(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`19 May 2011 (19.05.2011)
`
`PCT
`
`1111111111111111 IIIIII IIIII 111111111111111 II Ill 111111111111111 IIIII IIIII IIII IIIIIII IIII 11111111
`
`(10) International Publication Number
`WO 2011/057350 Al
`
`(51)
`
`International Patent Classification:
`C12N 15/113 (2010.01)
`A61K 31/7088 (2006.01)
`A61K 31/712 (2006.01)
`A61K 48/00 (2006.01)
`A61P 21/00 (2006.01)
`
`(21) International Application Number:
`PCT/AU20I0/001520
`
`(22) International Filing Date:
`12 November 2010 (12.l l.2010)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`2009905549 12 November 2009 (12.11.2009)
`
`AU
`(71) Applicant (for all designated States except US): THE
`UNIVERSITY OF WESTERN
`AUSTRALIA
`[AU/AU]; Stirling Highway, Nedlands, Western Australia
`6907 (AU).
`
`(72)
`(75)
`
`Inventors; and
`Inventors/Applicants (for US only): WILTON, Stephen
`[AU/AU]; 18 Spey Road, Applecross, Western Australia
`6153 (AU). FLETCHER, Sue [AU/AU]; 14 Roberts
`Street, Bayswater, Western Australia 6053
`(AU).
`ADAMS, Abbie [AU/AU]; IO Crayden Road, Kalamun(cid:173)
`da, Western Australia 6076 (AU). MELONI, Penny
`[AU/AU]; IO Purslowe Street, Mount Hawthorne, West(cid:173)
`ern Australia 6016 (AU).
`
`(74) Agent: WRAYS; Ground Floor, 56 Ord Street, West
`Perth, Western Australia 6005 (AU).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, VA, VG, US, UZ, VC, VN, ZA, ZM, ZW.
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG,
`ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`with international search report (Art. 21 (3))
`
`with sequence listing part of description (Rule 5.2(a))
`
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`,-.-1 <
`0 "' ~
`t---"' ~
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`0
`(57) Abstract: An antisense molecule capable of binding to a selected target site to induce exon skipping in the dystrophin gene,
`~
`as set forth in SEQ ID NO: I to 59.
`
`(54) Title: ANTISENSE MOLECULES AND METHODS FOR TREATING PATHOLOGIES
`
`

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`WO 2011/057350
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`PCT/AU2010/001520
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`1
`
`Antisense Molecules and Methods for Treating Pathologies
`
`Field of the Invention
`
`The present invention relates to novel antisense compounds and compositions
`suitable for facilitating exon skipping. It also provides methods for inducing exon
`skipping using
`the novel antisense compounds as well as
`therapeutic
`compositions adapted for use in the methods of the invention.
`
`5
`
`Background Art
`
`10
`
`15
`
`The following discussion of the background art is intended to facilitate an
`.
`understanding of the present
`invention only. The discussion
`is not an
`acknowledgement or admission that any of the material referred to is or was part
`of the common general knowledge as at the priority date of the application.
`
`Significant effort is currently .. being expended into . researching methods for
`suppressing or compensating for disease-causing mutations in genes. Antisense
`technologies are being developed ·using a range of chemistries to affect· gene
`expression at a variety of different levels · (transcription,. splicing, stability,
`translation). Much of that research has focused on the use of antisense
`compounds to correct or compensate for abnormal or disease-associated genes
`in a myriad of different conditions.
`
`20 Antisense molecules are able to inhibit gene expression with exquisite specificity
`and because of this many research efforts concerning oligonucleotides as
`modulators of gene expression · have focused on inhibiting the expression of
`targeted genes such as oncogenes or viral genes. The antisense oligonucieotides
`are directed either against RNA (sense strand) or against DNA where they form
`triplex structures inhibiting transcript!on by RNA polymerase II.
`
`25
`
`To achieve a desired effect in specific gene down-regulation, the oligonucleotides
`must either promote the decay of the targeted rnRNA or block translation of that
`
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`2
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`mRNA, thereby effectively preventing de nova synthesis of the undesirable target
`protein.
`
`Such techniques are not . useful where the object is to up-regulate production of
`the native protein or compensate for mutations which
`induce premature
`5 . termination of translation such as nonsense or frame-shifting mutations.
`
`Furthermore, in cases where a normally functional protein is prematurely
`terminated because of mutations therein, a means for restoring some functional
`protein production through antisense technology has been shown to be possible
`through. intervention during the splicing processes (Sierakowska H, et al., (1996)
`10 Proc Natl Acad Sci USA 93, 12840-12844; Wilton SD, et al., (1999) Neuromusc
`Disorders 9,330-338; van Deutekom JC et al., (2001) Human Mol Genet 10, 1547-
`1554). In these cases, the defective gene transcript should not be subjected to
`targeted degradation so the antisense oligonucleotide chemistry should not
`promote target mRNA decay.
`
`15
`
`20
`
`25
`
`30
`
`• In a variety of genetic diseases, the effects of mutations on the eventual
`expression of a gene can be modulated through a process of targeted exon
`skipping during the splicing process. The splicing process is directed by complex
`multi"'particle machinery that brings adjacent exon-intron junctions in pre-mRNA
`into close proximity and performs cleavage of phosphodiester bonds at the ends
`of the intrans with their subsequent reformation between exons that are to be
`spliced together. 'This complex and highly precise process is mediated by
`sequence motifs in the pre-mRNA that are relatively short semi-conserved RNA
`segments to which bind the various nuclear splicing factors that are then involved
`in the splicing reactions. By changing the way the splicing ma·chinery reads or
`recognises the motifs involved in pre-mRNA processing, it is possible to create
`differentially spliced mRNA molecules. It has now been recognised that the
`majority of human genes are alternatively spliced during normal gene expression,
`although the mechanisms invoked hav~ not been identified. Using antisense
`oligonucleotides, it has .been shown that errors and deficiencies in a coded mRNA
`could be bypassed or removed from the mature gene transcripts.
`
`

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`WO 2011/057350
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`
`In nature, the extent of genetic deletion or exon skipping in the splicing process is
`.
`not fully understood, although many instances have been documented to occur,
`generally at very low levels (Sherrat TG, et al., (1993) Am J Hum Genet 53, 1007-
`1015). However, it is recognised that if exons associated with disease- causing
`5 mutations can be specifically deleted from some genes, a. shortened protein
`product can sometimes be produced that has similar biological properties of the
`native protein or has sufficient biological activity to ameliorate the disease caused
`by mutations · associated with the target exon (Lu QL, et al., (2003) · Nature
`Medicine 9, 1009-1014; Aartsma-Rus A et al., (2004) Am J Hum Genet 74: 83-92).
`
`10 This process of targeted exon skipping is likely to be particularly useful in long
`genes where there are many axons and introns, where there is redundancy in the
`genetic constitution of the exons or where a protein is able to function without one
`or more particular exons (e.g. with the dystrophin gene, which consists of 79
`exons; or possibly some collagen genes which encode for repeated blocks of
`seqyence or the huge nebulin or titin genes which are comprised of -80 and over
`370 axons, respectively).
`
`15
`
`Efforts to redirect gene processing for the treatment of genetic diseases
`associated with truncations caused by mutations in various genes have focused
`on the use of antisense oligonucleotides that either: (1 ) fully or partially overlap
`20 with the elements involved. in the splicing process; or (2) bind to the pre-mRNA at
`a position sufficiently close to the element to disrupt the binding and function of
`the. splicing factors that would normally mediate a particular splicing reaction
`which occurs at that element (e.g., binds to the pre-mRNA at a position within 3,
`6, or 9 nucleotides of the element to be blocked).
`
`25
`
`30
`
`For example, modulation of mutant dystrophin pre-mRNA splicing with. antisense
`oligoribonucleotides has been reported both in vitro and in vivo. In one type of
`dystrophin mutation reported in Japan, a 52-base pair deletion mutation causes
`exon 19 to be removed with the flanking intrans during the splicing process
`(Matsuo et al., (1991) J Clin Invest. 87:2127-2131). An in vitro minigene splicing
`system has been used to show that a 31-mer 2'-O-methyl oligoribonucleotide
`.
`complementary to the 5' half of th~ deleted sequence in dystrophin Kobe exon 19
`
`'
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`
`inhibited splicing of wild-type pre-mRNA (Takashima et al. (1995), J. Clin. Invest.
`95:515-520). The same oligonucleotide was used to induce exon skipping from
`the native dystrophin gene transcript in human cultured lymphoblastoid cells.
`
`5
`
`Dunckley et al. (1997) Nucleosides & Nucleotides, 16, 1665-1668 described in
`vitro constructs for analysis of splicing around exon 23 of mutated dystrophin in
`the mdx mouse mutant, a model for muscular dystrophy. Plans to analyse these
`constructs in vitro using 2' modified oligonucleotides targeted to splice sites within
`and adjacent to mouse dystrophin exon 23 were discussed, though no target sites
`or sequences were given.
`
`1 0
`
`2'-O-methyl oligoribonucleotides were subsequently reported to correct dystrophin
`deficiency in myoblasts from the mdx mouse from this group. An antisense
`oligonucleotide targeted to the 3' splice site of murine dystrophin intron 22 was
`reported to cause skipping of the mutant exon as well as several flanking exons
`and created a novel in-frame dystrophin transcript with a novel internal deletion.
`15 This mutated dystrophin was expressed in 1-2% of. antisense treated mdx
`myotubes. Use of other oligonucleotide modifications such as 2'-0- methoxyethyl
`phosphodiesters are described (Dunckley et al.. (1998) Human Mol. Genetics,
`5:1083-90).
`
`20
`
`Thus, antisense molecules may provide a tool in the treatment of genetic
`disorders such as Duchenne Muscular Dystrophy (DMD). However, attempts to
`induce exon skipping using antisense moiecules have had mixed success.
`
`Studies on dystrophin exon 19, where successful skipping of that exon from the
`dystrophin pre-mRNA was · achieved using a variety of antisense molecules
`directed at the flanking splice sites or motifs within the exon · involved in exon
`definition as described by Errington et al. (2003) J Gen Med 5: 518-527).
`
`I
`
`25
`
`In contrast to the apparent ease of exon 19 skipping, the first report of exon 23
`skipping in the mdx mouse by Dunckley et al., (1998) is now considered to be
`. reporting only a naturally occurring revertant transcript or artefact rather than any
`true antisense activity. In addition to not consistently generating transcripts
`30 missing exon 23, Dunckley et al, (1998) did not show any time course of induced
`
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`5
`
`exon skipping, or even titration of antisense oligonucleotides, to demonstrate dose
`dependent effects where the levels of exon skipping corresponded with increasing
`or decreasing amounts of antisense oligonucleot_ide. Furthermore, this work could
`not be replicated by other researchers.
`
`I
`
`5 The first example of specific and reproducible exon skipping in the mdx mouse
`model was reported by Wilton et al., (1999) Neuromuscular'Disorders 9,330- 338.
`By directing an antisense molecule to the donor splice site, consistent and
`efficient exon 23 skipping was induced in the dystrophin mRNA within 6 hours of
`treatment of the cultured cells. Wilton et al., (1999), also describe targeting the
`acceptor region of the mouse dystrophin pre-mRNA with longer antisense
`oligonucleotides and being unable to repeat the published results of Dunckley et
`al. (1998). No exon skipping, either 23 alone or multiple removal of several
`flanking exons, could be reproducibly detected using a selection of antisense
`oligonucleotides directed at the acceptor splice site of intron 22.
`
`10
`
`15 While the first antisense oligonucleotide directed at the intron 23 donor splice site
`induced consistent exon skipping in primary cultured myoblasts, this compound
`was found to be rryuch less efficient in immortalized cell cultures expressing higher
`level_s of dystrophin. However, with refined targeting and antisense oligonucleotide
`design, the efficiency of specific exon removal was increased by almost an order
`of magnitude (see Mann CJ et al., (2002) J Gen Med 4,644-654).
`
`20
`
`Thus, there remains a need to provide antisense oligonucleotides capable of
`binding to and modifying the splicing of a target nucleotide sequence. Simply
`directing the antisense oligonucleotides to motifs presumed to be crucial for
`splicing is no guarantee of the efficacy of that compound in a therapeutic setting.
`
`The preceding discussion of the background to the invention is intended only to
`facilitate.an understanding of the present invention. It should be appreciated that
`the discussion is not an acknowledgment or admission that any of the material
`referred to was part of the common general knowledge as at the priority date of
`the application.
`
`25
`
`30
`
`

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`WO 2011/057350
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`6
`
`Summary of the Invention
`
`The present invention provides antisense molecule compounds and compositions
`suitable for binding to RNA motifs involved in the splicing of pre-mRNA that are
`able to induce specific and efficient exon skipping and a method for their use
`thereof.
`
`5
`
`The choice of target selection plays a crucial role in the efficiency of exon skipping
`and hence its subsequent application· of a potential therapy. Simply designing
`antisense molecules to target regions of pre-mRNA presumed to be involved in
`splicing is no guarantee of inducing efficient and specific exon skipping. The most
`10 obvious or readily defined targets for splicing intervention are the donor and
`acceptor splice sites although there are less defined or conserved motifs including
`exonic splicing enhancers, silencing elements and branch points. The acceptor
`and donor splice sites have consensus sequences of about 16 and. 8 bases
`respectively (see Figure 1 for schematic representation of motifs and domains
`involved in exon recognition, intron removal and the splicing process).
`
`15
`
`According to a first aspect, the invention provides antisense molecules capable of
`binding to a selected target to induce exon skipping.
`
`For example, to induce exon skipping in exons 5, 12, 17, 21, 22, 24, 43-47, 49, 50,
`54-64, 66, 67, 70 and 72 in the Dystrophin gene transcript the antisense molecules
`20 are preferably selected from the group listed in Table 1A.
`
`In a further example,
`it is possible to combine two or niore antisense
`oligonucleotides of the present invention together to induce more efficient exon
`skipping in exons 3, 4, a·, 10, 26, 36, 48, 60, 66 and 68. A combinatic;m 1or
`"cocktail" of antisense oligonucleotides are directed at exons to induce efficient
`25 exon skipping.
`According to a second aspect, the present invention provides antisense molecules
`selected and or adapted to aid in the prophylactic or therapeutic treatment of a
`genetic disorder comprising at least an antisense molecule in a form suitable for
`delivery to a patient.
`
`

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`WO 2011/057350
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`7
`
`According to a third aspect, the invention provides a method for treating a patient
`suffering from a genetic disease wherein there is a mutation in a gene encoding a
`particular protein and the affect of the mutation can be abrogated by exon
`skipping, comprising the steps of: (a) ,selecting an antisense molecule in
`accordance with the methods described herein; and (b) administering the
`molecule to a patient in need of such treatment.
`
`5
`
`The
`invention also addresses the use of purified and
`isolated antisense
`oligonucleotides of the invention, for the manufacture of a medicament for treatment
`of a genetic disease.
`
`1 O The invention further provides a method of treating a condition characterised by
`Duchenne muscular dystrophy, which method comprises administering to a
`patient in need of treatment an effective amount of an appropriately designed
`antisense oligonucleotide of the invention, relevant to the p_articular genetic lesion
`in that patient. Further, the invention provides a method for prophylactically
`treating a patient to prevent or· at least minimise Duchene muscular dystrophy,
`comprising the step of: administering to the patient an effective amount of an
`antisense oligonucleotide or a pharmaceutical composition comprising one or
`more of these biological molecules.
`
`15
`
`The invention also provides kits for treating a genetic disease, which kits comprise
`20 · at least a antisense oligonucleotide of the present invention, packaged in a
`suitable container and instructions for its use.
`
`Other aspects and advantages of the invention will become apparent to those skilled
`in the art from a review of the ensuing description, which proceeds with reference to
`the following figures.
`
`25
`
`Brief Description of the Drawings
`
`Figure 1
`
`Schematic representation of motifs and domains involved in exon
`recognition, intron removal and the splicing process.
`
`

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`WO 2011/057350
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`8
`
`Figure 2.
`
`Figure 3.
`
`Figure 4.
`
`Figure 5
`
`Figure 6.
`
`Figure 7.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`the concept of antisense
`representation of
`Diagrammatic
`oligonucleotide induced exon skipping to by-pass disease-causing
`mutations (not drawn to scale). The hatched box represents an
`exon carrying a mutation that prevents .the translation of the rest of
`the mRNA into a protein. The solid black bar represents an
`antisense oligonucleotide that prevents inclusion of that exon in the
`mature mRNA.
`
`Gel electrophoresis showing a "cocktail" of antisense molecules
`directed at exon 3 which induce strong and consistent exon skipping
`at a transfection concentration of 10 nanomolar in cultured normal
`human muscle cells.
`
`Gel electrophoresis showing a "cocktail" of antisense molecules
`directed at exon 4 which induce strong and consistent exon skipping
`at a transfection concentration of 25 nanomolar in cultured normal
`human muscle cells.
`
`Gel electrophoresis showing strong and efficient human exon 5
`skipping using an antisense molecules [H5A(+35+65)] directed at an
`exon 5 internal domain, presumably an exon splicing enhancer. This
`induces consistent exon skipping at a
`preferred compound
`transfection concentration of 25 nanomolar in cultured human
`muscle cells.
`
`Gel electrophoresis showing a "cocktail" of antisense molecules
`directed at exon 8 which induce strong and consistent exon skipping
`of both exon 8 and exon8/9 at a transfection concentration of 10
`nanomolar in cultured normal human muscle cells.
`
`Gel electrophoresis showing various cocktails and single antisense
`molecules wich induce skipping of exon 10 and surrounding exons.
`A combination of [H10A(-05+16)] and [H10A(+98+119)] qr [H10A(-
`05+16)] and [H10A(+130+149)] induces skipping of exon 10 and
`exons 9-12, whilst [H10A(-05+16)] alone induces skipping of exons
`9-14.
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`9
`
`Figure 8.
`
`Figure 9.
`
`Gel electrophoresis showing exon 14 skipping using antisense
`molecule H14A(+31+61) directed at exon 14.
`
`Gel electrophoresis showing exon 17 skipping using antisense
`molecule H17A(+10+35) directed at exon 17.
`
`Figure 10. Gel electrophoresis showing two cocktails of antisense molecules
`directed at exon 26. The double cocktail of [H26A(-07+19)] and
`[H26A(+24+50)] induces good skipping of,exon 26, and the addition
`of a further antisense molecule to the cocktail does not affect the
`efficiency of skipping.
`
`Figure 11. Gel electrophoresis showing a "cocktail" of antisense molecules
`directed at exon 36 which induce strong and consistent exon
`skipping at a transfection concentration of 25 nanomolar in cultured
`normal human muscle cells.
`
`5
`
`10
`
`15
`
`Figure 12. Gel electrophoresis showing strong and consistent exon 43 skipping
`to 25 nanomolar in cultured normal human muscle cells using
`antisense molecule H43A(+92+117).
`
`Figure 13. Gel electrophoresis showing dose dependant exon 55 skipping using
`antisense molecule H44A(+65+90).
`
`Figure 14. Gel electrophoresis showing ~trong and consistent exon 45 skipping
`using antisense molecule H45A(-09+25).
`
`20
`
`Figure 15. Gel electrophoresis showing strong and consistent exon 46 skipping
`using antisense molecule H46A(+81+109).
`.
`
`Figure 16. Gel electrophoresis showing strong and consistent exon 47 skipping
`using antisense molecule H47A(+01+29).
`
`25
`
`Figure 17. Gel electrophoresis showing a "cocktail" of al'.ltisense molecules
`directed at exon 47 which induce strong and consistent exon
`skipping.
`
`Figure 18. Gel electrophoresis showing strong and consistent exon 49 skipping
`using antisense molecule H49A(+45+70).
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`Figure 19. Gel electrophoresis showing strong and consistent exon 50 skipping
`using antisense molecule H50A(+48+74).
`Figure 20. Gel electrophoresis showing strong and consistent exon 51 skipping
`using antisense molecule H51A(+66+95).
`5 Figure 21. Gel electrophoresis showing strong and consistent exon 54 skipping
`using antisense molecule H54A(+67+97).
`Figure 22. Gel electrophoresis showing antisense, molecule H55A(-10+20)
`induced dose dependant exon 55 skipping.
`Figure 23. Gel electrophoresis showing strong and consistent exon 56 skipping
`using antisense molecule H56A(+92+121).
`Figure 24. Gel electrophoresis showing antisense molecule H57A(-10+20)
`induced dose dependant exon 57 skipping.
`Figure 25. Gel electrophoresis showin~ exon 59 and exon 58/59 skipping using
`antisense molecule H59A(+96+120) directec;1 at exon 59.
`15 Figure 26. Gel electrophoresis showing two different cocktails which induce
`exon skipping of exon 60.
`
`10
`
`Figure 27. Gel electrophoresis showing exon 63 skipping using antisense
`molecule H63A(+20+49).
`
`Figure 28. Gel electrophoresis showing exon 64 skipping using antisense
`molecule H64A( +34+62).
`
`20
`
`Figure 29. Gel electrophoresis showing a "cocktail" of antisense molecules
`directed at exon 66 which induce dose dependant exon skipping.
`Figure 30. Gel electrophoresis showing exon 67 skipping using antisense
`molecule H67A(+17+47).
`
`25
`
`Figure 31. Gel .electrophoresis showing a 11cocktail'_' of antisense molecules
`directed at exon 68 which induce dose dependant exon skipping.
`Figure 32. Gel electrophoresis showing a "cocktail" of antisense molecules
`which induce strong and consistent exon skipping of exons 69/70 at
`a transfection concentration pf 25 nanomolar.
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`11
`
`Figure 33. Gel electrophoresis showing various "cocktails" of antisense
`molecules which induce various levels of skipping in exon 50.
`Figure 34. Gel electrophoresis showing a cocktail of three antisense molecules
`which induce efficient skipping of exons 50/51.
`
`5
`
`10
`
`Figure 35. Graph of densitometry results showing various efficiencies of exon ·
`skipping.
`The antisense molecules
`tested were Exon 3
`[H3A(+30+60) & H3A(+61+85}]; Exon 4
`[H4O(+14-11) &
`H4A(+11+40)]; Exon 14 [H14A(+32+61)]; Exon 17 [H17A(+10+35)];
`Exon 26 [H26A(-07+19), H26A(+24+50} & H26A(+68+92)]; Exon 36
`[H36A(~16+09) & H36A(+22+51)].
`
`Figure 36. Graph of densitometry results showing various efficiencies of exon
`skipping.
`The antisense molecules
`tested were Exon 46
`[H46A(+81+109)];
`Exon
`47
`[H47A(+01+29)];
`Exon
`48
`. [H48A(+q1+28) & H48A(+40+67)]; Exon 49 [H49A(+45+70)].
`
`15
`
`Figure 37. Gel electrophoresis showing exon 11 skipping using antisense
`molecule H11A(+50+79).
`
`Figure 38. Gel electrophoresis showing exon 12 skipping using antisense
`molecule H12A{+30+57).
`
`Figure 39. Gel electrophoresis showing exon 44 skipping using antisense
`molecule H44A(+59+85).
`
`20
`
`Figure 40. Gel electrophoresis showing exon 45 skipping using, antisense
`molecule H45A(-03+25).
`
`F_igure 41. Gel electrophoresis showing exon 51 skipping -using antisense
`molecule H51A(+71+~00).
`
`25
`
`Figure 42. Gel electrophoresis showing exon 52 skipping using antisense
`molecule H52A(+09+38).
`
`Figure 43. Gel electrophoresis showing exon 53 skipping · using antisense
`molecule H53A(+33+65).
`
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`
`Figure 44. Gel electrophoresi.s showing exon 46 skipping using anti sense
`molecule H46A(+93+122).
`
`Figure 45. Gel electrophoresis showing exon . 73 skipping using anti sense
`molecule H73A(+02+26).
`
`5
`
`Figure 46. Sequences of antisense molecules.
`
`Detailed Description
`
`BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS
`
`Table 1A: Single antisense molecules
`
`SEQID
`
`1
`
`52
`
`2
`53
`
`3
`4
`
`5
`6
`
`7
`
`8
`
`9
`
`10
`54
`
`11
`55
`61
`62
`
`12
`56
`
`13
`
`14
`
`Exon
`Exon 5
`H5A(+35+65)
`Exon 11
`H11A(+50+79)
`Exon 12
`H12A(+52+75)
`H12A(+30+57)
`Exon 17
`H17A(-07+23)
`H17A(+61+86)
`Exon 21
`H21A(+86+114)
`H21A(+90+119)
`Exon 22
`H22A(+125+146)
`Exon 24
`H24A(+51+73)
`Exon43
`H43A(+92 +117)
`Exon 44
`H44A(+65+90)
`H44A( +59+85)
`Exon 45
`H45A (-09+25)
`H45A(-03+25)
`H45A(-06+25)
`H45A(-12+19)
`Exon46
`H46A(+81+109) ·
`H46A(+93+122)
`Exon 47
`H47A(+01+29)
`Exon 49
`H49A(+45+ 70)
`Exon 50
`
`Sequence
`
`AAA CCA AGA GUC AGU UUA UGA UUU GCA UCU A
`
`CUG UUC CAA UCA GCU UAC UUC CCA AUU GUA
`
`UCU UCU GUU UUU GUU AGC CAG UCA
`CAG UCA UUC AAC UCU UUC AGU UUC UGA U
`
`GUG GUG GUG ACA GCC UGU GAA ALIC UGU GAG
`UGU UCC CUU GUG GUC ACC GUA GUU AC
`
`CAC AAA GUC UGC AUC CAG GAA GAU GGG UC
`AAG GCC ACA AAG UCU GCA UCC AGG AAC AUG
`
`CUG CAA UUC CCC GAG UCU CUG C
`
`CAA GGG CAG GCC AUU CCU CCU UC
`
`GAG AGC UUC CUG UAG CUU CAC CCU UU
`
`UGU UCA GCU UCU GUU AGC CAC UGA
`CUG UUC AGC UUC UGU UAG CCA CUG AUU
`
`GCU GCC CM UGC CAU CCU GGA GUU CCU GUA AGA U
`GCU GCC CM UGC CAU CCU GGA GUU CCU G
`GCU GCC CM UGC GAU CCU GGA GUU CCU GUA A
`CM UGC GAU CCU GGA GUU CCU GUA AGA UAC C
`
`UCC AGG UUC AAG UGG GAU ACU AGC AAU GU
`GUU GCU GCU CUU UUC CAG GUU CAA GUG GGA
`
`UGG CGC AGG GGC AAC UCU UGC ACC AGU AA
`
`ACA AAU GCU GCC CUU UAG ACA AAA UC
`
`

`

`WO 2011/057350
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`PCT/AU2010/001520
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`13
`
`15
`
`57
`
`58
`
`59
`
`16
`
`17
`
`18
`19
`
`20
`
`21
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`29
`
`30
`
`60
`
`·' H50A(+48+74)
`Exon 51
`H51A(+71+100)
`Exon 52
`H52A(+09+38)
`Exon 53
`H53A(+33+65)
`Exon54
`H54A(+67+97)
`Exon 55
`H55A(-10 +20)
`Exon 56
`H56A{+92+121)
`H56A{+112+141)
`Exon 57
`H57A(-10+20)
`Exon 58
`H58A(+34+64)
`H58D(+17-07)
`Exon 59
`H59A(+96 +120)
`Exon 60
`H60A{+33+62)
`Exon 61
`H61A(+10+40)
`Exon 62
`H62A(23+52)
`Exon 63
`H63A(+20+49)
`Exon64
`'
`H64A(+34+62)
`Exon 66
`H66A(-8+19)
`Exon 67
`H67A(+17+47)
`Exon 73
`H73A(+02+26)
`
`GGC UGC uuu GCC cue AGC ucu UGA AGU
`
`AGC AGG UAC CUC CAA CAU CAA GGA AGA UG
`
`UCC AAC UGG GGA CGC CUC UGU UCC AAA UCC UGC
`
`uuc AAC UGU UGC cue CGG uuc UGA AGG UGU ucu
`
`UGG UCU GAU CUG GAG AAU MU CCC GGA GAA G
`
`CAG CCU CUC GCU CAC UCA CCC UGC AAA GGA
`
`CCA AAC GUC UUU GUA ACA GGA CUG GAU
`CCA CUU GAA GUU GAU GUU AUC CM ACG UCU
`
`AAC UGG CUU CCA AAU GGG ACC UGA AAA AGA
`
`UUC GUA CAG UCU CAA GAG UAC UCA UGA UUA C
`CM UUA CCU CUG GGC UCC UGG UAG
`
`CUA UUU UUC UCU GCC AGU CAG CGG A
`
`CGA GCA AGG UCA UUG ACG UGG CUC ACG UUC
`
`GGG cuu GAU GCA GCU GCC UGA cue GGU CCU C
`
`UAG GGC ACU UUG UUU GGC GAG AUG GCU CUC
`
`GAG cue UGU CAU uuu GGG AUG GUC CCA GCA
`
`CUG CAG UCU UCG GAG UUU CAU GGC AGU CC
`
`GAU CCU CCC UGU UCG UCC CCU AUU AUG
`
`GCG CUG GUC ACA AM UCC UGU UGA ACU UGC
`
`CAU UGC UGU UUU CCA UUU CUG GUA G
`
`Table 1 B: Cocktails of antisense molecules
`
`SEQID
`
`31
`32
`
`33
`34
`
`35
`36
`
`37
`38
`
`Exon
`Exon 3 cocktails
`H3A(+30+60)
`H3A(+61+85)
`Exon 4 cocktails
`H4A( + 11 +40)
`H4D{+14-11)
`Exon 8 cocktails
`H8A(-06+24)
`H8A(+134+156)
`Exon 10 cocktails
`H10A(-05+16)
`H10A(+98+119)
`Exon 26 cocktails
`
`Sequence
`
`UAG GAG GCG CCU CCC AUC CUG UAG GUC ACU G
`G CCC UGU CAG GCC UUC GAG GAG GUC
`
`UGU UCA GGG GAU GM cue UUG UGG AUC cuu
`GUA CUA CUU ACA UUA UUG UUC UGC A
`
`UAU CUG GAU AGG UGG UAU CM CAU CUG UAA
`AUG UAA CUG AAA AUG UUC UUC UUU A
`
`CAG GAG CUU CCA AAU GCU GCA
`UCC UCA GCA GM AGA AGC CAC G
`
`

`

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`14
`
`39
`40
`41
`
`42
`43
`
`44
`45
`
`46
`47
`
`48
`49
`
`50
`51
`
`H26A(-07+19)
`H26A(+24+50)
`H26A( +68+92)
`Exon 36 cocktails
`H36A(-16+09)
`H36A(+22+51)
`Exon 48 cocktails
`H48A(+01+28)
`H48A{+40+67)
`Exon 60 cocktails
`H60A(+87+116)
`HB0A(+37 +66)
`Exon 66 cocktails
`H66A(-02+28)
`H66O(+13~17)
`Exon 68 cocktails
`H68A(+48+ 72)
`H68D(+23-03)
`
`CCU CCU UUC UGG CAU AGA CCU UCC AC
`CUU ACA GUU UUC UCC AAA CCU CCC UUC
`UGU GUC AUC CAU UCG UGC AUC UCU G
`
`CUG GUA UUC CUU MU UGU ACA GAG A
`UGU GAU GUG GUC CAC AUU CUG GUC AM AGU
`
`cuu GUU ucu CAG GUA AAG cue UGG AMC
`CM GCU GCC CM GGU CUU UUA UUU GAG C
`
`UCC AGA GUG CUG AGG UUA UAC GGU GAG AGC
`CUG GCG AGC AAG GUC CUU GAC GUG GCU CAC
`
`CAG GAC ACG GAU CCU CCC UGU UCG UCC CCU
`UAA UAU ACA GGA CUU ACA UCU GUA GUU GUC
`
`GAG GAU GGA CUG GGG UUC CAG UCU C
`UAC CUG MU CCA AUG AUU GGA CAG UC
`
`·
`
`GENERAL
`
`Those skilled in the art will appreciate that the invention described herein is
`susceptible to variations and modifications other than those specifically described.
`It is · to be understood that the invention includes all such variations and
`modifications. The invention also includes all of the steps, features, compositjons
`and compounds referred to or indicated in the specification, individually or
`collectively and any and all combinations or any two or more of the steps or
`features.
`
`The pre~ent invention is not to be limited in scope by the specific embodiments
`described herein, which are intended for the purpose of exemplification only.
`Functionally equivalent products, compositions and methods are clearly within the
`scope of the invention as described herein.
`
`Sequence identity numbers (SEQ ID NO:) containing nucleotide and amino acid
`sequence information incluqed in this specification are collected at the end of the
`description and have been prepared using the programme Patentln Version 3.0.
`Each nucleotide or amino :acid sequence is identified in the sequence listing by
`the numeric indicator <210> followed by the sequence identifier (e.g. <210>1,
`<210>2, etc.). The length, type of sequence and source organism for each
`nucleotide or amino acid sequence are indicated by information provided in the
`
`5
`
`10
`
`15
`
`20
`
`

`

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`15
`
`numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide and
`amino acid sequences referred to in the specification ·· are defined by the ·
`information provided in numeric indicator _field <400> followed by the sequence
`identifier (e.g. <400>1', <400>2, etc.).
`
`5 An antisense molecule nomenclature system was proposed and published to
`distinguish between the different antisense molecules (see Mann et al., (2002) .J
`Gen Med 4, 644-654). This nomenclature became especially relevant when
`testing several slightly different antisense molecules, all directed at the same
`target region, as shown below:
`
`10
`
`15
`
`20
`
`25
`
`H #AID (x: y).
`
`The first letter designates the species
`
`(e.g. H: human, M: murine, C: canine)
`
`"#" designates target dystrophin exon number.
`
`"AID" indicates acceptor or donor splice site at the beginning and end of the exon,
`respectively.
`
`(x y) represents the annealing coordinates where "-" or "+" indicate intronic or
`exonic sequences respectively. As a