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(12) United States Patent (10) Patent No.: US 7,534.879 B2

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(12) United States Patent (10) Patent No.: US 7,534.879 B2 USOO7534879B2 (12) United States Patent (10) Patent No.: US 7,534.879 B2 Van Deutekom 45) Date of Patent: MaV 19,9 2009 (54) MODULATION OF EXON RECOGNITION IN WO 2004/083432 A1 9, 2004 PRE-MRNABY INTERFERING WITH THE OTHER PUBLICATIONS SECONDARY RNASTRUCTURE Aartsma-Rus et al., Targeted exon skipping as a potential gene cor rection therapy for Duchenne muscular dystrophy, Neuromuscular (75) Inventor: Judith C.T. van Deutekom, Dordrecht Disorders, 2002, S71-S77, vol. 12. (NL) Dickson et al., Screening for antisense modulation of dystrophin pre-mRNA splicing, Neuromuscul. Disord., 2002, S67-70, Suppl. 1. De Angelis et al., Chimeric SnRNA molecules carrying antisense (73) Assignee: Academisch Ziekenhuis Leiden, Leiden sequences against the splice junctions of exon 51 of the dystrophin (NL) pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in delta 48-50 DMD cells, PNAS, Jul. 9, 2002, pp. 9456 (*) Notice: Subject to any disclaimer, the term of this 9461, vol.99, No. 14. patent is extended or adjusted under 35 Dunckley et al., Modifications of splicing the dystrophin gene in U.S.C.M YW- 154(b) by 0 days S.olecular M Genetics,muscle t ,by pp. antigslides , Vol. , No. 1. Human Lu et al., Massive Idiosyncratic Exon Skipping Corrects the Non This patent is Subject to a terminal dis- sense Mutation in Dystrophic Mouse Muscle and Produces Func claimer. tional Revertant Fibers by Clonal Expansion. The Journal of Cell Biology, Mar. 6, 2000, pp.985-995, vol. 148, No. 5. (21) Appl. No.: 12/198,007 Mann et al., Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse, PNAS, Jan. 2, 2001, pp. 42-47, vol.98. 1-1. No. 1. (22) Filed: Aug. 25, 2008 Mann et al., Improved antisense oligonucleotide induced exon skip O O ping in the mdx mouse model of muscular dystrophy, The Journal of (65) Prior Publication Data Gene Medicine, 2002, pp. 644-654, vol. 4. US 2009/0076246A1 Mar. 19, 2009 Suter et al., Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human B-thalassemic muta tions, Human Molecular Genetics, 1999, pp. 2415-2423, vol. 8, No. Related U.S. Application Data 13. Van Deutekom et al., Antisense-induced exon skipping restores (63) Continuation of application No. 1 1,233,495, filed on dystrophin expression in DMD patient derived muscle cells, Human Sep. 21, 2005, which is a continuation of application Molecular Genetics, 2001, pp. 1547-1554, vol. 10, No. 15. No. PCT/NL2003/000214, filed on Mar. 21, 2003. Primary Examiner Kimberly Chong (51) Int. Cl (74) Attorney, Agent, or Firm TraskBritt C7H 2L/04 (2006.01) (57) ABSTRACT (52) U.S. Cl. ....................... 55.5 S. The invention provides a method for generating an oligo s s nucleotide with which an exon may be skipped in a pre (58) Field of Classification Search ............... r None mRNA and thus excluded from a produced mRNA thereof. See application file for complete search history. Further provided are methods for altering the secondary (56) References Cited structure of an mRNA to interfere with splicing processes and uses of the oligonucleotides and methods in the treatment of FOREIGN PATENT DOCUMENTS disease. Further provided are pharmaceutical compositions and methods and means for inducing skipping of several EP 1054 058 A1 5, 2000 exons in a pre-mRNA. EP 1133993 A2 9, 2001 EP 1 191097 A1 3, 2002 3 Claims, 7 Drawing Sheets U.S. Patent May 19, 2009 Sheet 1 of 7 US 7,534.879 B2 s Netan:ear es i. 32 s is s It is Is ISIS - i. ". Si -- GURE . U.S. Patent US 7.534.879 B2 A B C E F FIGURE 2 . U.S. Patent May 19, 2009 Sheet 3 of 7 US 7.534.879 B2 A B C S 3 * wn a xt & - dystrophin in - myosin D E F a 2 2 e st as 9 & 9 - dystrophin - myosin FIGURE 3. U.S. Patent May 19, 2009 Sheet 4 of 7 US 7,534.879 B2 cy-Sarcoglycan 3-Sarcoglycan 1-SarCOglycan 6-dystroglycan 2 FIGURE 4. U.S. Patent May 19, 2009 Sheet 5 Of 7 US 7,534.879 B2 FIGURE 5. U.S. Patent May 19, 2009 Sheet 6 of 7 US 7,534.879 B2 in NGt pe O O O 9 3 m 55 S2 t is a 781 bp - - 4243 44 45-46 633 bp - - 424345.46 460 bp - - 4245.46 FIGURE 6. U.S. Patent May 19, 2009 Sheet 7 Of 7 US 7,534.879 B2 A Y O. KM109 DL 470.2 50685.1 9 NTmix U NTmix U NT mix U (, s - 44 - 47 - 44 - 4752 4445 5152 - N As ZEE7 -44 5052 44 52- -44 52 B KM109 D 470.2 50685.1 FIGURE 7. US 7,534,879 B2 1. 2 MODULATION OF EXON RECOGNITION IN mentary or partly complementary stretches within the same PRE-MRNA BY INTERFERING WITH THE RNA. It has long since been thought that structures in the SECONDARY RNASTRUCTURE RNA play a role in the function of the RNA. Without being bound by theory, it is believed that the secondary structure of CROSS-REFERENCE TO RELATED the RNA of an exon plays a role in structuring the splicing APPLICATIONS process. Through its structure, an exon is recognized as a part that needs to be included in the pre-mRNA. Herein, this This application is a continuation of patent application Ser. signaling function is referred to as an “exon inclusion signal.” No. 1 1/233,495, filed Sep. 21, 2005, pending, which is a A complementary oligonucleotide of the invention is capable continuation of PCT International Patent Application No. 10 of interfering with the structure of the exon and thereby PCT/NL2003/000214, filed on Mar. 21, 2003, designating the capable of interfering with the exon inclusion signal of the United States of America, and published, in English, as PCT exon. It has been found that many complementary oligo International Publication No. WO 2004/083432 A1 on Sep. nucleotides indeed comprise this capacity, Some more effi 30, 2004, the contents of the entirety of each of which is ciently than others. Oligonucleotides of the invention, i.e., incorporated by this reference. 15 those with the overlap directed toward open and closed struc tures in the native exon RNA, are a selection from all possible TECHNICAL FIELD oligonucleotides. The selection encompasses oligonucle otides that can efficiently interfere with an exon inclusion The invention relates to the fields of molecular biology and signal. medicine. More in particular, the invention relates to the Without being bound by theory, it is thought that the over restructuring of mRNA produced from pre-mRNA, and thera lap with an open structure improves the invasion efficiency of peutic uses thereof. the oligonucleotide (i.e., increases the efficiency with which the oligonucleotide can enter the structure), whereas the over BACKGROUND lap with the closed structure subsequently increases the effi 25 ciency of interfering with the secondary structure of the RNA The central dogma of biology is that genetic information of the exon, and thereby interfere with the exon inclusion resides in the DNA of a cell and is expressed upon transcrip signal. It is found that the length of the partial complementa tion of this information, where after production of the rity to both the closed and the open structure is not extremely encoded protein follows by the translation machinery of the restricted. We have observed high efficiencies with oligo cell. This view of the flow of genetic information has 30 nucleotides with variable lengths of complementarity in prompted the predominantly DNA-based approach for inter either structure. The term “complementarity” is used herein fering with the protein content of a cell. This view is slowly to refer to a stretch of nucleic acids that can hybridize to changing and alternatives for interfering at the DNA level are another stretch of nucleic acids under physiological condi being pursued. tions. It is thus not absolutely required that all the bases in the In higher eukaryotes, the genetic information for proteins 35 region of complementarity are capable of pairing with bases in the DNA of the cell is encoded in exons that are separated in the opposing strand. For instance, when designing the from each other by intronic sequences. These introns are in oligonucleotide, one may want to incorporate, for example, a Some cases very long. The transcription machinery generates residue that does not base pair with the base on the comple a pre-mRNA that contains both exons and introns, while the mentary strand. Mismatches may to some extent be allowed, splicing machinery, often already during the production of the 40 if under the circumstances in the cell, the stretch of nucle pre-mRNA, generates the actual coding region for the protein otides is capable of hybridizing to the complementary part. In by splicing together the exons present in the pre-mRNA. a preferred embodiment, a complementary part (either to the Although much is known about the actual processes open or to the closed structure) comprises at least three and, involved in the generation of an mRNA from a pre-mRNA, more preferably, at least four, consecutive nucleotides. The much also remains hidden. In the invention it has been shown 45 complementary regions are preferably designed such that, possible to influence the splicing process Such that a different when combined, they are specific for the exon in the pre mRNA is produced. The process allows for the predictable mRNA. Such specificity may be created with various lengths and reproducible restructuring of mRNA produced by a splic of complementary regions as this depends on the actual ing machinery.
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