., Nature Cell Biol., vol. 5, p. 834-9, 2003.
`
`
`
`
`Priority Document of U.S. Appl. No. 60/117,635, Double-Stranded
`
`
`
`
`
`
`
`Hornung et al., Nature Medicine, vol. 11, No. 3, pp. 263-270, Mar.
`
`
`
`
`RNA Blocks Specific Gene Expression in Multicellular Settings in
`2005.
`
`
`in Vivo and in Vitro, Mar. 14, 2000.
`
`
`
`
`
`
`
`Judge et al, Nature Biotechnology, vol. 23, No. 4, pp. 457-462, Apr.
`
`
`tion) DE 0000244 (IPER), Jul. 7, 2003.
`2005.
`Andrew J. Hamilton and David C. Balcombe, Species of Small
`
`
`
`
`
`
`Applicant's Response to the Written Opinion in the Examination
`
`
`
`Antisense RNA in Posttranscriptional Gene Silencing in Plants,
`
`proceedings Mar. 28, 2001.
`
`
`Science, vol. 286. Dec. 1999, pp. 950-951.
`
`
`
`Brennicke et al., FEMS Microbiol. Rev., vol. 23, p. 297-316 1999.
`Andrew Fire. et al., Potent and specific genetic interference by
`
`
`
`
`
`
`Perler, Nucl. Acids Res., vol. 30, No. 1, pp. 383-384, 2002.
`
`
`
`double-stranded RNA in Caenorhabditis elegans, Nature Macmilian
`
`
`
`
`Zhao, et al., Developmental Biol., vol. 229, pp. 215-223, 2001.
`
`
`
`Publishers Ltd. 1993, vol. 391/191, pp. 806-811.
`
`Hu-Lieskovan, et al, Cancer Res., vol. 65, No. 19, pp. 8984-8992,
`Peter M. Waterhouse, et al., Virus resistance and gene silencing in
`
`
`
`
`Oct. 1, 2005.
`
`
`
`
`plants can be induced by simultaneous expression of sense and
`
`
`
`Caplen, et al, PNAS, vol. 98, No. 17, pp. 9742-9747, Aug. 14, 2001.
`
`
`
`antisense RNA, Communicated by W. James Peacock, Common­
`
`
`
`Hunter, et al., JBC, vol. 250, No. 2, pp. 409-417, Jan. 25, 1975.
`
`
`
`wealth Scientific and Industrial Research Organization, Canberra,
`
`
`
`Manche et al., Mo!. Cell, Biol., vol. 12, No. 11, p. 5238-5248, Nov.
`
`Australia, Aug. 17, 1998, vol. 95, pp. 13959-13964.
`1992.
`
`
`
`Mohanunad B. Baluamian. et al., Transcriptional and Post­
`Zeng, et al., RNA, vol. 8, pp. 855-860, 2002.
`
`
`
`
`
`transcriptional Silencing of Rodent 01(1) collagen by a Homolo­
`Induce Patrick J. Paddison, et al., Short Hairp in RNAs (shRNAs)
`
`
`
`
`
`
`
`gous Transcriptionally Self-Silenced Transgene, Molecular and
`
`
`
`Sequence-Specific Silencing in Manunalian Cells, Genes & Devel­
`
`
`
`Cellular Biology, Jan. 1999, vol. 19, No. 1. pp. 274-283.
`
`
`opment vol. 16, pp. 948-958, 2002.
`
`
`RNA, Genes & Devel­Phillip A. Sharp , RNAi and Double-strand
`
`
`Zheng, et al., RNA, vol. 10, pp. 1934-1945, 2004.
`
`
`
`
`
`opment, vol. 13, pp. 139-141, Cold Spring Harbor Laboratory Press,
`
`
`
`Martinez et al., Cell, vol. 110, pp. 563-574. Jun. 9, 2002.
`1999.
`
`
`Chien, et al., Cancer Gene Therapy, 1-8, 2004.
`Thomas Tusch, et al., Targeted mRNA Degradation by double­
`
`
`
`
`Soutschek, et al., Nature, vol. 432, 1-8, 2004.
`
`
`
`stranded RNA in vitro, Genes & Development, vol. 13, pp. 3191-
`
`
`Karberg, Mach <loch ma! das Gen aus, Die Zeit, No. 41, May 10,
`
`3197; Cold Spring Harbor Laboratory . 1999.
`2006.
`
`
`
`News of the Week, Science, Candidate Gene Silencers Found, Fetal
`
`
`
`
`
`
`Cells Help Parkinson's Patients, Science, vol. 286, p. 886, Dec.
`2007.
`1999.
`Feb. 2, 2007. Patent, v. Opposed Comparative (Pachuk) Figu re: Dl0
`
`
`
`
`
`
`
`
`
`
`
`
`Elbashir, et al., RNA Interference Is Mediated by 21-and
`
`402, pp. 128-129, Nov. 11, 1999.
`
`
`22-Nucleotide RNAs, Genes & Development vol. 15, pp. 188-200,
`
`
`
`Michael T. McManus, et al. Gene Silencing in Mammals by Small
`Jan. 2001.
`
`
`
`
`Interfering RNAs, Center for Cancer Research Massachusetts, vol.
`
`
`
`
`
`Napoli, et al., Introduction of a Chimeric Chalcone Synthase Gene
`
`3, pp. 737-750,Oct. 2002.
`
`
`
`
`into Petunia Results in Reversible Co-Suppression of Homologous
`Mary K. Montgomery , et al., Double-stranded RNA as a Mediator
`
`
`
`Genes in trans, The Plant Cell, vol. 2, pp. 279-289, Apr. 1990.
`
`
`
`
`in Sequence-Specific Genetic Silencing and Co-Suppression, TIG,
`
`
`
`Oates, et al., Too Much Interference: Injection of Double-Stranded
`
`
`
`vol. 14 No. 7, pp. 255-256 and 256, Jul. 1998.
`
`
`
`RNA Has Nonspecific Effects in the Zebrafish Embryo, Develop­
`
`
`
`
`Robert Barstead, "Genome-wide RNAi", Current Opinion in
`
`
`
`mental Biology, Aug. 2000, pp. 20-28, vol. 224, Issue 1.
`
`
`
`Chemical Biology, 2001, pp. 63-66, vol. 5.
`
`
`
`Caplen et al; "Specific inhibition of gene expression by small
`
`
`David C. Baulcombe, "Fast forward genetics based on virus­
`
`
`
`
`double-stranded RN As in invertebrate and vertebrate systems" Proc
`
`
`
`
`
`induced gene silencing", Current Opinion in Plant Biology, 1999,
`Natl Acad Sci U S A. Aug. 14, 2001;98(17):9742-7.
`
`pp. 109-113, vol. 2.
`
`
`
`
`Kraynack et al.,"Small interfering RNAs containing full 2'-O­
`Emily Bernstein et al., "Role for a bidentate ribonuclease in the
`
`
`
`
`
`
`methylribonucleotide-modified sense strands display Argonaute2/
`
`
`
`initiation step of RNA interference", Nature, Jan. 18, 2001, p.
`
`
`eIF2C2-dependent activity." RNA. Jan. 2006;12(1):163-76.
`
`363-366, vol. 409.
`
`
`
`Chiu et al. "siRNA function in RNAi: a chemical modification
`Eric Billy et al., "Specific Interference with Gene Expression
`
`
`
`analysis." RNA. 2003 ep;9(9):1034-48.
`
`
`Induced by Long, Double-Stranded RN A in Mouse Embry onal
`Griinweller et al. "Comparison of different antisense strategies in
`
`
`
`
`
`
`
`
`
`Teratocarcinoma Cell Lines", Proceedings of the National Academy
`
`
`
`
`mammalian cells using locked nucleic acids, 2'-O-methyl RNA,
`
`
`
`of Sciences of the United States of America, Dec. 4, 2001, pp.
`
`
`
`phosphorothioates and small interfering RNA." Nucleic Acids Res.
`
`14428-14433, vol. 98, No. 25.
`Jun. 15, 2003;31(12):3185-93.
`
`
`
`
`
`Feb. 2,v. Opposed Patent, Comparative (Agrawal) Figu re: D60
`
`
`
`Rueyling Lin and Leon Avery, Policing Rogue Genes, Nature, vol.
`
`iv
`
`

`

`US 9,695.423 B2
`Page 5
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`Christian Cazenave et al., “Rate of degradation of a- and LB
`oligodeoxynucleotides in Xenopus oocytes. implications for anti
`messenger strategies'. Nucleic Acids Research, 1987, pp. 10507
`10521, vol. 15, No. 24.
`Sayda M. Elbashir et al., “Duplexes of 21-nucleotide RNAs mediate
`RNA interference in cultured mammalian cells', Nature, May 24,
`2001, pp. 494-498, vol. 411.
`Sudhir Agrawal, et al., Self-Stabilized Oligonucleotides as Novel
`Antisense Agents, Nucleic Acids, Res., vol. 21, No. 11, 1993, pp.
`2729-2735.
`Jason R. Kennerdell et al., “Heritable gene silencing in Drosophila
`using doublestranded RNA'. Nature Biotechnology, Jul. 2000, pp.
`896-898, vol. 17.
`Jason R. Kennerdell et al., “Use of dsRNA-Mediated Genetic
`Interference to Demonstrate that frizzled and frizzled 2 Act in the
`Wingless Pathway”. Cell, Dec. 23, 1998, pp. 1017-1026, vol. 95.
`Stuart K. Kim, "Functional genomics: The worm scores a knock
`out'. 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
`ogy, 2001, pp. 171-176, vol. 11.
`Mary K. Montgomery et al., “RNA as a Target of Double-Stranded
`RNA-Mediated Genetic Interference in Caenorhabditis elegans',
`Proceedings of the National Academy of Sciences 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, Nov. 2,
`2001, pp. 309-321, vol. 107.
`Phillip A. Sharp, “RNA interference—2001'. Genes & Develop
`ment, 2001, pp. 485-490, vol. 15.
`Maria Sternberger et al., “GeneBlocs Are Powerful Tools to Study
`and Delineate A15 Signal Transduction Processes That Regulate
`Cell Growth and Transformation'. Antisense & Nucleic Acid Drug
`Development, 2002, pp. 131-143, vol. 12.
`Thomas Tuschl et al., “Targeted mRNA degradation by double
`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 Biophysi
`cal Methods, 1986, pp. 97-102, vol. 13.
`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.
`R. Schlingenslepen et al., Antisense—From Technology to Therapy,
`EX L1BRIS Roche), 1997, vol. 6, pp. 30-58.
`Iiho Ha et al., A Bulged Lin-4/lin-14 RNA Duplex is Sufficient for
`Caenorhabditis Elegans Lin-14 Temporal Gradient Formation,
`Genes & Development, 1996, vol. 10, pp. 3041-3050.
`Glenn D. Hoke et al., Effects of Phosphorothioate Capping on
`Antisense Oligonucleotide Stability, Hybridization and Antiviroal
`Efficacy Versus Herpes Simplex Virus Infection, Nucleic Acids
`Research, 1991, vol. 19, No. 20, pp. 5743-5748.
`Richard H. Griffey, et al., 2'-O-Aminoprophyll Ribonucleotides; A
`Zwitterionic Modification that Enhances the Exonuclease Resis
`tance and Biological Activity of Antisense Oligonucleotides, J.
`Med. Chem, 1996, vol. 39, pp. 5100-5109.
`Theo T. Nikiforov. et al., Oligodeoxynucleotides Containing
`4-Thiothymidine and 6-Thiodeoxyguanosine as Affinity Labels for
`the Eco RV Restriction Endonuclease and Modification Methylase.
`Nucleic Acids Research, 1992, vol. 20, pp. 1209-1214.
`
`Jane A. Grasby, et al., Purine Functional Groups in Essential
`Residues of the Hairpin Ribozyme Required for Catalytic Cleavage
`of RNA, Biochemistry 1995, vol. 34, pp. 4068-4076.
`Thomas Horn, et al., Chemical Synthesis and Characterization of
`Branched Oligodeoxyribonucleotides (bONA) for Use as Signal
`Amplifiers in Nucleic Acid Quantification Assays, Nucleic Acids
`Research, 1997, vol. 25, No. 23, pp. 4842-4849.
`Reiko Iwase et al., Gene Regulation by Decoy Approach (I):
`Synthesis and Properties of Photo-crosslinked Oligonucleotides,
`Nucleic Acids Symposium Series, 1997, No. 37, pp. 203-204.
`Eugene Skripkin. et at, 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 of Vicinal 2',5'(or 2',3'-) and
`3',5'Phosphodiester Linkages on the Formation of Hairepin DNA,
`Bioconjugate Chern., 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.
`Poui Nielsen, et al., A Novel Class of Conformationally Restricted
`Oligonucleotide Analogues: Synthesis of 2',3'-Bridged Monomers
`and RNA-Selective Hybridisation, Chern. Commun., 1997, pp.
`825-826.
`Mark D. Pegram, et al., Phase II Study of Receptor-Enhanced
`Chemosensitivity. Using Recombinant Humanized Anti-p185her2/
`NEU Monoclonal Antibody Plus Cisplatin in Patients With HER2/
`neu-Overexpressing Metastatic Breast Cancer Refractory to Che
`motherapy Treatment. Journal of Clinical Oncology, vol. 16, No. 8
`Aug. 1998: pp. 2659-2671.
`Sayda M. Eibashir, et al., Functional Anatomy of siRNAs for
`Mediating 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
`fications of Synthetic siRNAs in Mammalian 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 Drosophila 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. Iee, 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. 86, pp. 637-646, Mar. 7, 1997.
`Yang Shi, et al