(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`1111111111111111 IIIIII IIIII 11111111111111111111 lllll 111111111111111 lllll 11111111111111111111111
`
`( 43) International Publication Date
`30 September 2004 (30.09.2004)
`
`PCT
`
`(10) International Publication Number
`WO 2004/083432 Al
`
`(51) International Patent Classification 7:
`A61K 31/7088, 48/00
`
`C12N 15/11,
`
`(74) Agent: PRINS, A., W.; Nieuwe Parklaan 97, NL-2587 BN
`Den Haag (NL).
`
`(21) International Application Number:
`PCT /NL2003/000214
`
`(22) International Filing Date: 21 March 2003 (21.03.2003)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(71) Applicant
`(for all designated States except US):
`ACADEMISCH ZIEKENHUIS LEIDEN [NL/NL];
`Albinusdreef 2, NL-2333 ZA Leiden (NL).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): VAN OMMEN,
`Garrit-Jan, Boudewijn
`[NL/NL]; Westerstraat 73,
`NL-1015 LW Amsterdam (NL). VAN DEUTEKOM,
`Judith, Christina, Theodora [NL/NL]; Van Ravensteyn(cid:173)
`erf 133, NL-3315 DK Dordrecht (NL). DEN DUNNEN,
`Johannes, Theodorus [NL/NL]; Boomheide 2, NL-3069
`LA Rotterdam (NL). AARTSMA-RUS, Annemieke
`[NL/NL]; Zijldonk 20, NL-2317 XX Leiden (NL).
`
`(81) Designated States (national): AE, AG, AL, AM, AT (util(cid:173)
`ity model), AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA,
`CH, CN, CO, CR, CU, CZ (utility model), CZ, DE (util(cid:173)
`ity model), DE, DK (utility model), DK, DM, DZ, EC, EE
`(utility model), EE, ES, FI (utility model), FI, GB, GD, GE,
`GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ,
`LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN,
`MW, MX, MZ, NI, NO, NZ, OM, PH, PL, PT, RO, RU,
`SC, SD, SE, SG, SK (utility model), SK, SL, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, Fl, FR, GB, GR, HU, IE, IT, LU, MC, NL, PT, RO,
`SE, SI, SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`with international search report
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`---iiiiiiiiiiii
`iiiiiiiiiiii -----
`
`---i
`
`iiiiiiiiiii
`iiiiiiiiiiii
`
`M
`~
`"'1'
`~
`Q0
`Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`~ (54) Title: MODULATION OF EXON RECOGNITION IN PRE-MRNA BY INTERFERING WITH THE SECONDARY RNA
`Q STRUCTURE
`Q
`M (57) Abstract: The invention provides a method for generating an oligonucleotide with which an exon may be skipped in a pre-
`0 mRNA and thus excluded from a produced mRNA thereof. Further provided are methods for altering the secondary structure of
`
`: , an mRNA to interfere with splicing processes and uses of the oligonucleotides and methods in the treatment of disease. Further
`;;, provided are pharmaceutical compositions and methods and means for inducing skipping of several exons in a pre-mRNA.
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`1
`
`Title:
`
`Modulation of exon recognition in pre-mRNA by interfering with the
`
`secondary RNA structure.
`
`The invention relates to the fields of molecular biology and medicine.
`More in particular the invention relates to the restructuring of mRNA
`produced from pre-mRNA, and therapeutic uses thereof.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`The central dogma of biology is that genetic information resides in
`the DNA of a cell and is expressed upon transcription of this information,
`where after production of the encoded protein follows by the translation
`machinery of the cell. This view of the flow of genetic information has
`prompted the pre-dominantly DNA based approach for interfering with the
`protein content of a cell. This view is slowly changing and alternatives for
`interfering at the DNA level are being pursued.
`In higher eukaryotes the genetic information for proteins in the
`DNA of the cell is encoded in exons which are separated from each other by
`intronic sequences. These intrans are in some cases very long. The
`transcription machinery generates a pre-mRNA which contains both exons and
`intrans, while the splicing machinery, often already during the production of
`the pre-mRNA, generates the actual coding region for the protein by splicing
`
`together the exons present in the pre-mRNA.
`Although much is known about the actual processes involved in the
`generation of an mRNA from a pre-mRNA, much also remains hidden. In the
`present invention it has been shown possible to influence the splicing process
`such that a different mRNA is produced. The process allows for the predictable
`and reproducible restructuring of mR~A produced by a splicing machinery. An
`oligonucleotide capable of hybridising to pre-mRNA at a location of an exon
`that is normally included in the mature mRNA can direct the exclusion of the
`thus targeted exon or a part thereof.
`In the present invention means and methods are provided for the
`design of appropriate complementary oligonucleotides. To this end the
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`2
`
`invention provides a method for generating an oligonucleotide comprising
`determining, from a (predicted) secondary structure of RNA from an exon, a
`region that assumes a structure that is hybridised to another part of said RNA
`(closed structure) and a region that is not hybridised in said structure (open
`structure), and subsequently generating an oligonucleotide, which at least in
`part is complementary to said closed structure and which at least in part is
`complementary to said open structure. RNA molecules exhibit strong
`secondary structures, mostly due to base pairing of complementary or partly
`complementary stretches within the same RNA. It has long since been thought
`that structures in the RNA play a role in the function of the RNA. Without
`being bound by theory, it is believed that the secondary structure of the RNA
`of an exon plays a role in structuring the splicing process. Through its
`structure, an exon is recognized as a part that needs to be included in the pre(cid:173)
`mRNA. Herein this signalling function is referred to as an exon inclusion
`signal. A complementary oligonucleotide of the invention is capable of
`interfering with the structure of the exon and thereby capable of interfering
`with the exon inclusion signal of the exon. It has been found that many
`complementary oligonucleotides indeed comprise this capacity, some more
`efficient than others. Oligonucleotides of the invention, i.e. those with the said
`overlap directed toward open and closed structures in the native exon RNA,
`are a selection from all possible oligonucleotides. The selection encompasses
`oligonucleotides that can efficiently interfere with an exon inclusion signal.
`Without being bound by theory it is thought that the overlap with an open
`structure improves the invasion efficiency of the oligonucleotide (i.e. increases
`the efficiency with which the oligonucleotide can enter the structure), whereas
`the overlap with the closed structure subsequently increases the efficiency of
`interfering with the secondary structure of the RNA of the exon, and thereby
`interfere with the exon inclusion signal. It is found that the length of the
`partial complementarity to both the closed and the open structure is not
`extremely restricted. We have observed high efficiencies with oligonucleotides
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`3
`
`with variable lengths of complementarity in either structure. The term
`complementarity is used herein to refer to a stretch of nucleic acids that can
`hybridise to another stretch of nucleic acids under physiological conditions. It
`is thus not absolutely required that all the bases in the region of
`complementarity are capable of pairing with bases in the opposing strand. For
`instance, when designing the oligonucleotide one may want to incorporate for
`instance a residue that does not base pair with the base on the complementary
`strand. Mismatches may to some extent be allowed, if under the circumstances
`in the cell, the stretch of nucleotides is capable of hybridising to the
`complementary part. In a preferred embodiment a complementary part (either
`to said open or to said closed structure) comprises at least 3, and more
`preferably at least 4 consecutive nucleotides. The complementary regions are
`preferably designed such that, when combined, they are specific for the exon in
`the pre-mRNA. Such specificity may be created with various lengths of
`complementary regions as this depends on the actual sequences in other (pre(cid:173)
`)mRNA in the system. The risk that also one or more other pre-mRNA will be
`able to hybridise to the oligonucleotide decreases with increasing size of the
`oligonucleotide. It is clear that oligonucleotides comprising mismatches in the
`region of complementarity but that retain the capacity to hybridise to the
`targeted region(s) in the pre-mRNA, can be used in the present invention.
`However, preferably at least the complementary parts do not comprise such
`mismatches as these typically have a higher efficiency and a higher specificity,
`than oligonucleotides having such mismatches in one or more complementary
`regions. It is thought that higher hybridisation strengths, (i.e. increasing
`number of interactions with the opposing strand) are favourable in increasing
`the efficiency of the process of interfering with the splicing machinery of the
`system.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`The secondary structure is best analysed in the context of the pre-mRNA
`30 wherein the exon resides. Such structure may be analysed in the actual RNA.
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`4
`
`However, it is currently possible to predict the secondary structure of an RNA
`
`molecule (at lowest energy costs) quite well using structure-modelling
`
`programs. A non-limiting example of a suitable program is RNA mfold version
`3.1 server (Mathews et al 1999, J. Mol. Biol. 288: 911-940). A person skilled in
`the art will be able to predict, with suitable reproducibility, a likely structure
`
`5
`
`of the exon, given the nucleotide sequence. Best predictions are obtained when
`
`providing such modelling programs with both the exon and flanking intron
`sequences. It is typically not necessary to model the structure of the entire pre(cid:173)
`
`mRNA.
`
`10
`
`The open and closed structure to which the oligonucleotide is directed, are
`preferably adjacent to one another. It is thought that in this way the annealing
`of the oligonucleotide to the open structure induces opening of the closed
`
`structure, annealing progresses into this closed structure. Through this action
`
`15
`
`the previously closed structure assumes a different conformation. The different
`
`conformation may result in the disruption of the exon inclusion signal.
`
`However, when potential (cryptic) splice acceptor and/or donor sequences are
`
`present within the targeted exon, occasionally a new exon inclusion signal is
`
`generated defining a different (neo) exon, i.e. with a different 5' end, a different
`
`20
`
`3' end, or both. This type of activity is within the scope of the present invention
`
`as the targeted exon is excluded from the mRNA. The presence of a new exon,
`
`containing part of the targeted exon, in the mRNA does not alter the fact that
`
`the targeted exon, as such, is excluded. The inclusion of a neo-exon can be seen
`
`as a side effect which occurs only occasionally. There are two possibilities when
`
`25
`
`exon skipping is used to restore (part of) an open reading frame that was
`
`disrupted as a result of a mutation. One is that the neo-exon is functional in
`
`the restoration of the reading frame, whereas in the other case the reading
`
`frame is not restored. When selecting oligonucleotides for restoring reading
`
`frames by means of exon-skipping it is of course clear that under these
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`5
`
`conditions only those oligonucleotides are selected that indeed result in exon(cid:173)
`skipping that restores the open reading frame, with or without a neo-exon.
`
`5
`
`Pre-mRNA can be subject to various splicing events, for instance through
`alternative splicing. Such events may be induced or catalysed by the
`environment of a cell or artificial splicing system. Thus, from the same pre(cid:173)
`mRNA several different mRNA's may be produced. The different mRNA's all
`included exonic sequences, as that is the definition of an exon. However, the
`fluidity of the mRNA content necessitates a definition of the term exon in the
`present invention. An exon according to the invention is a sequence present in
`both the pre-mRNA and mRNA produced thereof, wherein the sequence
`included in the mRNA is, in the pre-mRNA, flanked on one side (first and last
`exon) or both sides (any other exon then the first and the last exon) by
`sequences not present in the mRNA. In principle any mRNA produced from
`the pre-mRNA qualifies for this definition. However, for the present invention,
`so-called dominant mRNA's are preferred, i.e. mRNA that makes up at least
`5% of the mRNA produced from the pre-mRNA under the set conditions.
`Human immuno-deficiency virus in particular uses alternative splicing to an
`extreme. Some very important protein products are produced from mRNA
`20 making up even less than 5% of the total mRNA produced from said virus. The
`genomic RNA of retroviruses can be seen as pre-mRNA for any spliced product
`derived from it. As alternative splicing may vary in different cell types the
`exons are defined as exons in the context of the splicing conditions used in that
`system. As a hypothetical example; an mRNA in a muscle cell may contain an
`exon that as absent in an mRNA produced from the same pre-mRNA in a
`nerve cell. Similarly, mRNA in a cancer cell may contain an exon not present
`in mRNA produced from the same mRNA in a normal cell.
`
`10
`
`15
`
`25
`
`Alternative splicing may occur by splicing from the same pre-mRNA. However,
`alternative splicing may also occur through a mutation in the pre-mRNA for
`
`30
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`6
`
`5
`
`10
`
`15
`
`20
`
`25
`
`instance generating an additional splice acceptor and/or splice donor sequence.
`Such alternative splice sequences are often referred to as cryptic splice
`acceptor/donor sequences. Such cryptic splice sites can result in new exons
`(neo-exons). Inclusion of neo-exons into produced mRNA can be at least in part
`prevented using a method of the invention. In case a neo-exon is flanked by a
`cryptic and a "normal" splice donor/acceptor sequence, the neo-exon
`encompasses the old (paleo) exon. If in this case the original splice
`donor/acceptor sequence, for which the cryptic splice donor/acceptor has taken
`its place, is still present in the pre-mRNA, it is possible to enhance the
`production of mRNA containing the paleo-exon by interfering with the exon(cid:173)
`recognition signal of the neo-exon. This interference can be both in the part of
`the neo-exon corresponding to the paleo-exon, or the additional part of such
`neo-exons. This type of exon skipping can be seen as splice correction.
`
`The exon skipping technique can be used for many different purposes.
`Preferably, however, exon skipping is used for restructuring mRNA that is
`produced from pre-mRNA exhibiting undesired splicing in a subject. The
`restructuring may be used to decrease the amount of protein produced by the
`cell. This is useful when the cell produces a particular undesired protein. In a
`preferred embodiment however, restructuring is used to promote the
`production of a functional protein in a cell, i.e. restructuring leads to the
`generation of a coding region for a functional protein. The latter embodiment is
`preferably used to restore an open reading frame that was lost as a result of a
`mutation. Preferred genes comprise a Duchenne muscular dystrophy gene, a
`collagen VI alpha 1 gene (COL6Al), a myotubular myopathy 1 gene (MTMl), a
`dysferlin gene (DYSF), a laminin-alpha 2 gene (LAMA2), an emery-dreyfuss
`muscular dystrophy gene (EMD), and/or a calpain 3 gene (CAPN3). The
`invention is further delineated by means of examples drawn from the
`Duchenne muscular dystrophy gene. Although this gene constitutes a
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`7
`
`particularly preferred gene in the present invention, the invention is not
`limited to this gene.
`
`5
`
`10
`
`Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD)
`are both caused by mutations in the DMD gene, that is located on the X
`chromosome and codes for dystrophin (1-6). DMD has an incidence of 1:3500
`newborn males. Patients suffer from progressive muscle weakness, are
`wheelchair bound before the age of 13 and often die before the third decade of
`their life (7). The generally milder BMD has an incidence of 1:20,000. BMD
`patients often remain ambulant for over 40 years and have longer life
`expectancies when compared to DMD patients (8).
`
`15
`
`Dystrophin is an essential component of the dystrophin-glycoprotein complex
`(DGC), which amongst others maintains the membrane stability of muscle
`fibers (9, 10). Frame-shifting mutations in the DMD gene result in dystrophin
`deficiency in muscle cells. This is accompanied by reduced levels of other DGC
`proteins and results in the severe phenotype found in DMD patients (11, 12).
`Mutations in the DMD gene that keep the reading frame intact, generate
`shorter, but partly functional dystrophins, associated with the less severe
`20 BMD (13, 14).
`
`Despite extensive efforts, no clinically applicable and effective therapy for
`DMD patients has yet been developed (15), although a delay of the onset
`and/or progression of disease manifestations can be achieved by glucocorticoid
`therapy (16). Promising results have recently been reported by us and others
`on a genetic therapy aimed at restoring the reading frame of the dystrophin
`pre-mRNA in cells from the mdx mouse model and DMD patients (17-23). By
`the targeted skipping of a specific exon, a DMD phenotype can be converted
`into a milder BMD phenotype. The skipping of an exon can be induced by the
`binding of antisense oligoribonucleotides (AONs) targeting either one or both
`
`25
`
`30
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`8
`
`5
`
`of the splice sites, or exon-internal sequences. Since an exon will only be
`included in the mRNA when both the splice sites are recognised by the
`spliceosome complex, splice sites are obvious targets for AONs. This was
`shown to be successful, albeit with variable efficacy and efficiency (17, 18, 20,
`21). We hypothesised that targeting exon-internal sequences might increase
`specificity and reduce interference with the splicing machinery itself. Some
`exons have weak splice sites and appear to require binding of a SR protein to
`an exon recognition sequence (ERS) or an exonic splicing enhancer (ESE) to be
`properly recognised by the splicing machinery (24). SR proteins are a highly
`conserved family of arginine/serine rich, spliceosome associated
`phosphoproteins essential for pre-mRNA splicing (50, 51). SR proteins appear
`to act early in splicing by promoting splice site recognition and spliceosome
`assembly. SR proteins also play a regulatory role, because they can determine
`alternative splice site usage in vivo and in vitro. SR proteins appear to be
`recruited from nuclear "speckles", in which they are concentrated, to sites of
`transcription in order to spatially coordinate transcription and pre-mRNA
`splicing within the cell nucleus (49, 52). Disruptive point mutations or AONs
`that block these sequences have been found to result in exon skipping (19, 22,
`24-28). Using e:xon-internal AONs specific for an ERS-like sequence in exon 46,
`20 we were previously able to modulate the splicing pattern in cultured myotubes
`from two different DMD patients with an exon 45 deletion (19). Following AON
`treatment, exon 46 was skipped, which resulted in a restored reading frame
`and the induction of dystrophin synthesis in at least 75% of the cells. We have
`recently shown that exon skipping can also efficiently be induced in human
`control muscle cells for 15 different DMD exons using exon-internal AONs (23,
`unpublished results). In contrast to the previous opinion that skipping can
`only be achieved with weak splice sites or exons containing ERS-like
`sequences, we have seen that of the exons that were skipped in the present
`invention most do not have weak splice sites nor do they contain ERS-like
`sequences. Thus binding of the AONs to the targeted exon per se is sufficient
`
`10
`
`15
`
`25
`
`30
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`9
`
`to cause exon skipping, either by interfering with one or more components of
`
`the splicing machinery or by altering the secondary structure of the RNA in
`
`such a manner that the splicing machinery no longer recognizes the exon. In a
`
`preferred embodiment the exon to be skipped comprises exons 2, 8, 9, 17, 19,
`
`5
`
`29, 40-46, 49-53, 55 or 59. More preferably, exons 2, 8, 9, 17, 40, 41, 42, 44, 49-
`
`52 or 59. In yet another embodiment the exon to be skipped comprises exons 2,
`
`29, 40, 41, 42, 43, 44, 45, 46, 49, 50, 51 or 53.
`
`Any oligonucleotide fulfilling the requirements of the invention may be used to
`
`10
`
`induce exon skipping in the DMD gene. In a preferred embodiment an
`
`oligonucleotide comprises a sequence as depicted as active in exon-skipping in
`
`table 2, or a functional equivalent thereof comprising a similar, preferably the
`
`same hybridisation capacity in kind, not necessarily in amount. Preferably an
`
`oligonucleotide comprising a sequence as depicted in table 2, derived from the
`
`15
`
`exons 2, 40, 41, 42, 43, 44, 45, 49, 50, 51 or 53, demonstratably active in exon
`
`skipping.
`
`Reading frame correction can be achieved by skipping one or two exons
`
`flanking a deletion, by skipping in-frame exons containing a nonsense
`
`20 mutation, or by skipping duplicated exons. This results in proteins similar to
`
`those found in various BMD patients (2, 29). A survey of the Leiden DMD
`
`mutation database [www.dmd.nl: (30)] learns that we can thus correct over
`
`75% of DMD causing mutations (see Table 4). We show the actual therapeutic
`
`effect of exon skipping for 7 different mutations. In all patient muscle cell
`
`25
`
`cultures, we were able to restore dystrophin synthesis in 75% to 80% of treated
`
`cells.
`
`The complementary oligonucleotide generated through a method of the
`
`invention is preferably complementary to a consecutive part of between 16 and
`
`30
`
`50 nucleotides of said exon RNA. Different types of nucleic acid may be used to
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`generate the oligonucleotide. Preferably, the oligonucleotide comprises RNA,
`
`as RNA/RNA hybrids are very stable. Since one of the aims of the exon
`
`skipping technique is to direct splicing in subjects it is preferred that the
`
`oligonucleotide RNA comprises a modification providing the RNA with an
`
`5
`
`additional property, for instance resistance to endonucleases and RNaseH,
`
`additional hybridisation strength, increased stability (for instance in a bodily
`
`fluid), increased or decreased flexibility, reduced toxicity, increased
`
`intracellular transport, tissue-specificity, etc. Preferably said modification
`
`comprises a 2' -O-methyl-phosphorothioate oligoribonucleotide modification.
`
`10
`
`With the advent of nucleic acid mimicking technology it has become possible to
`generate molecules that have a similar, preferably the same hybridisation
`
`characteristics in kind not necessarily in amount as nucleic acid itself. Such
`
`equivalents are of course also part of the invention. Examples of such mimics
`
`15
`
`equivalents are peptide nucleic acid, locked nucleic acid and/or a morpholino
`phosphorodiamidate. Suitable but non-limiting examples of equivalents of
`
`oligonucleotides of the invention can be found in (W ahlestedt, C. et al. Potent
`and non-toxic antisense oligonucleotides containing locked nucleic acids. Proc
`
`Natl Acad Sci US A 97, 5633-8. (2000). Elayadi, A.N. & Corey, D.R.
`
`20 Application of PNA and LNA oligomers to chemotherapy. Curr Opin Investig
`Drugs 2, 558-61. (2001). Larsen, H.J., Bentin, T. & Nielsen, P.E. Antisense
`
`properties of peptide nucleic acid. Biochim Biophys Acta 1489, 159-66. (1999).
`
`Braasch, D.A. & Corey, D.R. Novel antisense and peptide nucleic acid
`
`25
`
`strategies for controlling gene expression. Biochemistry 41, 4503-10. (2002).
`Summerton, J. & Weller, D. Morpholino antisense oligomers: design,
`preparation, and properties. Antisense Nucleic Acid Drug Dev 7, 187-
`
`95.(1997)). Hybrids between one or more of the equivalents among each other
`and/or together with nucleic acid are of course also part of the invention. In a
`
`preferred embodiment an equivalent comprises locked nucleic acid, as locked
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`11
`
`nucleic acid displays a higher target affinity and reduced toxicity and therefore
`
`shows a higher efficiency of exon skipping.
`
`An oligonucleotide of the invention typically does not have to overlap with a
`
`splice donor or splice acceptor of the exon.
`
`5
`
`An oligonucleotide of the invention, or equivalent thereof, may of course be
`
`combined with other methods for interfering with the structure of an mRNA. It
`
`is for instance possible to include in a method at least one other oligonucleotide
`
`that is complementary to at least one other exon in the pre-mRNA. This can be
`
`10
`
`used to prevent inclusion of two or more exons of a pre-mRNA in mRNA
`
`produced from this pre-mRNA. In a preferred embodiment, said at least one
`
`other oligonucleotide is an oligonucleotide, or equivalent thereof, generated
`
`through a method of the invention. This part of the invention is further
`
`referred to as double-or multi-exon skipping. In most cases double-exon
`
`15
`
`skipping results in the exclusion of only the two targeted (complementary)
`
`exons from the pre-mRNA. However, in other cases it was found that the
`
`targeted exons and the entire region in between said exons in said pre-mRNA
`
`were not present in the produced mRNA even when other exons (intervening
`
`exons) were present in such region. This multi-skipping was notably so for the
`
`20
`
`combination of oligonucleotides derived from the DMD gene, wherein one
`
`oligonucleotide for exon 45 and one oligonucleotide for exon 51 was added to a
`
`cell transcribing the DMD gene. Such a set-up resulted in mRNA being
`
`produced that did not contain exons 45 to 51. Apparently, the structure of the
`
`pre-mRNA in the presence of the mentioned oligonucleotides was such that the
`
`25
`
`splicing machinery was stimulated to connect exons 44 and 52 to each other.
`
`In the present invention it has found possible to specifically promote the
`
`skipping of also the intervening exons by providing a linkage between the two
`
`complementary oligonucleotides. To this end the invention provides a
`
`30
`
`compound capable of hybridising to at least two exons in a pre-mRNA encoded
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`12
`
`by a gene, said compound comprising at least two parts wherein a first part
`comprises an oligonucleotide having at least 8 consecutive nucleotides that are
`complementary to a first of said at least two exons, and wherein a second part
`comprises an oligonucleotide having at least 8 consecutive nucleotides that are
`complementary to a second exon in said pre-mRNA. The at least two parts are
`linked in said compound so as to form a single molecule. The linkage may be
`through any means but is preferably accomplished through a nucleotide
`linkage. In the latter case the number of nucleotides that not contain an
`overlap between one or the other complementary exon can be zero, but is
`preferably between 4 to 40 nucleotides. The linking moiety can be any type of
`moiety capable of linking oligonucleotides. Currently, many different
`compounds are available that mimic hybridisation characteristics of
`oligonucleotides. Such a compound is also suitable for the present invention if
`such equivalent comprises similar hybridisation characteristics in kind not
`necessarily in amount. Suitable equivalents were mentioned earlier in this
`description. One or preferably, more of the oligonucleotides in the compound
`are generated by a method for generating an oligonucleotide of the present
`invention. As mentioned, oligonucleotides of the invention do not have to
`consist of only oligonucleotides that contribute to hybridisation to the targeted
`exon. There may be additional material and/or nucleotides added.
`
`As mentioned, a preferred gene for restructuring mRNA is the DMD gene. The
`DMD gene is a large gene, with many different exons. Considering that the
`gene is located on the X-chromosome, it is mostly boys that are affected,
`although girls can also be affected by the disease, as they may receive a bad
`copy of the gene from both parents, or are suffering from a particularly biased
`inactivation of the functional allele due to a particularly biased X chromosome
`inactivation in their muscle cells. The protein is encoded by a plurality of exons
`(79) over a range of at least 2,6 Mb. Defects may occur in any part of the DMD
`gene. Skipping of a particular exon or particular exons can, very often, result
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`13
`
`in a restructured mRNA that encodes a shorter than normal but at least
`partially functional dystrophin protein. A practical problem in the
`development of a medicament based on exon-skipping technology is the
`plurality of mutations that may result in a deficiency in functional dystrophin
`protein in the cell. Despite the fact that already multiple different mutations
`can be corrected for by the skipping of a single exon, this plurality of
`mutations, requires the generation of a large number of different
`pharmaceuticals as for different mutations different exons need to be skipped.
`An advantage of a compound of the invention, i.e. a compound capable of
`inducing skipping of two or more exons, is that more than one exon can be
`skipped with a single pharmaceutical. This property is not only practically
`very useful in that only a limited number of pharmaceuticals need to be
`generated for treating many different Duchenne or Becker mutations. Another
`option now open to the person skilled in the art is to select particularly
`functional restructured dystrophin proteins and produce compounds capable of
`generating these preferred dystrophin proteins. Such preferred end results are
`further referred to as mild phenotype dystrophins. The structure of the normal
`dystrophin protein can be schematically represented as two endpoints having
`structural function (the beads), which are connected to each other by a long at
`least partly flexible rod. This rod is shortened in many Becker patients.
`
`This led the field to the conclusion that not so much the length of the rod but
`the presence of a rod and the composition thereof (with respect to particular
`hinge regions in the protein), is crucial to the function per se of the dystrophin
`protein. Though the size of the rod may have an impact on the amount of
`functionality of the resulting (Becker) protein, there are many notable
`exceptions. These exceptions will be detailed below. There are especially
`benign mutations that can have a very short rod. It was noted by the inventors
`that many more different types of Becker patients should have been detected
`in the patient population. However, some types of shortened dystrophin
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2004/083432
`
`PCT /NL2003/000214
`
`14
`
`5
`
`15
`
`proteins, that according to this hypothesis should have a Becker phenotype,
`are not detected in human population. For some of these "theoretical" Becker
`forms, this could just be a matter of chance. However, in the present invention
`it has been found, that at least some of these "potential" Becker patients have
`such a benign phenotype that subjects having these types of mutations do not
`present themselves to a doctor, or are not diagnosed as suffering from Becker's
`disease. With a compound of the invention it is possible to restructure DMD
`pre-mRNA of many different Duchenne and even Becker patients such that a
`mild phenotype dystrophin is generated after transla