Compositions and method for inhibiting hepcidin antimicrobial peptide (HAMP) or HAMP-related gene expression
| Title: | Compositions and method for inhibiting hepcidin antimicrobial peptide (HAMP) or HAMP-related gene expression |
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| Patent Number: | 9,228,188 |
| Publication Date: | January 05, 2016 |
| Appl. No: | 14/118489 |
| Application Filed: | June 21, 2012 |
| Abstract: | The invention relates to lipid formulated double-stranded ribonucleic acid (dsRNA) targeting a hepcidin antimicrobial peptide (HAMP) and/or HAMP-related gene, and methods of using the dsRNA to inhibit expression of HAMP and/or HAMP-related genes. |
| Inventors: | Bettencourt, Brian (Groton, MA, US); Akinc, Akin (Needham, MA, US); Sehgal, Alfica (Allston, MA, US); Foster, Don (Attleboro, MA, US); Milstein, Stuart (Cambridge, MA, US); Kuchimanchi, Satyanarayana (Acton, MA, US); Maier, Martin A. (Belmont, MA, US); Charisse, Klaus (Acton, MA, US); Rajeev, Kallanthottathil (Wayland, MA, US) |
| Assignees: | ALNYLAM PHARMACEUTICALS, INC. (Cambridge, MA, US) |
| Claim: | 1. A double-stranded ribonucleic acid (dsRNA) compound, comprising a sense strand and an antisense strand that are each equal to or less than 30 nucleotides in length, wherein the compound is targeted to a nucleic acid molecule encoding transferrin receptor 2(TRF2), wherein the compound specifically hybridizes with the nucleic acid molecule so that the abundance of TRF2 mRNA is inhibited by at least 20% relative to a PBS control, and wherein the antisense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:38. |
| Claim: | 2. The dsRNA of claim 1 , wherein the nucleotide sequence of the sense strand comprises 15 or more contiguous nucleotides of SEQ ID NO:35 with a start position of position 239 of the TFR2 mRNA transcript and the nucleotide sequence of the antisense strand comprises 15 or more contiguous nucleotides of SEQ ID NO:38 with a start position complementary to position 239 of the TFR2 mRNA transcript. |
| Claim: | 3. The dsRNA of claim 1 , wherein the nucleotide sequence of the sense strand comprises 16, 17, 18, 19, 20, or 21 contiguous nucleotides of SEQ ID NO:35 with a start position of position 239 of the TFR2 mRNA transcript and the nucleotide sequence of the antisense strand comprises 16, 17, 18, 19, 20, or 21 contiguous nucleotides of SEQ ID NO:38 with a start position complementary to position 239 of the TFR2 mRNA transcript. |
| Claim: | 4. The dsRNA of claim 1 , wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:35 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:38. |
| Claim: | 5. The dsRNA of claim 1 , further comprising a phosphorothioate at the first internucleotide linkage at the 3′ end of the sense strand, the antisense strand or both the sense strand and the antisense strand. |
| Claim: | 6. The dsRNA of claim 1 , wherein the sense strand, the antisense strand or both the sense and antisense strand further comprises at least one 3′n-overhang wherein the 3′-overhang comprises from 1 to 6 nucleotides. |
| Claim: | 7. The dsRNA of claim 1 , wherein the dsRNA further comprises a non-nucleotide moiety. |
| Claim: | 8. The dsRNA of claim 1 , wherein said dsRNA further comprises at least one modified nucleotide. |
| Claim: | 9. The dsRNA of claim 1 , further comprising a 2′-modified nucleotide in the sense strand, r the antisense strand or both the sense and antisense strand. |
| Claim: | 10. The dsRNA of claim 8 , wherein at least one of said modified nucleotides is selected from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. |
| Claim: | 11. The dsRNA of claim 8 , wherein the modified nucleotide is selected from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. |
| Claim: | 12. The dsRNA of claim 1 , wherein the dsRNA is formulated in a nucleic acid lipid particle formulation. |
| Claim: | 13. The dsRNA of claim 1 , wherein the nucleic acid lipid particle formulation comprises a cationic lipid, a non-cationic lipid, and a cholesterol/ polyethyleneglycol-lipid conjugate. |
| Claim: | 14. The dsRNA of claim 1 , wherein the dsRNA is selected from the group consisting of AD-52551, AD-52552, AD-52557, AD-52558, AD-52563, AD-52564, AD-52569, AD-52570, AD-52574, AD-52575, AD-52579, AD-52580, AD-52584, AD-52585, AD-52589, AD-52590, and AD-47826. |
| Claim: | 15. A cell comprising the dsRNA of claim 1 . |
| Claim: | 16. A vector encoding at least one of the antisense strand and the sense strand of the dsRNA of claim 1 . |
| Claim: | 17. A cell comprising the vector of claim 15 . |
| Claim: | 18. A pharmaceutical composition comprising the dsRNA of claim 1 and a pharmaceutically acceptable carrier. |
| Claim: | 19. The pharmaceutical composition of claim 17 further comprising a lipid formulation. |
| Claim: | 20. A method of inhibiting TFR2 expression in a cell, the method comprising: (a) introducing into the cell the dsRNA of claim 1 and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TFR2 gene, thereby inhibiting expression of the TFR2 gene in the cell. |
| Claim: | 21. A method of treating a disorder associated with TFR2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of the dsRNA of claim 1 . |
| Claim: | 22. The dsRNA of claim 1 , wherein the sense strand consists of the nucleotide sequence set forth in SEQ ID NO:35 and the antisense strand consists of the nucleotide sequence set forth in SEQ ID NO:38. |
| Patent References Cited: | 6054299 April 2000 Conrad 7374927 May 2008 Palma et al. 7427605 September 2008 Davis et al. 7718629 May 2010 Bumcrot et al. 8163711 April 2012 Nakayama et al. 8268799 September 2012 Nakayama et al. 8470799 June 2013 Nakayama et al. 8791250 July 2014 Nakayama et al. 2003/0143732 July 2003 Fosnaugh et al. 2003/0170891 September 2003 McSwiggen 2004/0259247 December 2004 Tuschl et al. 2005/0004026 January 2005 Kasibhatla et al. 2005/0272080 December 2005 Palma et al. 2006/0263435 November 2006 Davis et al. 2007/0004664 January 2007 McSwiggen et al. 2007/0031844 February 2007 Khvorova et al. 2007/0224186 September 2007 Kulaksiz et al. 2007/0281899 December 2007 Bumcrot et al. 2008/0213277 September 2008 Sasu et al. 2009/0149403 June 2009 MacLachlan 2009/0209478 August 2009 Nakayama et al. 2010/0204307 August 2010 Nakayama et al. 2011/0015250 January 2011 Bumcrot et al. 2012/0244207 September 2012 Fitzgerald et al. 2014/0294936 October 2014 Nakayama et al. 10100586 April 2002 2001149083 June 2001 WO 99/32619 July 1999 WO 99/53050 October 1999 WO 99/61631 December 1999 WO 00/22113 April 2000 WO 00/22114 April 2000 WO 00/44895 August 2000 WO 02/098444 December 2002 WO 2004/080406 September 2004 WO 2004/090108 October 2004 WO 2007/120883 October 2007 WO 2007/120883 October 2007 WO 2008/036933 March 2008 WO 2008/089795 July 2008 WO 2009/073809 June 2009 WO 2010/147992 December 2010 |
| Other References: | PCT International Search Report and Written Opinion for PCT/US2012/043603, Dec. 7, 2012, 14 Pages. cited by applicant Agrawal, S., et al., “Antisense oligonucleotides: towards clinical trials.” Trends in Biotechnology. Oct. 1996, vol. 14, pp. 376-387. cited by applicant Andrews, N. C., “Anemia of Inflammation: the Cytokine-Hepcidin Link,” J. Clin. Invest., vol. 113, No. 9, May 1, 2004. cited by applicant Bass, B., “The short answer,” Nature, May 24, 2001, pp. 428-429, vol. 411. cited by applicant Boese et al., “Mechanistic Insights Aid Computational Short Interfering RNA Design,” Methods in Enzymology, vol. 392, pp. 73-96, 2005. cited by applicant Couture, A., et al., “Anti-Gene Therapy: The Use of Ribozymes to Inhibit Gene Function,” TIG, vol. 12, No. 12, pp. 510-515, Dec. 1996. cited by applicant Crosby, J., et al., “Targeting Hepcidin with Antisense Oligonucleotides Improves Anemia Endpoints in Mice,” Antisense Drug Discovery, Abstract No. 269, American Society of Hematology Annual Meeting, Nov. 16, 2006, vol. 108, No. 11, pp. 83a-84a. cited by applicant Donovan, A., et al., “Positional Cloning of Zebrafish ferroporin 1 Identifies a Conserved Vertebrate Iron Exporter,” Nature, vol. 403, pp. 776-781, Feb. 2000. cited by applicant Elbashir, S., et al., “Analysis of gene function in somatic mammalian cells using small interfering RNAs,” Methods, 2002, pp. 199-213, vol. 26. cited by applicant Elbashir, S., et al., “Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture,” Nature, May 24, 2001, p. 494-498, vol. 411. cited by applicant Elbashir, S., et al., “Functional Anatomy of siRNAs for Mediating Efficient RNAi in Drosophila melanogaster Embryo Lysate”, The EMBO Journal, 2001, pp. 6877-6888, vol. 20, No. 23. cited by applicant Elbashir, S., et al., “RNA Interference is Mediated by 21- and 22 Nucleotide RNAs,” Genes & Development, 2001, pp. 188-200, vol. 15. cited by applicant Fire, A., “RNA-triggered Gene Silencing,” Trends in Genetics, Sep. 1999, pp. 358-363, vol. 15, No. 9. cited by applicant Fire, A., et al., “Potent and Specific Genetic Interference by Double Stranded RNA in Caenorhabditis elegans,” Nature, Feb. 19, 1998, pp. 806-811, vol. 391. cited by applicant Fleming, R.E., et al., “Hepcidin: A Putative Iron-Regulatory Hormone Relevant to Hereditary Hemochromatosis and the Anemia of Chronic Disease,” Proceedings of the National Academy of Science, vol. 98, No. 15, pp. 8160-8162, 2001. cited by applicant Fleming, R.E., “Advances in Understanding the Molecular Basis for the Regulation of Dietary Iron Absorption,” Curr. Opin. Gastrolenterol, vol. 21, pp. 201-206, 2005. cited by applicant Ganz, T., “Hepcidin, a Key Regulator of Iron Metabolism and Mediator of Anemia of Inflammation,” Blood, vol. 102, No. 3, pp. 783-788, Aug. 2003. cited by applicant Gassmann, M., et al., “Maintenance of an Extrachromosomal Plasmid Vector in Mouse Embryonic Stem Cells,” Proc. Nat'l. Acad. Sci., USA, vol. 92, pp. 1292-1296, 1995. cited by applicant Genbank Accession No. NM—021175.2, Feb. 19, 2006. cited by applicant GenBank Accession No. NM—032541.1 (Oct. 22, 2011), NCBI Sequence Viewer, [online] [Retrieved on Nov. 18, 2011] Retrieved from the internet <http://www.ncbi.nlm.nih.gov/nuccore/NM.sub.--032541.1>. cited by applicant Holen et al., “Positional Effects of Short Interfering RNAs Targeting the Human Coagulation Trigger Tissue Factor,” Nucleic Acid Research, vol. 30, pp. 1757-1766, 2002. cited by applicant Hornung, V., et al., “Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells throughTLR7,” Nature Medicine, Mar. 2005, pp. 263-270, vol. 11, No. 3. cited by applicant Li, S., et al., “Folate-Mediated Targeting of Antisense Oligodeoxynucleotides to Ovarian Cancer Cells,” Pharm. Res., vol. 15, No. 10, pp. 1540-1545, 1998. cited by applicant Manoraran, M., “Oligonucleotide Conjugates as Potential Antisense Drugs with Improved Uptake, Biodistribution, Targeted Delivery, and Mechanism of Action,” Antisense & Nucleic Acid Drug Development, vol. 12, pp. 103-128, 2002. cited by applicant Nemeth, E., et al., “Hepcidin Regulates Cellular Iron Efflux by Binding to Ferroportin and Inducing its Internalization,” Science, vol. 306, pp. 2090-2093, 2004. cited by applicant Nicholas et al., “Lack of Hepcidin Gene Expression and Severe Tissue Iron Overload in Upstream Stimulatory Factor 2 (USF2) Knockout Mice,” Proceedings of the National Academy of Science, vol. 98, vol. 15, pp. 8780-8785, 2001. cited by applicant Nicholas, G., et al., “The Gene Encoding the Iron Regulatory Peptide Hepcidin is Regulated by Anemia, Hypoxia and Inflammation,” The J. of Clin. Invest., vol. 110, No. 7, pp. 1037-1044, Oct. 2002. cited by applicant Office Action for Australian Patent Application No. 2007299629, mailed Mar. 8, 2011, 2 pages. cited by applicant Office Action for Canadian Patent Application No. 2,663,581, mailed May 14, 2012, 2 pages. cited by applicant Office Action for Canadian Patent Application No. 2,663,581, mailed Jan. 18, 2011, 4 pages. cited by applicant Papanikolaou, G., et al., “Hepcidin in Iron Overload Disorders,” Blood, vol. 105, pp. 4103-4105, 2005. cited by applicant Park, C.H., et al., “Hepcidin, a Urinary Antimicrobial Peptide Synthesized in the Liver,” J. Biol. Chem., vol. 276, pp. 7806-7810, 2001. cited by applicant Patent Examination Report No. 1 for Australian Patent Application No. AU 2011250816, Dec. 21, 2012, 2 Pages. cited by applicant PCT International Search Report and Written Opinion, PCT/US2007/079212, Aug. 14, 2008, 11 pages. cited by applicant Pietrangelo, A., “Hereditary Hemochromatosis—A New Look at an Old Disease,” New England Journal of Medicine, Jun. 3, 2004, pp. 2383-2397, vol. 350, No. 23. cited by applicant Pigeon, C., et al., “A New Mouse Liver-Specific Gene, Encoding a Protein Homologous to Human Antimicrobial Peptide Hepcidin, is Overexpressed During Iron Overload,” J. Biol. Chem., vol. 276, pp. 7811-7819, 2001. cited by applicant Reynolds, et al. (2004) “Rational siRNA design for RNA interference,” Nature Biotechnology, vol. 22, No. 3, pp. 326-330. cited by applicant Robbins, M., et al., “Stable expression of shRNAs in human CD34+ progenitor cells can avoid induction of interferon responses to siRNAs in vitro,” Nature Biotechnology, May 2006, pp. 566-571, vol. 24, No. 5. cited by applicant Roetto, A., et al., “Mutant Antimicrobial Peptide Hepcidin Is Associated With Severe Juvenile Hemochromatosis,” Nature Genetics, Jan. 2003, pp. 21-22, vol. 33. cited by applicant Rose, S., et al., “Functional polarity is introduced by Dicer processing of short substrate RNAs,” Nucleic Acids Research, 2005, pp. 4140-4156, vol. 33, No. 13. cited by applicant Schwarz et al., “Asymmetry in the Assembly of the RNAi Enzyme Complex,” Cell, vol. 115, pp. 199-208, 2003. cited by applicant Supplementary European Search Report, European Patent Application No. EP 07853594, Nov. 5, 2010, 7 Pages. cited by applicant Tuschl, T., “Functional genomics: RNA sets the standard,” Nature, Jan. 16, 2003, vol. 421, No. 6920, pp. 220-221. cited by applicant Tuschl T., “RNA Interference and Small Interfering RNAs” Chembiochem, 2001, pp. 239-245, vol. 2. cited by applicant Tuschl, T., et al., “Small Interfering RNAs: A Revolutionary Tool for the Analysis of Gene Function and Gene Therapy,” Molecular Interventions, 2002, pp. 158-167, vol. 2, No. 3. cited by applicant Tuschl, T., “Mammalian RNA Interference,” RNAi, A Guide to Gene Silencing, Chapter 13, G.J. Hannon (ed,), 2003, pp. 265-295. cited by applicant Tuschl, T., et al., “Targeted mRNA Degradation by Double-Stranded RNA In Vitro,” Genes & Development, 1999, pp. 3191-3197, vol. 13. cited by applicant Tuschl, T., “Expanding small RNA interference,” Nature Biotechnology, May 2002, pp. 446-448, vol. 20. cited by applicant Vickers, T., et al., “Efficient Reduction of Target RNAs by Small Interfering RNA and RNase H-dependent Antisense Agents,” The Journal of Biological Chemistry, Feb. 28, 2003, pp. 7108-7118, vol. 278, No. 9. cited by applicant Weil, et al (2002) “Targeting the Kinesin Eg5 to Monitor siRNA Transfection in Mammalian Cells,” Biotechniques 33(6):1244-1248. cited by applicant Weiss, G., et al., “Anemia of Chronic Disease,” N. E. J. of Med., vol. 352, pp. 1011-1023, 2005. cited by applicant Yang, D., et al., “Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos,” Curr. Biol., vol. 10, No. 19, pp. 1191-1200, 2000. cited by applicant Zimmerman, et al. (2006) “RNAi-mediated gene silencing in non-human primates,” Nature, vol. 441, May 4: 111-114. cited by applicant European Extended Search Report, European Application No. 12801896.7, Nov. 11, 2014, 7 pages. cited by applicant Ganz, T. et al., “Hepcidin and Disorders of Iron Metabolism,” Annual Review of Medicine, Feb. 18, 2011, pp. 347-360, vol. 62, No. 1. cited by applicant Theurl, I. et al., “Autocrine Formation of Hepcidin Induces Iron Retention in Human Monocytes,” Blood, Dec. 11, 2007, pp. 2392-2399, vol. 111, No. 4. cited by applicant Bartolomei, G. et al., “Modulation of Hepatitis C Virus Replication by Iron and Hepcidin in Huh7 Hepatocytes,” Journal of General Virology, May 18, 2011, pp. 2072-2081, vol. 92, No. 9. cited by applicant United States Office Action, U.S. Appl. No. 14/303,921, Oct. 6, 2014, 13 pages. cited by applicant United States Office Action, U.S. Appl. No. 13/900,854, Sep. 24, 2013, 11 pages. cited by applicant United States Office Action, U.S. Appl. No. 13/590,783, Oct. 15, 2012, 11 pages. cited by applicant United States Office Action, U.S. Appl. No. 13/184,087, Jan. 10, 2012, 14 pages. cited by applicant United States Office Action, U.S. Appl. No. 12/757,497, May 6, 2011, 9 pages. cited by applicant United States Office Action, U.S. Appl. No. 11/859,288, Dec. 21, 2009, 12 pages. cited by applicant Office Action for Canadian Patent Application No. 2,663,581, mailed Apr. 23, 2013, 2 pages. cited by applicant European Examination Report, European Application No. 07853594.5, Mar. 13, 2014, 4 pages. cited by applicant |
| Assistant Examiner: | Poliakova, Kate |
| Primary Examiner: | Vivlemore, Tracy |
| Attorney, Agent or Firm: | Fenwick & West LLP |
| Accession Number: | edspgr.09228188 |
| Database: | USPTO Patent Grants |
| Language: | English |
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