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`US 20100168212Al
`
`c19) United States
`c12) Patent Application Publication
`POPPLEWELL et al.
`
`c10) Pub. No.: US 2010/0168212 Al
`Jul. 1, 2010
`(43) Pub. Date:
`
`(54) OLIGOMERS
`
`(75)
`
`Inventors:
`
`Linda POPPLEWELL, Surrey
`(GB); Ian Graham, Cambridge
`(GB); John George Dickson,
`Surrey (GB)
`
`(51)
`
`Int. Cl.
`A61K 3117088
`C07H 21104
`C12N 15163
`
`(2006.01)
`(2006.01)
`(2006.01)
`
`Publication Classification
`
`Correspondence Address:
`BANNER & WITCOFF, LTD.
`1100 13th STREET, N.W., SUITE 1200
`WASHINGTON, DC 20005-4051 (US)
`
`(52) U.S. Cl. .................... 514/44 R; 536/23.1; 435/320.1
`
`(73) Assignee:
`
`Royal Holloway, University of
`London, Surrey (GB)
`
`(21) Appl. No.:
`
`12/556,626
`
`(22) Filed:
`
`Sep.10,2009
`
`Related U.S. Application Data
`
`(60) Provisional application No. 61/096,073, filed on Sep.
`11, 2008, provisional application No. 61/164,978,
`filed on Mar. 31, 2009.
`
`(57)
`
`ABSTRACT
`
`Molecules are provided for inducing or facilitating exon skip(cid:173)
`ping in forming spliced mRNA products from pre-mRNA
`molecules in cells. The molecules may be provided directly as
`oligonucleotides or expression products of vectors that are
`administered to a subject. High rates of skipping can be
`achieved. High rates of skipping reduce the severity of a
`disease like Duchene Muscular Dystrophy so that the disease
`is more like Becker Muscular Dystrophy. This is a severe
`reduction in symptom severity and mortality.
`
`

`

`Patent Application Publication
`
`Jul. 1, 2010 Sheet 1 of 11
`
`US 2010/0168212 Al
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`FIG.1
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`Patent Application Publication
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`Jul. 1, 2010 Sheet 2 of 11
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`Patent Application Publication
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`Jul. 1, 2010 Sheet 3 of 11
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`

`Patent Application Publication
`
`Jul. 1, 2010 Sheet 11 of 11
`
`US 2010/0168212 Al
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`US 2010/0168212 Al
`
`Jul. 1, 2010
`
`1
`
`OLIGOMERS
`
`TECHNICAL FIELD OF THE INVENTION
`
`[0001] The present invention relates to molecules which are
`capable of causing exon skipping and, in particular, relates to
`molecules which are capable of causing exon skipping in the
`dystrophin gene.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Duchenne muscular dystrophy (DMD) is a severe
`X-linked muscle wasting disease, affecting 1:3500 boys.
`Prognosis is poor: loss of mobility by the age of 12, compro(cid:173)
`mised respiratory and cardiac function by late teens, and
`probable death by the age of 30. The disease is caused by
`mutations within the large dystrophin gene, such that the
`reading frame is disrupted leading to lack of dystrophin pro(cid:173)
`tein expression and breakdown of muscle fibre integrity [1].
`The dystrophin gene is large, with 79 exons. The most com(cid:173)
`mon DMD mutation is genomic deletion of one or more
`exons, generally centred around hotspots involving exons 44
`to 55 and the 5' end of the gene [2]. Mutations of the dystro(cid:173)
`phin gene that preserve the reading frame result in the milder,
`non-life threatening Becker muscular dystrophy (BMD).
`[0003] Exon skipping induced by anti sense oligoribonucle(cid:173)
`otides (AOs ), generally based on an RNA backbone, is a
`future hope as a therapy for DMD in which the effects of
`mutations in the dystrophin 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-intronjunctions in pre(cid:173)
`mRNA into close proximity and performs cleavage of phos(cid:173)
`phodiester bonds at the ends of the intrans with their subse(cid:173)
`quent reformation between exons that are to be spliced
`together. This complex and highly precise process is medi(cid:173)
`ated by sequence motifs in the pre-mRNA that are relatively
`short semi-conserved RNA segments to which bind the vari(cid:173)
`ous nuclear splicing factors that are then involved in the
`splicing reactions. By changing the way the splicing machin(cid:173)
`ery reads or recognises the motifs involved in pre-mRNA
`processing, it is possible to create differentially spliced
`mRNA molecules.
`[0004]
`It has now been recognised that the majority of
`human genes are alternatively spliced during normal gene
`expression, although the mechanisms involved have 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.
`Indeed, by skipping out-of-frame mutations of the dystrophin
`gene, the reading frame can be restored and a truncated, yet
`functional, Becker-like dystrophin protein is expressed. Stud(cid:173)
`ies in human cells in vitro [3, 4] and in animal models of the
`disease in vivo [5-9] have proven the principle of exon skip(cid:173)
`ping as a potential therapy for DMD (reviewed in [1 OJ). Initial
`clinical trials using two different AO chemistries (phospho(cid:173)
`rodiamidate morpholino oligomer (PMO) and phospho(cid:173)
`rothioate-linked 2'-~-methyl RNA (2'OMePS)) [11] have
`recently been performed, with encouraging results. Indisput(cid:173)
`ably impressive restoration of dystrophin expression in the
`TA muscle of four DMD patients injected with a 2'OMePS
`AO to exon 51 has been reported by van Deutekom et al. [ 11].
`[0005] However, it should be noted that, relative to
`2'OMePS AOs, PMOs have been shown to produce more
`consistent and sustained exon skipping in the mdx mouse
`
`model of DMD [12-14; A. Malerba et al, manuscript submit(cid:173)
`ted], in human muscle explants [ 15], and in dystrophic canine
`cells in vitro [16]. Most importantly, PMOs have excellent
`safety profiles from clinical and pre-clinical data [17].
`[0006] The first step to a clinical trial is the choice of the
`optimal AO target site for skipping of those dystrophin exons
`most commonly deleted in DMD. In depth analysis of arrays
`of 2'OMePS AOs have been reported [18, 19], and relation(cid:173)
`ships between skipping bioactivity and AO variables exam(cid:173)
`ined.
`[0007] One problem associated with the prior art is that the
`antisense oligonucleotides of the prior art do not produce
`efficient exon skipping. This means that a certain amount of
`mRNA produced in the splicing process will contain the
`out-of-frame mutation which leads to protein expression
`associated with DMD rather than expression of the truncated,
`yet functional, Becker-like dystrophin protein associated
`with mRNA in which certain exons have been skipped.
`[0008] Another problem associated with the prior art is that
`antisense oligonucleotides have not been developed to all of
`the exons in the dystrophin gene in which mutations occur in
`DMD.
`[0009] An aim of the present invention is to provide mol(cid:173)
`ecules which cause efficient exon skipping in selected exons
`of the dystrophin gene, thus being suitable for use in amelio(cid:173)
`rating the effects of DMD.
`
`SUMMARY OF THE INVENTION
`
`[0010] The present invention relates to molecules which
`can bind to pre-mRNA produced from the dystrophin gene
`and cause a high degree of exon skipping in a particular exon.
`These molecules can be administered therapeutically.
`[0011] The present invention provides a molecule for ame(cid:173)
`liorating DMD, the molecule comprising at least a 25 base
`length from a base sequence selected from:
`
`(SEQ ID NO, 1)
`XGA AAA CGC CGC CAX XXC XCA ACA GAX CXG;
`
`(SEQ ID NO, 2)
`CAX AAX GAA AAC GCC GCC AXX XCX CAA CAG;
`
`(SEQ ID NO, 3)
`XGX XCA GCX XCX GXX AGC CAC XGA XXA AAX;
`
`(SEQ ID NO, 4)
`CAG XXX GCC GCX GCC CAA XGC CAX CCX GGA;
`
`(SEQ ID NO, 5)
`XXG CCG CXG CCC AAX GCC AXC CXG GAG XXC;
`
`(SEQ ID NO, 6)
`XGC XGC XCX XXX CCA GGX XCA AGX GGG AXA;
`
`(SEQ ID NO, 7)
`CXX XXA GXX GCX GCX CXX XXC CAG GXX CAA;
`
`(SEQ ID NO, 8)
`CXX XXC XXX XAG XXG CXG CXC XXX XCC AGG;
`
`(SEQ ID NO, 9)
`XXA GXX GCX GCX CXX XXC CAG GXX CAA GXG;
`
`(SEQ ID NO, 10)
`CXG XXG CCX CCG GXX CXG AAG GXG XXC XXG;
`
`(SEQ ID NO, 11)
`CAA CXG XXG CCX CCG GXX CXG AAG GXG XXC;
`
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`
`i)
`
`j)
`
`k)
`
`

`

`US 2010/0168212 Al
`
`Jul. 1, 2010
`
`2
`
`-continued
`
`[0015] The molecule that causes skipping in exon 53 com(cid:173)
`prises at least a 25 base length from a base sequence selected
`from:
`
`(SEQ ID NO, 10)
`CXG XXG CCX CCG GXX CXG AAG GXG XXC XXG;
`
`(SEQ ID NO, 11)
`CAA CXG XXG CCX CCG GXX CXG AAG GXG XXC;
`
`(SEQ ID NO, 12)
`XXG CCX CCG GXX CXG AAG GXG XXC XXG XAC,
`
`j)
`
`k)
`or
`
`1)
`
`wherein the molecule's sequence can vary from the above
`sequence at up to two base positions, and wherein the mol(cid:173)
`ecule can bind to a target site to cause exon skipping in exon
`53 of the dystrophin gene.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0016] FIG. 1 shows a scheme summarizing the tools used
`in the design of PMOs to exon 53. (a) Results ofESEfinder
`analysis, showing the location and values above threshold for
`SF2/ASF, SF2/ASF (BRCAl), SC35, SRp40 and SRp55,
`shown as grey and black bars, as indicated in the legend
`above. (b) Output of PESX analysis, showing the location of
`exonic splicing enhancers as solid lines, and exonic splicing
`silencer as a dashed line. ( c) Rescue ESE analysis for exon 53,
`showing predicted ES Es by lines, and where they overlap, by
`a ladder of lines. (d) AccessMapper analysis of in vitro
`hybridization. Synthetic pre-mRNA containing exon 53 and
`surrounding intrans was subjected to a hybridization screen
`against a random hexamer oligonucleotide array, as described
`in Materials and Methods. Areas of hybridization, suggestive
`of areas of open conformation, are indicated by peaks on the
`graph. ( e) The position of the target sites of two 2'OMePS
`AOs studied previously [18] are shown for comparison. (f)
`The location of the target sites for all the 25mer and 30mer
`PM Os to exon 53 used in this study are indicated by lines, and
`numbered according to the scheme used in Table 1, except for
`exclusion of the prefix "h53";
`[0017] FIG. 2 shows a comparison of active (effective) and
`inactive (ineffective) PMOs. RT-PCR analysis of mRNA
`from normal human skeletal muscle cells treated with PM Os
`to exon 53 demonstrates a wide variation in the efficiency of
`exon skipping. Over 75% exon skipping is seen with
`h53A30/2 (lane 5) and h53A30/3 (lane 6). h53A30/1 (lane 4)
`produced around 50% skipping, while the 25-mer h53Al
`(lane 3) produced just over 10% skipping. In contrast, h53Cl
`(lane 2) was completely inactive. Lane 1 contains a negative
`control in which cells were treated with lipofectin but no
`PMO.
`[0018] FIG. 3 shows an Mfold secondary structure predic(cid:173)
`tion for exon 53 of the human dystrophin gene. MFOLD
`analysis [25] was performed using exon 53 plus 50 nt of the
`upstream and downstream intrans, and with a maximum
`base-pairing distance of 100 nt. The intron and exon bound(cid:173)
`aries are indicated, as are the positions of the target sites of the
`bioactive PMO h53A30/2 (87 .2% skip) and an inactive PMO
`(h53B2). Examples ofopen and closed RNA secondary struc(cid:173)
`ture are arrowed.
`[0019] FIG. 4 shows boxplots of parameters significant to
`strong PMO bioactivity. Comparisons were made between
`inactive PM Os and those inducing skipping at levels in excess
`of7 5%. Boxplots are shown for parameters which are signifi-
`
`or
`
`1)
`
`(SEQ ID NO, 12)
`XXG CCX CCG GXX CXG AAG GXG XXC XXG XAC,
`
`wherein the molecule's base sequence can vary from the
`above sequence at up to two base positions, and wherein the
`molecule can bind to a target site to cause exon skipping in an
`exon of the dystrophin gene.
`[0012] The exon of the dystrophin gene is selected from
`exons 44, 45, 46 or 53. More specifically, the molecule that
`causes skipping in exon 44 comprises at least a 25 base length
`from a base sequence selected from:
`
`(SEQ ID NO, 1)
`XGA AAA CGC CGC CAX XXC XCA ACA GAX CXG;
`
`(SEQ ID NO, 2)
`CAX AAX GAA AAC GCC GCC AXX XCX CAA CAG;
`
`(SEQ ID NO, 3)
`XGX XCA GCX XCX GXX AGC CAC XGA XXA AAX,
`
`a)
`
`b)
`or
`
`c)
`
`wherein the molecule's sequence can vary from the above
`sequence at up to two base positions, and wherein the mol(cid:173)
`ecule can bind to a target site to cause exon skipping in exon
`44 of the dystrophin gene.
`[0013] The molecule that causes skipping in exon 45 com(cid:173)
`prises at least a 25 base length from a base sequence selected
`from:
`
`(SEQ ID NO, 4)
`CAG XXX GCC GCX GCC CAA XGC CAX CCX GGA;
`
`(SEQ ID NO, 5)
`XXG CCG CXG CCC AAX GCC AXC CXG GAG XXC,
`
`d)
`or
`
`e)
`
`wherein the molecule's sequence can vary from the above
`sequence at up to two base positions, and wherein the mol(cid:173)
`ecule can bind to a target site to cause exon skipping in exon
`45 of the dystrophin gene.
`[0014] The molecule that causes skipping in exon 46 com(cid:173)
`prises at least a 25 base length from a base sequence selected
`from:
`
`(SEQ ID NO, 6)
`XGC XGC XCX XXX CCA GGX XCA AGX GGG AXA;
`
`(SEQ ID NO, 7)
`CXX XXA GXX GCX GCX CXX XXC CAG GXX CAA;
`
`(SEQ ID NO, 8)
`CXX XXC XXX XAG XXG CXG CXC XXX XCC AGG;
`
`(SEQ ID NO, 9)
`XXA GXX GCX GCX CXX XXC CAG GXX CAA GXG,
`
`f)
`
`g)
`
`h)
`or
`
`i)
`
`wherein the molecule's sequence can vary from the above
`sequence at up to two base positions, and wherein the mol(cid:173)
`ecule can bind to a target site to cause exon skipping in exon
`46 of the dystrophin gene.
`
`

`

`US 2010/0168212 Al
`
`Jul. 1, 2010
`
`3
`
`cant on a Mann-Whitney rank sum test: PMO to target bind(cid:173)
`ing energy, distance of the target site from the splice acceptor
`site, the percentage overlap with areas of open conformation,
`as predicted by MF OLD software, and the percentage overlap
`of the target site with the strongest area accessible to binding,
`as revealed by hexamer hybridization array analysis. Degrees
`of significance are indicated by asterisks.*: p<0.05; **: p<0.
`01; ***: p<0.001.
`[0020] FIG. 5 shows boxplots of parameters significantly
`different between bioactive (effective) and inactive (ineffec(cid:173)
`tive) PMOs. Comparisons were made between PMOs deter(cid:173)
`mined as bioactive (those that induced skipping at greater
`than 5%) and those that were not. Boxplots are shown for
`parameters which are significant from a Mann-Whitney rank
`sum test: PMO to target binding energy, distance of the target
`site from the splice acceptor site, the score over threshold for
`a predicted binding site for the SR protein SF2/ ASF, and the
`percentage overlap of the target site with the strongest area
`accessible to binding, as revealed by hexamer hybridization
`array analysis. Degrees of significance are indicated by aster(cid:173)
`isks. *: p<0.05; **: p<0.01; ***: p<0.001.
`[0021] FIG. 6 shows a comparison ofbioactivity of PM Os
`targeted to exon 53 in normal hSkMCs. Myoblasts were
`transfected with each of the 25mer (panel a) and 30mer (panel
`b) PM Os indicated at 500 nM using lipofectin (1:4 ). RNA was
`harvested after 24 hours and subjected to nested RT-PCR and
`products visualised by agarose gel electrophoresis.
`[0022] FIG. 7 shows low dose efficacy and timecourse of
`skipping of the most bioactive PMOs in normal hSkMCs. (a)
`hSkMC myoblasts were transfected with the PM Os indicated
`over a concentration range of 25 nM to 100 nM using lipo(cid:173)
`fectin (1 :4). RNA was harvested after 24 hours and subjected
`to nested RT-PCR, and products visualised by agarose gel
`electrophoresis. (b) hSkMC myoblasts were transfected with
`100 nM and 500 nM concentrations of PMO-G ( +30+59)
`using lipofectin. RNA was harvested at the timepoints indi(cid:173)
`cated following transfection and subjected to nested RT-PCR,
`and products visualised by agarose gel electrophoresis.
`Skipped (248 bp) and unskipped ( 460 bp) products are shown
`schematically.
`[0023] FIG. 8 shows blind comparison of 13 PMO oligo(cid:173)
`nucleotide sequences to skip human exon 53. Myoblasts
`derived from a DMD patient carrying a deletion of dystrophin
`exons 45-52 were transfected at 300 nM in duplicate with
`each of the PM Os by nucleofection. RNA was harvested 3
`days following transfection, and amplified by nested RT(cid:173)
`PCR. (a) Bars indicate the percentage of exon skipping
`achieved for each PMO, derived from Image J analysis of the
`electropherogram of the agarose gel (b ). Skipped ( 4 77 bp)
`and unskipped ( 689 bp) products are shown schematically.
`[0024] FIG. 9 shows the dose-response of the six best(cid:173)
`performing PMOs. (a) Myoblasts derived from a DMD
`patient carrying a deletion of dystrophin exons 45-52 were
`transfected with the six best-performing PMOs by nucleofec(cid:173)
`tion, at doses ranging from 25 nM to 400 nM. RT-PCR prod(cid:173)
`ucts derived from RNA isolated from cells 3 days post-trans(cid:173)
`fection were separated by agarose gel electrophoresis. (b) The
`percentage of exon skipping observed is expressed for each
`concentration of each PMO as a comparison of the percentage
`OD of skipped and unskipped band, as measured using Image
`J.
`[0025] FIG.10 shows persistence of dystrophin expression
`in DMD cells following PMO treatment. (a) Myoblasts
`derived from a DMD patient carrying a deletion of dystrophin
`
`exons 45-52 were transfected by nucleofection at 300 nM
`with each of the six best-performing PMOs, and were cul(cid:173)
`tured for 1 to 10 days before extracting RNA. The percentage
`of exon skipping was compared using the percentage OD of
`skipped and unskipped bands, measured using Image J analy(cid:173)
`sis of the agarose gel of the nested RT-PCR products shown in
`(b ). The experiment was repeated, but this time using the two
`best-performing PMOs from the previous analysis, and con(cid:173)
`tinuing the cultures for 21 days post-transfection ( c and d). ( e)
`Western blot analysis was performed on total protein extracts
`from de! 45-52 DMD cells 7 days after transfection with the
`six best PMOs (300 nM). Blots were probed with antibodies
`to dystrophin, to dysferlin as a muscle-specific loading con(cid:173)
`trol, and protogold for total protein loading control. CHQ5B
`myoblasts, after 7 days of differentiation were used as a
`positive control for dystrophin protein (normal).
`[0026] FIG. 11 shows a comparison of most active PM Os in
`hDMD mice. PM Os were injected in a blind experiment into
`the gastrocnemius muscle ofhDMD mice. RT-PCR analysis
`of RNA harvested from isolated muscle (L=left, R =right) was
`performed and products visualised by agarose gel electro(cid:173)
`phoresis. Quantification of PCR products was performed
`using a DNA LabChip.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0027] Without being restricted to any particular theory, it
`is thought by the inventors that the binding of the molecules to
`the dystrophin pre-mRNA interacts with or interferes with the
`binding of SR proteins to the exon of interest. SR proteins are
`involved in the slicing process of adjacent exons. Therefore, it
`is thought that interacting or interfering with the binding of
`the SR proteins interferes with the splicing machinery result(cid:173)
`ing in exon skipping.
`[0028] The base "X" in the above base sequences is defined
`as being thymine (T) oruracil (U). The presence of either base
`in the sequence will still allow the molecule to bind to the
`pre-mRNA of the dystrophin gene as it is a complementary
`sequence. Therefore, the presence of either base in the mol(cid:173)
`ecule will cause exon skipping. The base sequence of the
`molecule may contain all thymines, all uracils or a combina(cid:173)
`tion of the two. One factor that can determine whether Xis T
`or U is the chemistry used to produce the molecule. For
`example, if the molecule is a phosphorodiamidate mor(cid:173)
`pholino oligonucleotide (PMO), X will be T as this base is
`used when producing PM Os. Alternatively, if the molecule is
`a phosphorothioate-linked 2'-O-methyl oligonucleotide
`(2'OMePS), X will be U as this base is used when producing
`2'OMePSs. Preferably, the base "X" is only thymine (T).
`[0029] The advantage provided by the molecule is that it
`causes a high level of exon skipping. Preferably, the molecule
`causes an exon skipping rate of at least 50%, more preferably,
`at least 60%, even more preferably, at least 70%, more pref(cid:173)
`erably still, at least 76%, more preferably, at least 80%, even
`more preferably, at least 85%, more preferably still, at least
`90%, and most preferably, at least 95%.
`[0030] The molecule can be any type of molecule as long as
`it has the selected base sequence and can bind to a target site
`of the dystrophin pre-mRNA to cause exon skipping. For
`example, the molecule can be an oligodeoxyribonucleotide,
`an oligoribonucleotide, a phosphorodiamidate morpholino
`oligonucleotide (PMO) or a phosphorothioate-linked 2'-O(cid:173)
`methyl oligonucleotide (2'OMePS). Preferably, the oligo(cid:173)
`nucleotide is a PMO. The advantage of a PMO is that it has
`excellent safety profiles and appears to have longer lasting
`
`

`

`US 2010/0168212 Al
`
`Jul. 1, 2010
`
`4
`
`effects in vivo compared to 2'OMePS oligonucleotides. Pref(cid:173)
`erably, the molecule is isolated so that it is free from other
`compounds or contaminants.
`[0031] The base sequence of the molecule can vary from
`the selected sequence at up to two base positions. If the base
`sequence does vary at two positions, the molecule will still be
`able to bind to the dystrophin pre-mRNA to cause exon skip(cid:173)
`ping. Preferably, the base sequence of the molecule varies
`from the selected sequence at one base position and, more
`preferably, the base sequence does not vary from the selected
`sequence. The less that the base sequence of the molecule
`varies from the selected sequence, the more efficiently it
`binds to the specific exon region in order to cause exon
`skipping.
`[0032] The molecule is at least 25 bases in length. Prefer(cid:173)
`ably, the molecule is at least 28 bases in length. Preferably, the
`molecule is no more than 35 bases in length and, more pref(cid:173)
`erably, no more than 32 bases in length. Preferably, the mol(cid:173)
`ecule is between 25 and 35 bases in length, more preferably,
`the molecule is between 28 and 32 bases in length, even more
`preferably, the molecule is between 29 and 31 bases in length,
`and most preferably, the molecule is 30 bases in length. It has
`been found that a molecule which is 30 bases in length causes
`efficient exon skipping. If the molecule is longer than 35
`bases in length, the specificity of the binding to the specific
`exon region is reduced. If the molecule is less than 25 bases in
`length, the exon skipping efficiency is reduced.
`[0033] The molecule may be conjugated to or complexed
`with various entities. For example, the molecule may be con(cid:173)
`jugated to or complexed with a targeting protein in order to
`target the molecule to muscle tissue. Alternatively, the mol(cid:173)
`ecule may be complexed with or conjugated to a drug or
`another compound for treating DMD. If the molecule is con(cid:173)
`jugated to an entity, it may be conjugated directly or via a
`linker. In one embodiment, a plurality of molecules directed
`to exon skipping in different exons may be conjugated to or
`complexed with a single entity. Alternatively, a plurality of
`molecules directed to exon skipping in the same exon may be
`conjugated to or complexed with a single entity. For example,
`an arginine-rich cell penetrating peptide (CPP) can be conju(cid:173)
`gated to or complexed with the molecule. In particular,
`(R-Ahx-R)( 4)AhxB can be used, where Ahx is 6-aminohex(cid:173)
`anoic acid and B is beta-alanine [35], or alternatively
`(RXRRBR)2XB can be used [36]. These entities have been
`complexed to known dystrophin exon-skipping molecules
`which have shown sustained skipping of dystrophin exons in
`vitro and in vivo.
`In another aspect, the present invention provides a
`[0034]
`vector for ameliorating DMD, the vector encoding a molecule
`of the invention, wherein expression of the vector in a human
`cell causes the molecule to be expressed. For example, it is
`possible to express anti sense sequences in the form of a gene,
`which can thus be delivered on a vector. One way to do this
`would be to modify the sequence of a U7 snRNA gene to
`include an antisense sequence according to the invention. The
`U7 gene, complete with its own promoter sequences, can be
`delivered on an adeno-associated virus (AAV) vector, to
`induce bodywide exon skipping. Similar methods to achieve
`exon skipping, by using a vector encoding a molecule of the
`invention, would be apparent to one skilled in the art.
`[0035] The present invention also provides a pharmaceuti(cid:173)
`cal composition for ameliorating DMD, the composition
`comprising a molecule as described above or a vector as
`described above and any pharmaceutically acceptable carrier,
`
`adjuvant or vehicle. Pharmaceutical compos1t10ns of this
`invention comprise any molecule of the present invention,
`and pharmaceutically acceptable salts, esters, salts of such
`esters, or any other compound which, upon administration to
`a human, is capable of providing ( directly or indirectly) the
`biologically active molecule thereof, with any pharmaceuti(cid:173)
`cally acceptable carrier, adjuvant or vehicle. Pharmaceuti(cid:173)
`cally acceptable carriers, adjuvants and vehicles that may be
`used in the pharmaceutical compositions of this invention
`include, but are not limited to, ion exchangers, alumina, alu(cid:173)
`minum stearate, lecithin, serum proteins, such as human
`serum albumin, buffer substances such as phosphates, gly(cid:173)
`cine, sorbic acid, potassium sorbate, partial glyceride mix(cid:173)
`tures of saturated vegetable fatty acids, water, salts or elec(cid:173)
`trolytes, such as protamine sulfate, disodium hydrogen
`phosphate, potassium hydrogen phosphate, sodium chloride,
`zinc salts, colloidal silica, magnesium trisilicate, polyvinyl
`pyrrolidone, cellulose-based substances, polyethylene gly(cid:173)
`col, sodium carboxymethylcellulose, polyacrylates, waxes,
`polyethylene-polyoxypropylene-block polymers, polyethyl(cid:173)
`ene glycol and wool fat.
`[0036] The pharmaceutical compositions of this invention
`may be administered orally, parenterally, by inhalation spray,
`topically, rectally, nasally, buccally, vaginally, intradermally
`or via an implanted reservoir. Oral administration or admin(cid:173)
`istration by injection is preferred. The pharmaceutical com(cid:173)
`positions of this invention may contain any conventional non(cid:173)
`toxic pharmaceutically-acceptable carriers, adjuvants or
`vehicles. The term parenteral as used herein includes subcu(cid:173)
`taneous, intracutaneous, intravenous, intramuscular, intra-ar(cid:173)
`ticular, intrasynovial, intrasternal, intrathecal, intralesional
`and intracranial injection or infusion techniques. Preferably,
`the route of administration is by injection, more preferably,
`the route of administration is intramuscular, intravenous or
`subcutaneous injection and most preferably, the route of
`administration is intravenous or subcutaneous injection.
`[0037] The pharmaceutical compositions may be in the
`form of a sterile injectable preparation, for example, as a
`sterile injectable aqueous or oleaginous suspension. This sus(cid:173)
`pension may be formul