# Patent

## Title
Antisense oligonucleotides for inducing exon skipping and methods of use thereof

## 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: 1 to 202.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 11/570,691, filed Jan. 15, 2008 (now U.S. Pat. No. 7,807,816), which is a 35 U.S.C. §371 National Phase Application of PCT/AU2005/000943, filed Jun. 28, 2005, which claims priority to Australian Patent Application No. 2004903474, filed Jun. 28, 2004; these applications are incorporated herein by reference in their entireties.
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is SequenceListing.txt. The text file is 61 KB, was created on Feb. 4, 2013, and is being submitted electronically via EFS-Web.


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.
BACKGROUND ART
Significant effort is currently being expended 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.
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 oligonucleotides are directed either against RNA (sense strand) or against DNA where they form triplex structures inhibiting transcription by RNA polymerase II. To achieve a desired effect in specific gene down-regulation, the oligonucleotides must either promote the decay of the targeted mRNA or block translation of that mRNA, thereby effectively preventing de novo 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 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) Proc Natl Acad Sci USA 93, 12840-12844; Wilton S D, et al., (1999) Neuromusc Disorders 9, 330-338; van Deutekom J C 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.
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 introns 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 machinery 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 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.
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 T G, et al., (1993) Am J Hum Genet 53, 1007-1015). However, it is recognised that if exons associated with disease-causing 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 Q L, et al., (2003) Nature Medicine 9, 1009-1014; Aartsma-Rus A et al., (2004) Am J Hum Genet 74: 83-92).
This process of targeted exon skipping is likely to be particularly useful in long genes where there are many exons 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 sequence or the huge nebulin or titin genes which are comprised of  ̃80 and over 370 exons, respectively).
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 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).
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 introns 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 the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mRNA (Takeshima 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.
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.
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. This mutated dystrophin was expressed in 1-2% of antisense treated mdx myotubes. Use of other oligonucleotide modifications such as 2′-O-methoxyethyl phosphodiesters are described (Dunckley et al. (1998) Human Mol. Genetics, 5, 1083-90).
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 molecules 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”.
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 missing exon 23, Dunckley et al., (1998) did not show any time course of induced 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 oligonucleotide. Furthermore, this work could not be replicated by other researchers.
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 of 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.
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 much less efficient in immortalized cell cultures expressing higher levels 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 C J et al., (2002) J Gen Med 4, 644-654).
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.
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.
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 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 FIG. 1 for schematic representation of motifs and domains involved in exon recognition, intron removal and the splicing process).
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 3 to 8, 10 to 16, 19 to 40, 42 to 44, 46, 47, and 50 to 53 in the Dystrophin gene transcript the antisense molecules are preferably selected from the group listed in Table 1A.
In a further example, it is possible to combine two or more antisense oligonucleotides of the present invention together to induce multiple exon skipping in exons 19-20, and 53. This is a similar concept to targeting of a single exon. A combination or “cocktail” of antisense oligonucleotides are directed at adjacent exons to induce efficient exon skipping.
In another example, to induce exon skipping in exons 19-20, 31, 34 and 53 it is possible to improve exon skipping of a single exon by joining together two or more antisense oligonucleotide molecules. This concept is termed by the inventor as a “weasel”, an example of a cunningly designed antisense oligonucleotide. A similar concept has been described in Aartsma-Rus A et al., (2004) Am J Hum Genet. 74:83-92).
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.
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.
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.
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 particular 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.
The invention also provides kits for treating a genetic disease, which kits comprise at least an 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.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Schematic representation of motifs and domains involved in exon recognition, intron removal and the splicing process (SEQ ID NO: 213 and SEQ ID NO: 214).
FIG. 2 Diagrammatic representation of the concept of antisense 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.
FIG. 3 Gel electrophoresis showing differing efficiencies of two antisense molecules directed at exon 8 acceptor splice site. The preferred compound [H8A(−06+18)] induces strong and consistent exon skipping at a transfection concentration of 20 nanomolar in cultured normal human muscle cells. The less preferred antisense oligonucleotide [H8A(−06+14)] also induces efficient exon skipping, but at much higher concentrations. Other antisense oligonucleotides directed at exon 8 either only induced lower levels of exon skipping or no detectable skipping at all (not shown).
FIG. 4 Gel electrophoresis showing differing efficiencies of two antisense molecules directed at internal domains within exon 7, presumably exon splicing enhancers. The preferred compound [H7A(+45+67)] induces strong and consistent exon skipping at a transfection concentration of 20 nanomolar in cultured human muscle cells. The less preferred antisense oligonucleotide [H7A(+2+26)] induces only low levels of exon skipping at the higher transfection concentrations. Other antisense oligonucleotides directed at exon 7 either only induced lower levels of exon skipping or no detectable skipping at all (not shown).
FIG. 5 Gel electrophoresis showing an example of low efficiency exon 6 skipping using two non-preferred antisense molecules directed at human exon 6 donor splice site. Levels of induced exon 6 skipping are either very low [H6D(+04−21)] or almost undetectable [H6D(+18−04)]. These are examples of non-preferred antisense oligonucleotides to demonstrate that antisense oligonucleotide design plays a crucial role in the efficacy of these compounds.
FIG. 6 Gel electrophoresis showing strong and efficient human exon 6 skipping using an antisense molecule [H6A(+69+91)] directed at an exon 6 internal domain, presumably an exon splicing enhancer. This preferred compound induces consistent exon skipping at a transfection concentration of 20 nanomolar in cultured human muscle cells.
FIG. 7 Gel electrophoresis showing strong human exon 4 skipping using an antisense molecule H4A(+13+32) directed at an exon 6 internal domain, presumably an exon splicing enhancer. This preferred compound induces strong and consistent exon skipping at a transfection concentration of 20 nanomolar in cultured human muscle cells.
FIG. 8 Gel electrophoresis showing (8B) strong human exon 11 skipping using antisense molecule H11A(+75+97) directed at an exon 11 internal domain; and (8A) strong human exon 12 skipping using antisense molecule H12A(+52+75) directed at exon 12 internal domain.
FIG. 9 Gel electrophoresis showing (9A) strong human exon 15 skipping using antisense molecules H15A (+48+71) H15A(−12+19) directed at an exon 15 internal domain; and (9B) strong human exon 16 skipping using antisense molecules H16A(−12+19) H16A(−06+25).
FIG. 10 Gel electrophoresis showing human exon 19/20 skipping using antisense molecules H20A(+44+71) and H20A(+149+170) directed at an exon 20 and a “cocktail” of antisense oligonucleotides H19A(+35+65, H20A (+44+71) H20A (+149+170) directed at exons 19/20.
FIG. 11 Gel electrophoresis showing human exon 19/20 skipping using “weasels” directed at exons 19 and 20.
FIG. 12 Gel electrophoresis showing exon 22 skipping using antisense molecules H22A (+125+106), H22A(+47+69), H22A(+80+101) and H22D(+13−11) directed at exon 22.
FIG. 13 Gel electrophoresis showing exon 31 skipping using antisense molecules H31D(+01−25) and H31D(+03−22); and a “cocktail” of antisense molecules directed at exon 31.
FIG. 14 Gel electrophoresis showing exon 33 skipping using antisense molecules H33A(+30+56) H33A(+64+88) at exon 33.
FIG. 15 Gel electrophoresis showing exon 35 skipping using antisense molecules H35A(+141+161), H35A(+116+135), and H35A(+24+43) and a “cocktail of two antisense molecules, directed at exon 35.
FIG. 16 Gel electrophoresis showing exon 36 skipping using antisense molecules H32A(+49+73) H36A(+26+50) at exon 36.
FIG. 17 Gel electrophoresis showing exon 37 skipping using antisense molecules H37A(+82+105) and H37A(+134+157) directed at exon 37.
FIG. 18 Gel electrophoresis showing exon 38 skipping using antisense molecule H38A(+88+112) directed at exon 38.
FIG. 19 Gel electrophoresis showing exon 40 skipping using antisense molecule H40A(−05+17) directed at exon 40.
FIG. 20 Gel electrophoresis showing exon 42 skipping using antisense molecule H42A(−04+23) at exon 42.
FIG. 21 Gel electrophoresis showing exon 46 skipping using antisense molecule H46A(+86+115) directed at exon 46
FIG. 22 Gel electrophoresis showing exon 51, exon 52 and exon 53 skipping using various antisense molecules directed at exons 51, 52 and 53, respectively. A “cocktail” of antisense molecules is also shown directed at exon 53.



BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS







TABLE 1A







Description of 2′-O-methyl phosphorothioate


antisense oligonucleotides that have been used


to date to study induced exon skipping during


the processing of the dystrophin pre-mRNA.


Since these 2'-O-methyl antisense


oligonucleotides are more RNA-like, U represents


uracil. With other antisense chemistries


such as peptide nucleic acids or morpholinos,


these U bases may be shown as “T”.









SEQ




ID
SEQUENCE
NUCLEOTIDE SEQUENCE (5′-3′)





  1
H8A(−06+18)
GAU AGG UGG UAU CAA CAU CUG UAA


 


  2
H8A(−03+18)
GAU AGG UGG UAU CAA CAU CUG


 


  3
H8A(−07+18) 
GAU AGG UGG UAU CAA CAU CUG UAA




G


 


  4
H8A(−06+14)
GGU GGU AUC AAC AUC UGU AA


 


  5
H8A(−10+10)
GUA UCA ACA UCU GUA AGC AC


 


  6
H7A(+45+67)
UGC AUG UUC CAG UCG UUG UGU GG


 


  7
H7A(+02+26)
CAC UAU UCC AGU CAA AUA GGU CUG




G


 


  8
H7D(+15−10)
AUU UAC CAA CCU UCA GGA UCG AGU




A


 


  9
H7A(−18+03) 
GGC CUA AAA CAC AUA CAC AUA


 


 10
C6A(−10+10)
CAU UUU UGA CCU ACA UGU GG


 


 11
C6A(−14+06)
UUU GAC CUA CAU GUG GAA AG


 


 12
C6A(−14+12) 
UAC AUU UUU GAC CUA CAU GUG GAA




AG


 


 13
C6A(−13+09)
AUU UUU GAC CUA CAU GGG AAA G


 


 14
CH6A(+69+91)
UAC GAG UUG AUU GUC GGA CCC AG


 


 15
C6D(+12−13)
GUG GUC UCC UUA CCU AUG ACU GUG




G


 


 16
C6D(+06−11)
GGU CUC CUU ACC UAU GA


 


 17
H6D(+04−21)
UGU CUC AGU AAU CUU CUU ACC UAU


 


 18
H6D(+18−04)
UCU UAC CUA UGA CUA UGG AUG AGA


 


 19
H4A(+13+32)
GCA UGA ACU CUU GUG GAU CC


 


 20
H4D(+04−16)
CCA GGG UAC UAC UUA CAU UA


 


 21
H4D(−24−44)
AUC GUG UGU CAC AGC AUC CAG


 


 22
H4A(+11+40)
UGU UCA GGG CAU GAA CUC UUG UGG




AUC CUU


 


 23
H3A(+30+60)
UAG GAG GCG CCU CCC AUC CUG UAG




GUC ACU G


 


 24
H3A(+35+65)
AGG UCU AGG AGG CGC CUC CCA UCC




UGU AGG U


 


 25
H3A(+30+54)
GCG CCU CCC AUC CUG UAG GUC ACU




G


 


 26
H3D(+46−21)
CUU CGA GGA GGU CUA GGA GGC GCC




UC


 


 27
H3A(+30+50)
CUC CCA UCC UGU AGG UCA CUG


 


 28
H3D(+19−03)
UAC CAG UUU UUG CCC UGU CAG G


 


 29
H3A(−06+20)
UCA AUA UGC UGC UUC CCA AAC UGA




AA


 


 30
H3A(+37+61)
CUA GGA GGC GCC UCC CAU CCU GUA




G


 


 31
H5A(+20+50)
UUA UGA UUU CCA UCU ACG AUG UCA




GUA CUU C


 


 32
H5D(+25−05)
CUU ACC UGC CAG UGG AGG AUU AUA




UUC AAA A


 


 33
H5D(+10−15)
CAU CAG GAU UCU UAC CUG CCA GUG




G


 


 34
H5A(+10+34)
CGA UGU CAG UAC UUC CAA UAU UCA




C


 


 35
H5D(−04−21)
ACC AUU CAU CAG GAU UCU


 


 36
H5D(+16−02)
ACC UGC CAG UGG AGG AUU


 


 37
H5A(−07+20)
CCA AUA UUC ACU AAA UCA ACC UGU




UAA


 


 38
H5D(+18−12)
CAG GAU UCU UAC CUG CCA GUG GAG




GAU UAU


 


 39
H5A(+05+35)
ACG AUG UCA GUA CUU CCA AUA UUC




ACU AAA U


 


 40
H5A(+15+45)
AUU UCC AUC UAC GAU GUC AGU ACU




UCC AAU A


 


 41
H10A(−05+16)
CAG GAG CUU CCA AAU GCU GCA


 


 42
H10A(−05+24) 
CUU GUC UUC AGG AGC UUC CAA AUG




CUG CA


 


 43
H10A(+98+119) 
UCC UCA GCA GAA AGA AGC CAC G


 


 44
H10A(+130+149)
UUA GAA AUC UCU CCU UGU GC


 


 45
H10A(−33−14)
UAA AUU GGG UGU UAC ACA AU


 


 46
H11D(+26+49)
CCC UGA GGC AUU CCC AUC UUG AAU


 


 47
H11D(+11−09)
AGG ACU UAC UUG CUU UGU UU


 


 48
H11A(+118+140)
CUU GAA UUU AGG AGA UUC AUC UG


 


 49
H11A(+75+97)
CAU CUU CUG AUA AUU UUC CUG UU


 


 50
H12A(+52+75)
UCU UCU GUU UUU GUU AGC CAG UCA


 


 51
H12A(−10+10)
UCU AUG UAA ACU GAA AAU UU


 


 52
H12A(+11+30)
UUC UGG AGA UCC AUU AAA AC


 


 53
H13A(+77+100)
CAG CAG UUG CGU GAU CUC CAC UAG


 


 54
H13A(+55+75)
UUC AUC AAC UAC CAC CAC CAU


 


 55
H13D(+06−19)
CUA AGC AAA AUA AUC UGA CCU UAA




G


 


 56
H14A(+37+64)
CUU GUA AAA GAA CCC AGC GGU CUU




CUG U


 


 57
H14A(+14+35)
CAU CUA CAG AUG UUU GCC CAU C


 


 58
H14A(+51+73)
GAA GGA UGU CUU GUA AAA GAA CC


 


 59
H14D(−02+18)
ACC UGU UCU UCA GUA AGA CG


 


 60
H14D(+14−10)
CAU GAC ACA CCU GUU CUU CAG UAA


 


 61
H14A(+61+80)
CAU UUG AGA AGG AUG UCU UG


 


 62
H14A(−12+12)
AUC UCC CAA UAC CUG GAG AAG AGA


 


 63
H15A(−12+19)
GCC AUG CAC UAA AAA GGC ACU GCA




AGA CAU U


 


 64
H15A(+48+71)
UCU UUA AAG CCA GUU GUG UGA AUC


 


 65
H15A(+08+28)
UUU CUG AAA GCC AUG CAC UAA


 


 66
H15D(+17−08)
GUA CAU ACG GCC AGU UUU UGA AGA




C


 


 67
H16A(−12+19)
CUA GAU CCG CUU UUA AAA CCU GUU




AAA ACA A


 


 68
H16A(−06+25)
UCU UUU CUA GAU CCG CUU UUA AAA




CCU GUU A


 


 69
H16A(−06+19)
CUA GAU CCG CUU UUA AAA CCU GUU




A


 


 70
H16A(+87+109)
CCG UCU UCU GGG UCA CUG ACU UA


 


 71
H16A(−07+19)
CUA GAU CCG CUU UUA AAA CCU GUU




AA


 


 72
H16A(−07+13)
CCG CUU UUA AAA CCU GUU AA


 


 73
H16A(+12+37)
UGG AUU GCU UUU UCU UUU CUA GAU




CC


 


 74
H16A(+92+116)
CAU GCU UCC GUC UUC UGG GUC ACU




G


 


 75
H16A(+45+67)
G AUC UUG UUU GAG UGA AUA CAG U


 


 76
H16A(+105+126)
GUU AUC CAG CCA UGC UUC CGU C


 


 77
H16D(+05−20)
UGA UAA UUG GUA UCA CUA ACC UGU




G


 


 78
H16D(+12−11)
GUA UCA CUA ACC UGU GCU GUA C


 


 79
H19A(+35+53)
CUG CUG GCA UCU UGC AGU U


 


 80
H19A(+35+65)
GCC UGA GCU GAU CUG CUG GCA UCU




UGC AGU U


 


 81
H20A(+44+71)
CUG GCA GAA UUC GAU CCA CCG GCU




GUU C


 


 82
H20A(+147+168)
CAG CAG UAG UUG UCA UCU GCU C


 


 83
H20A(+185+203)
UGA UGG GGU GGU GGG UUG G


 


 84
H20A(−08+17)
AUC UGC AUU AAC ACC CUC UAG AAA




G


 


 85
H20A(+30+53)
CCG GCU GUU CAG UUG UUC UGA GGC


 


 86
H20A(−11+17)
AUC UGC AUU AAC ACC CUC UAG AAA




GAA A


 


 87
H20D(+08−20)
GAA GGA GAA GAG AUU CUU ACC UUA




CAA A


 


 88
H20A(+44+63)
AUU CGA UCC ACC GGC UGU UC


 


 89
H20A(+149+168)
CAG CAG UAG UUG UCA UCU GC


 


 90
H21A(−06+16)
GCC GGU UGA CUU CAU CCU GUG C


 


 91
H21A(+85+106)
CUG CAU CCA GGA ACA UGG GUC C


 


 92
H21A(+85+108)
GUC UGC AUC CAG GAA CAU GGG UC


 


 93
H21A(+08+31)
GUU GAA GAU CUG AUA GCC GGU UGA


 


 94
H21D(+18−07)
UAC UUA CUG UCU GUA GCU CUU UCU


 


 95
H22A(+22+45)
CAC UCA UGG UCU CCU GAU AGC GCA


 


 96
H22A(+125+146)
CUG CAA UUC CCC GAG UCU CUG C


 


 97
H22A(+47+69)
ACU GCU GGA CCC AUG UCC UGA UG


 


 98
H22A(+80+101)
CUA AGU UGA GGU AUG GAG AGU


 


 99
H22D(+13−11)
UAU UCA CAG ACC UGC AAU UCC CC


 


100
H23A(+34+59)
ACA GUG GUG CUG AGA UAG UAU AGG




CC


 


101
H23A(+18+39)
UAG GCC ACU UUG UUG CUC UUG C


 


102
H23A(+72+90)
UUC AGA GGG CGC UUU CUU C


 


103
H24A(+48+70)
GGG CAG GCC AUU CCU CCU UCA GA


 


104
H24A(−02+22)
UCU UCA GGG UUU GUA UGU GAU UCU


 


105
H25A(+9+36)
CUG GGC UGA AUU GUC UGA AUA UCA




CUG


 


106
H25A(+131+156)
CUG UUG GCA CAU GUG AUC CCA CUG




AG


 


107
H25D(+16−08)
GUC UAU ACC UGU UGG CAC AUG UGA


 


108
H26A(+132+156)
UGC UUU CUG UAA UUC AUC UGG AGU




U


 


109
H26A(−07+19)
CCU CCU UUC UGG CAU AGA CCU UCC




AC


 


110
H26A(+68+92)
UGU GUC AUC CAU UCG UGC AUC UCU




G


 


111
H27A(+82+106)
UUA AGG CCU CUU GUG CUA CAG GUG




G


 


112
H27A(−4+19)
GGG CCU CUU CUU UAG CUC UCU GA


 


113
H27D(+19−03)
GAC UUC CAA AGU CUU GCA UUU C


 


114
H28A(−05+19)
GCC AAC AUG CCC AAA CUU CCU AAG


 


115
H28A(+99+124)
CAG AGA UUU CCU CAG CUC CGC CAG




GA


 


116
H28D(+16−05)
CUU ACA UCU AGC ACC UCA GAG


 


117
H29A(+57+81)
UCC GCC AUC UGU UAG GGU CUG UGC




C


 


118
H29A(+18+42)
AUU UGG GUU AUC CUC UGA AUG UCG




C


 


119
H29D(+17−05)
CAU ACC UCU UCA UGU AGU UCC C


 


120
H30A(+122+147)
CAU UUG AGC UGC GUC CAC CUU GUC




UG


 


121
H30A(+25+50)
UCC UGG GCA GAC UGG AUG CUC UGU




UC


 


122
H30D(+19−04)
UUG CCU GGG CUU CCU GAG GCA UU


 


123
H31D(+06−18)
UUC UGA AAU AAC AUA UAC CUG UGC


 


124
H31D(+03−22)
UAG UUU CUG AAA UAA CAU AUA CCU




G


 


125
H31A(+05+25)
GAC UUG UCA AAU CAG AUU GGA


 


126
H31D(+04−20)
GUU UCU GAA AUA ACA UAU ACC UGU


 


127
H32D(+04−16)
CAC CAG AAA UAC AUA CCA CA


 


128
H32A(+151+170)
CAA UGA UUU AGC UGU GAC UG


 


129
H32A(+10+32)
CGA AAC UUC AUG GAG ACA UCU UG


 


130
H32A(+49+73)
CUU GUA GAC GCU GCU CAA AAU UGG




C


 


131
H33D(+09−11)
CAU GCA CAC ACC UUU GCU CC


 


132
H33A(+53+76)
UCU GUA CAA UCU GAC GUC CAG UCU


 


133
H33A(+30+56)
GUC UUU AUC ACC AUU UCC ACU UCA




GAC


 


134
H33A(+64+88)
CCG UCU GCU UUU UCU GUA CAA UCU




G


 


135
H34A(+83+104)
UCC AUA UCU GUA GCU GCC AGC C


 


136
H34A(+143+165)
CCA GGC AAC UUC AGA AUC CAA AU


 


137
H34A(−20+10)
UUU CUG UUA CCU GAA AAG AAU UAU




AAU GAA


 


138
H34A(+46+70)
CAU UCA UUU CCU UUC GCA UCU UAC




G


 


139
H34A(+95+120)
UGA UCU CUU UGU CAA UUC CAU AUC




UG


 


140
H34D(+10−20)
UUC AGU GAU AUA GGU UUU ACC UUU




CCC CAG


 


141
H34A(+72+96)
CUG UAG CUG CCA GCC AUU CUG UCA




AG


 


142
H35A(+141+161)
UCU UCU GCU CGG GAG GUG ACA


 


143
H35A(+116+135)
CCA GUU ACU AUU CAG AAG AC


 


144
H35A(+24+43)
UCU UCA GGU GCA CCU UCU GU


 


145
H36A(+26+50)
UGU GAU GUG GUC CAC AUU CUG GUC




A


 


146
H36A(−02+18)
CCA UGU GUU UCU GGU AUU CC


 


147
H37A(+26+50) 
CGU GUA GAG UCC ACC UUU GGG CGU




A


 


148
H37A(+82+105)
UAC UAA UUU CCU GCA GUG GUC ACC


 


149
H37A(+134+157)
UUC UGU GUG AAA UGG CUG CAA AUC


 


150
H38A(−01+19)
CCU UCA AAG GAA UGG AGG CC


 


151
H38A(+59+83)
UGC UGA AUU UCA GCC UCC AGU GGU




U


 


152
H38A(+88+112)
UGA AGU CUU CCU CUU UCA GAU UCA




C


 


153
H39A(+62+85)
CUG GCU UUC UCU CAU CUG UGA UUC


 


154
H39A(+39+58)
GUU GUA AGU UGU CUC CUC UU


 


155
H39A(+102+121)
UUG UCU GUA ACA GCU GCU GU


 


156
H39D(+10−10)
GCU CUA AUA CCU UGA GAG CA


 


157
H40A(−05+17)
CUU UGA GAC CUC AAA UCC UGU U


 


158
H40A(+129+153)
CUU UAU UUU CCU UUC AUC UCU GGG




C


 


159
H42A(−04+23)
AUC GUU UCU UCA CGG ACA GUG UGC




UGG


 


160
H42A(+86+109)
GGG CUU GUG AGA CAU GAG UGA UUU


 


161
H42D(+19−02)
A CCU UCA GAG GAC UCC UCU UGC


 


162
H43D(+10−15)
UAU GUG UUA CCU ACC CUU GUC GGU




C


 


163
H43A(+101+120)
GGA GAG AGC UUC CUG UAG CU


 


164
H43A(+78+100)
UCA CCC UUU CCA CAG GCG UUG CA


 


165
H44A(+85+104)
UUU GUG UCU UUC UGA GAA AC


 


166
H44D(+10−10)
AAA GAC UUA CCU UAA GAU AC


 


167
H44A(−06+14)
AUC UGU CAA AUC GCC UGC AG


 


168
H46D(+16−04)
UUA CCU UGA CUU GCU CAA GC


 


169
H46A(+90+109)
UCC AGG UUC AAG UGG GAU AC


 


170
H47A(+76+100)
GCU CUU CUG GGC UUA UGG GAG CAC




U


 


171
H47D(+25−02)
ACC UUU AUC CAC UGG AGA UUU GUC




UGC


 


172
H47A(−9+12)
UUC CAC CAG UAA CUG AAA CAG


 


173
H50A(+02+30)
CCA CUC AGA GCU CAG AUC UUC UAA




CUU CC


 


174
H50A(+07+33)
CUU CCA CUC AGA GCU CAG AUC UUC




UAA


 


175
H50D(+07−18)
GGG AUC CAG UAU ACU UAC AGG CUC




C


 


176
H51A(−01+25)
ACC AGA GUA ACA GUC UGA GUA GGA




GC


 


177
H51D(+16−07)
CUC AUA CCU UCU GCU UGA UGA UC


 


178
H51A(+111+134)
UUC UGU CCA AGC CCG GUU GAA AUC


 


179
H51A(+61+90)
ACA UCA AGG AAG AUG GCA UUU CUA




GUU UGG


 


180
H51A(+66+90)
ACA UCA AGG AAG AUG GCA UUU CUA




G


 


181
H51A(+66+95)
CUC CAA CAU CAA GGA AGA UGG CAU




UUC UAG


 


182
H51D(+08−17)
AUC AUU UUU UCU CAU ACC UUC UGC




U


 


183
H51A/D(+08−17)
AUC AUU UUU UCU CAU ACC UUC UGC



& (−15+30)
UAG GAG CUA AAA


 


184
H51A(+175+195)
CAC CCA CCA UCA CCC UCU GUG


 


185
H51A(+199+220)
AUC AUC UCG UUG AUA UCC UCA A


 


186
H52A(−07+14)
UCC UGC AUU GUU GCC UGU AAG


 


187
H52A(+12+41)
UCC AAC UGG GGA CGC CUC UGU UCC




AAA UCC


 


188
H52A(+17+37)
ACU GGG GAC GCC UCU GUU CCA


 


189
H52A(+93+112)
CCG UAA UGA UUG UUC UAG CC


 


190
H52D(+05−15)
UGU UAA AAA ACU UAC UUC GA


 


191
H53A(+45+69)
CAU UCA ACU GUU GCC UCC GGU UCU




G


 


192
H53A(+39+62)
CUG UUG CCU CCG GUU CUG AAG GUG


 


193
H53A(+39+69)
CAU UCA ACU GUU GCC UCC GGU UCU




GAA GGU G


 


194
H53D(+14−07)
UAC UAA CCU UGG UUU CUG UGA


 


195
H53A(+23+47)
CUG AAG GUG UUC UUG UAC UUC AUC




C


 


196
H53A(+150+176)
UGU AUA GGG ACC CUC CUU CCA UGA




CUC


 


197
H53D(+20−05)
CUA ACC UUG GUU UCU GUG AUU UUC




U


 


198
H53D(+09−18)
GGU AUC UUU GAU ACU AAC CUU GGU




UUC


 


199
H53A(−12+10)
AUU CUU UCA ACU AGA AUA AAA G


 


200
H53A(−07+18)
GAU UCU GAA UUC UUU CAA CUA GAA




U


 


201
H53A(+07+26)
AUC CCA CUG AUU CUG AAU UC


 


202
H53A(+124+145)
UUG GCU CUG GCC UGU CCU AAG A


 


203
H46A(+86+115)
CUC UUU UCC AGG UUC AAG UGG GAU




ACU AGC


 


204
H46A(+107+137)
CAA GCU UUU CUU UUA GUU GCU GCU




CUU UUC C


 


205
H46A(−10+20)
UAU UCU UUU GUU CUU CUA GCC UGG




AGA AAG


 


206
H46A(+50+77)
CUG CUU CCU CCA ACC AUA AAA CAA




AUU C


 


207
H45A(−06+20)
CCA AUG CCA UCC UGG AGU UCC UGU




AA


 


208
H45A(+91+110)
UCC UGU AGA AUA CUG GCA UC


 


209
H45A(+125+151)
UGC AGA CCU CCU GCC ACC GCA GAU




UCA


 


210
H45D(+16−04)
CUA CCU CUU UUU UCU GUC UG


 


211
H45A(+71+90)
UGU UUU UGA GGA UUG CUG AA
















TABLE 1B







Description of ‘a cocktail of 2′-O-methyl


phosphorothioate antisense oligonucleotides


that have been used to date to study induced


exon skipping during the processing of


the dystrophin pre-mRNA.









SEQ




ID
SEQUENCE
NUCLEOTIDE SEQUENCE (5′-3′)












81
H20A(+44+71)
CUG GCA GAA UUC GAU CCA CCG




GCU GUU C


82
H20A(+147+168)
CAG CAG UAG UUG UCA UCU GCU




C


 


80
H19A(+35+65)
GCC UGA GCU GAU CUG CUG GCA




UCU UGC


81
H20A(+44+71)
AGU U


82
H20A(+147+168)
CUG GCA GAA UUC GAU CCA CCG




GCU GUU C




CAG CAG UAG UUG UCA UCU GCU




C


 


194
H53D(+14−07)
UAC UAA CCU UGG UUU CUG UGA


195
H53A(+23+47)
CUG AAG GUG UUC UUG UAC UUC




AUC C


196
H53A(+150+176)
UGU AUA GGG ACC CUC CUU CCA




UGA CUC
















TABLE 1C







Description of a “weasel” of 2′-O-methyl


phosphorothioate antisense oligonucleotides


that have been used to date to study induced


exon skipping during the processing of the


dystrophin pre-mRNA.









SEQ




ID
SEQUENCE
NUCLEOTIDE SEQUENCE (5′-3′)





 81
H20A(+44+71)-
CUG GCA GAA UUC GAU CCA CCG




GCU GUU C-


 82
H20A(+147+168)
CAG CAG UAG UUG UCA UCU GCU




C


 


 80
H19A(+35+65)-
GCC UGA GCU GAU CUG CUG GCA


 88
H20A(+44+63)-
UCU UGC AGU U


 79
H20A(+149+168)
-AUU CGA UCC ACC GGC UGU




UC- CUG CUG GCA UCU UGC




AGU U


 


 80
H19A(+35+65)-
GCC UGA GCU GAU CUG CUG GCA


 88
H20A(+44+63)
UCU UGC AGU U




-AUU CGA UCC ACC GGC UGU




UC-


 


 80
H19A(+35+65)-
GCC UGA GCU GAU CUG CUG GCA


 79
H20A(+149+168)
UCU UGC AGU U




- CUG CUG GCA UCU UGC AGU




U


 


138
H34A(+46+70)-
CAU UCA UUU CCU UUC GCA UCU




UAC G-


139
H34A(+94+120)
UGA UCU CUU UGU CAA UUC CAU




AUC UG


 


124
H31D(+03−22)-
UAG UUU CUG AAA UAA CAU AUA



UU-
CCU G- UU-


144
H35A(+24+43)
UCA GGU GCA CCU UCU GU


 


195
H53A(+23+47)-
CUG AAG GUG UUC UUG UAC UUC



AA-
AUC C- UGU AUA GGG ACC CUC




CUU CCA UGA CUC-


196
H53A(+150+176)-
AA-



AA-
UAC UAA CCU UGG UUU CUG UGA


194
H53D(+14−07)



 


—
Aimed at exons
CAG CAG UAG UUG UCA UCU GCU


212
19/20/20
CAA CUG GCA GAA UUC GAU CCA




CCG GCU GUU CAA GCC UGA GCU




GAU CUG CUC GCA UCU UGC AGU









DETAILED DESCRIPTION OF THE INVENTION
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, compositions 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 present 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 included in this specification are collected at the end of the description and have been prepared using the programme PatentIn 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 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.).
An antisense molecules 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:

    
    
        H # A/D (x:y).
    
    


The first letter designates the species (e.g. H: human, M: murine, C: canine) “#” designates target dystrophin exon number.
“A/D” 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 an example, A(−6+18) would indicate the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. The closest splice site would be the acceptor so these coordinates would be preceded with an “A”. Describing annealing coordinates at the donor splice site could be D(+2−18) where the last 2 exonic bases and the first 18 intronic bases correspond to the annealing site of the antisense molecule. Entirely exonic annealing coordinates that would be represented by A(+65+85), that is the site between the 65th and 85th nucleotide from the start of that exon.
The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates.
As used necessarily herein the term “derived” and “derived from” shall be taken to indicate that a specific integer may be obtained from a particular source albeit not directly from that source.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.
DESCRIPTION OF THE PREFERRED EMBODIMENT
When antisense molecule (s) targeted to nucleotide sequences involved in splicing in exons within pre-mRNA sequences, normal splicing of the exon may be inhibited causing the splicing machinery to by-pass the entire mutated exon from the mature mRNA. The concept of antisense oligonucleotide induced exon skipping is shown in FIG. 2. In many genes, deletion of an entire exon would lead to the production of a non-functional protein through the loss of important functional domains or the disruption of the reading frame. In some proteins, however, it is possible to shorten the protein by deleting one or more exons, without disrupting the reading frame, from within the protein without seriously altering the biological activity of the protein. Typically, such proteins have a structural role and or possess functional domains at their ends. The present invention describes antisense molecules capable of binding to specified dystrophin pre-mRNA targets and re-directing processing of that gene.
Antisense Molecules
According to a first aspect of the invention, there is provided antisense molecules capable of binding to a selected target to induce exon skipping. To induce exon skipping in exons of the Dystrophin gene transcript, the antisense molecules are preferably selected from the group of compounds shown in Table 1A. There is also provided a combination or “cocktail” of two or more antisense oligonucleotides capable of binding to a selected target to induce exon skipping. To induce exon skipping in exons of the Dystrophin gene transcript, the antisense molecules in a “cocktail” are preferably selected from the group of compounds shown in Table 1B. Alternatively, exon skipping may be induced by antisense oligonucleotides joined together “weasels” preferably selected from the group of compounds shown in Table 1C.
Designing antisense molecules to completely mask consensus splice sites may not necessarily generate any skipping of the targeted exon. Furthermore, the inventors have discovered that size or length of the antisense oligonucleotide itself is not always a primary factor when designing antisense molecules. With some targets such as exon 19, antisense oligonucleotides as short as 12 bases were able to induce exon skipping, albeit not as efficiently as longer (20-31 bases) oligonucleotides. In some other targets, such as murine dystrophin exon 23, antisense oligonucleotides only 17 residues long were able to induce more efficient skipping than another overlapping compound of 25 nucleotides.
The inventors have also discovered that there does not appear to be any standard motif that can be blocked or masked by antisense molecules to redirect splicing. In some exons, such as mouse dystrophin exon 23, the donor splice site was the most amenable to target to re-direct skipping of that exon. It should be noted that designing and testing a series of exon 23 specific antisense molecules to anneal to overlapping regions of the donor splice site showed considerable variation in the efficacy of induced exon skipping. As reported in Mann et al., (2002) there was a significant variation in the efficiency of bypassing the nonsense mutation depending upon antisense oligonucleotide annealing (“Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy”. J Gen Med 4:644-654). Targeting the acceptor site of exon 23 or several internal domains was not found to induce any consistent exon 23 skipping.
In other exons targeted for removal, masking the donor splice site did not induce any exon skipping. However, by directing antisense molecules to the acceptor splice site (human exon 8 as discussed below), strong and sustained exon skipping was induced. It should be noted that removal of human exon 8 was tightly linked with the co-removal of exon 9. There is no strong sequence homology between the exon 8 antisense oligonucleotides and corresponding regions of exon 9 so it does not appear to be a matter of cross reaction. Rather the splicing of these two exons is inextricably linked. This is not an isolated instance as the same effect is observed in canine cells where targeting exon 8 for removal also resulted in the skipping of exon 9. Targeting exon 23 for removal in the mouse dystrophin pre-mRNA also results in the frequent removal of exon 22 as well. This effect occurs in a dose dependent manner and also indicates close coordinated processing of 2 adjacent exons.
In other targeted exons, antisense molecules directed at the donor or acceptor splice sites did not induce exon skipping while annealing antisense molecules to intra-exonic regions (i.e. exon splicing enhancers within human dystrophin exon 6) was most efficient at inducing exon skipping. Some exons, both mouse and human exon 19 for example, are readily skipped by targeting antisense molecules to a variety of motifs. That is, targeted exon skipping is induced after using antisense oligonucleotides to mask donor and acceptor splice sites or exon splicing enhancers.
To identify and select antisense oligonucleotides suitable for use in the modulation of exon skipping, a nucleic acid sequence whose function is to be modulated must first be identified. This may be, for example, a gene (or mRNA transcribed form the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. Within the context of the present invention, preferred target site(s) are those involved in mRNA splicing (i.e. splice donor sites, splice acceptor sites, or exonic splicing enhancer elements). Splicing branch points and exon recognition sequences or splice enhancers are also potential target sites for modulation of mRNA splicing.
Preferably, the present invention aims to provide antisense molecules capable of binding to a selected target in the dystrophin pre-mRNA to induce efficient and consistent exon skipping. Duchenne muscular dystrophy arises from mutations that preclude the synthesis of a functional dystrophin gene product. These Duchenne muscular dystrophy gene defects are typically nonsense mutations or genomic rearrangements such as deletions, duplications or micro-deletions or insertions that disrupt the reading frame. As the human dystrophin gene is a large and complex gene with the 79 exons being spliced together to generate a mature mRNA with an open reading frame of approximately 11,000 bases, there are many positions where these mutations can occur. Consequently, a comprehensive antisense oligonucleotide based therapy to address many of the different disease-causing mutations in the dystrophin gene will require that many exons can be targeted for removal during the splicing process.
Within the context of the present invention, preferred target site(s) are those involved in mRNA splicing (i.e. splice donor sites, splice acceptor sites or exonic splicing enhancer elements). Splicing branch points and exon recognition sequences or splice enhancers are also potential target sites for modulation of mRNA splicing.
The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridisable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense molecule need not be 100% complementary to that of its target sequence to be specifically hybridisable. An antisense molecule is specifically hybridisable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
While the above method may be used to select antisense molecules capable of deleting any exon from within a protein that is capable of being shortened without affecting its biological function, the exon deletion should not lead to a reading frame shift in the shortened transcribed mRNA. Thus, if in a linear sequence of three exons the end of the first exon encodes two of three nucleotides in a codon and the next exon is deleted then the third exon in the linear sequence must start with a single nucleotide that is capable of completing the nucleotide triplet for a codon. If the third exon does not commence with a single nucleotide there will be a reading frame shift that would lead to the generation of truncated or a non-functional protein.
It will be appreciated that the codon arrangements at the end of exons in structural proteins may not always break at the end of a codon, consequently there may be a need to delete more than one exon from the pre-mRNA to ensure in-frame reading of the mRNA. In such circumstances, a plurality of antisense oligonucleotides may need to be selected by the method of the invention wherein each is directed to a different region responsible for inducing splicing in the exons that are to be deleted.
The length of an antisense molecule may vary so long as it is capable of binding selectively to the intended location within the pre-mRNA molecule. The length of such sequences can be determined in accordance with selection procedures described herein. Generally, the antisense molecule will be from about 10 nucleotides in length up to about 50 nucleotides in length. It will be appreciated however that any length of nucleotides within this range may be used in the method. Preferably, the length of the antisense molecule is between 17 to 30 nucleotides in length.
In order to determine which exons can be connected in a dystrophin gene, reference should be made to an exon boundary map. Connection of one exon with another is based on the exons possessing the same number at the 3′ border as is present at the 5′ border of the exon to which it is being connected. Therefore, if exon 7 were deleted, exon 6 must connect to either exons 12 or 18 to maintain the reading frame. Thus, antisense oligonucleotides would need to be selected which redirected splicing for exons 7 to 11 in the first instance or exons 7 to 17 in the second instance. Another and somewhat simpler approach to restore the reading frame around an exon 7 deletion would be to remove the two flanking exons. Induction of exons 6 and 8 skipping should result in an in-frame transcript with the splicing of exons 5 to 9. In practise however, targeting exon 8 for removal from the pre-mRNA results in the co-removal of exon 9 so the resultant transcript would have exon 5 joined to exon 10. The inclusion or exclusion of exon 9 does not alter the reading frame. Once the antisense molecules to be tested have been identified, they are prepared according to standard techniques known in the art. The most common method for producing antisense molecules is the methylation of the 2′ hydroxyribose position and the incorporation of a phosphorothioate backbone produces molecules that superficially resemble RNA but that are much more resistant to nuclease degradation.
To avoid degradation of pre-mRNA during duplex formation with the antisense molecules, the antisense molecules used in the method may be adapted to minimise or prevent cleavage by endogenous RNase H. This property is highly preferred as the treatment of the RNA with the unmethylated oligonucleotides either intracellularly or in crude extracts that contain RNase H leads to degradation of the pre-mRNA:antisense oligonucleotide duplexes. Any form of modified antisense molecules that is capable of by-passing or not inducing such degradation may be used in the present method. An example of antisense molecules which when duplexed with RNA are not cleaved by cellular RNase H is 2′-O-methyl derivatives. 2′-O-methyl-oligoribonucleotides are very stable in a cellular environment and in animal tissues, and their duplexes with RNA have higher Tm values than their ribo- or deoxyribo-counterparts.
Antisense molecules that do not activate RNase H can be made in accordance with known techniques (see, e.g., U.S. Pat. No. 5,149,797). Such antisense molecules, which may be deoxyribonucleotide or ribonucleotide sequences, simply contain any structural modification which sterically hinders or prevents binding of RNase H to a duplex molecule containing the oligonucleotide as one member thereof, which structural modification does not substantially hinder or disrupt duplex formation. Because the portions of the oligonucleotide involved in duplex formation are substantially different from those portions involved in RNase H binding thereto, numerous antisense molecules that do not activate RNase H are available. For example, such antisense molecules may be oligonucleotides wherein at least one, or all, of the inter-nucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. For example, every other one of the internucleotide bridging phosphate residues may be modified as described. In another non-limiting example, such antisense molecules are molecules wherein at least one, or all, of the nucleotides contain a 2′ lower alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other one of the nucleotides may be modified as described.
While antisense oligonucleotides are a preferred form of the antisense molecules, the present invention comprehends other oligomeric antisense molecules, including but not limited to oligonucleotide mimetics such as are described below.
Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural inter-nucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their inter-nucleoside backbone can also be considered to be oligonucleosides.
In other preferred oligonucleotide mimetics, both the sugar and the inter-nucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleo-bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
Modified oligonucleotides may also contain one or more substituted sugar moieties. Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. Certain nucleo-bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds that are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense molecules, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the increased resistance to nuclease degradation, increased cellular uptake, and an additional region for increased binding affinity for the target nucleic acid.
Methods of Manufacturing Antisense Molecules
The antisense molecules used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). One method for synthesising oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066.
Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. In one such automated embodiment, diethyl-phosphoramidites are used as starting materials and may be synthesized as described by Beaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.
The antisense molecules of the invention are synthesised in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules. The molecules of the invention may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
Therapeutic Agents
The present invention also can be used as a prophylactic or therapeutic, which may be utilised for the purpose of treatment of a genetic disease.
Accordingly, in one embodiment the present invention provides antisense molecules that bind to a selected target in the dystrophin pre-mRNA to induce efficient and consistent exon skipping described herein in a therapeutically effective amount admixed with a pharmaceutically acceptable carrier, diluent, or excipient.
The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similarly untoward reaction, such as gastric upset and the like, when administered to a patient. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Martin, Remington's. Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa., (1990).
In a more specific form of the invention there are provided pharmaceutical compositions comprising therapeutically effective amounts of an antisense molecule together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength and additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). The material may be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 that are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilised form.
It will be appreciated that pharmaceutical compositions provided according to the present invention may be administered by any means known in the art. Preferably, the pharmaceutical compositions for administration are administered by injection, orally, or by the pulmonary, or nasal route. The antisense molecules are more preferably delivered by intravenous, intra-arterial, intraperitoneal, intramuscular, or subcutaneous routes of administration.
Antisense Molecule Based Therapy
Also addressed by the present invention is the use of antisense molecules of the present invention, for manufacture of a medicament for modulation of a genetic disease.
The delivery of a therapeutically useful amount of antisense molecules may be achieved by methods previously published. For example, intracellular delivery of the antisense molecule may be via a composition comprising an admixture of the antisense molecule and an effective amount of a block copolymer. An example of this method is described in US patent application US 20040248833.
Other methods of delivery of antisense molecules to the nucleus are described in Mann C J et al., (2001) [“Antisense-induced exon skipping and the synthesis of dystrophin in the mdx mouse”. Proc., Natl. Acad. Science, 98(1) 42-47] and in Gebski et al., (2003). Human Molecular Genetics, 12(15): 1801-1811.
A method for introducing a nucleic acid molecule into a cell by way of an expression vector either as naked DNA or complexed to lipid carriers, is described in U.S. Pat. No. 6,806,084.
It may be desirable to deliver the antisense molecule in a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposome formulations.
Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. These formulations may have net cationic, anionic or neutral charge characteristics and are useful characteristics with in vitro, in vivo and ex vivo delivery methods. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 φm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, and DNA can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981).
In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the antisense molecule of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).
The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
Alternatively, the antisense construct may be combined with other pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration.
The routes of administration described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and any dosage for any particular animal and condition. Multiple approaches for introducing functional new genetic material into cells, both in vitro and in vivo have been attempted (Friedmann (1989) Science, 244:1275-1280). Expression vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV), herpesvirus, bovine papilloma virus, and others offer features that may make them attractive for some applications of long-term genetic modification of mammalian cells. Human AAV viruses have a number of potential advantages over retrovirus vectors, including the fact that they are ubiquitous in humans and can be concentrated to titers exceeding 109 infectious units per milliliter. These approaches include integration of the gene to be expressed into modified retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl.: 5074S-5079S); integration into non-retrovirus vectors (Rosenfeld, et al. (1992) Cell, 68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434); or delivery of a transgene linked to a heterologous promoter-enhancer element via liposomes (Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281; Nabel, et al. (1990) Science, 249:1285-1288; Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855); coupled to ligand-specific, cation-based transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624) or the use of naked DNA, expression vectors (Nabel et al. (1990), supra); Wolff et al. (1990) Science, 247:1465-1468). Direct injection of transgenes into tissue produces only localized expression (Rosenfeld (1992) supra); Rosenfeld et al. (1991) supra; Brigham et el. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra). The Brigham et al. group (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) have reported in vivo transfection only of lungs of mice following either intravenous or intratracheal administration of a DNA liposome complex. An example of a review article of human gene therapy procedures is: Anderson, Science (1992) 256:808-813.
The antisense molecules of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such pro-drugs, and other bioequivalents.
The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, (including by nebulizer, intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Kits of the Invention
The invention also provides kits for treatment of a patient with a genetic disease which kit comprises at least an antisense molecule, packaged in a suitable container, together with instructions for its use.
In a preferred embodiment, the kits will contain at least one antisense molecule as shown in Table 1A, or a cocktail of antisense molecules as shown in Table 1B or a “weasel” compound as shown in Table 1C. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.
Those of ordinary skill in the field should appreciate that applications of the above method has wide application for identifying antisense molecules suitable for use in the treatment of many other diseases.
EXAMPLES
The following Examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these Examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. The references cited herein are expressly incorporated by reference.
Methods of molecular cloning, immunology and protein chemistry, which are not explicitly described in the following examples, are reported in the literature and are known by those skilled in the art. General texts that described conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, included, for example: Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Glover ed., DNA Cloning: A Practical Approach, Volumes I and II, MRL Press, Ltd., Oxford, U.K. (1985); and Ausubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. Current Protocols in Molecular Biology. Greene Publishing Associates/Wiley Intersciences, New York (2002).
Determining Induced Exon Skipping in Human Muscle Cells
Attempts by the inventors to develop a rational approach in antisense molecules design were not completely successful as there did not appear to be a consistent trend that could be applied to all exons. As such, the identification of the most effective and therefore most therapeutic antisense molecules compounds has been the result of empirical studies.
These empirical studies involved the use of computer programs to identify motifs potentially involved in the splicing process. Other computer programs were also used to identify regions of the pre-mRNA which may not have had extensive secondary structure and therefore potential sites for annealing of antisense molecules. Neither of these approaches proved completely reliable in designing antisense oligonucleotides for reliable and efficient induction of exon skipping.
Annealing sites on the human dystrophin pre-mRNA were selected for examination, initially based upon known or predicted motifs or regions involved in splicing. 2OMe antisense oligonucleotides were designed to be complementary to the target sequences under investigation and were synthesised on an Expedite 8909 Nucleic Acid Synthesiser. Upon completion of synthesis, the oligonucleotides were cleaved from the support column and de-protected in ammonium hydroxide before being desalted. The quality of the oligonucleotide synthesis was monitored by the intensity of the trityl signals upon each deprotection step during the synthesis as detected in the synthesis log. The concentration of the antisense oligonucleotide was estimated by measuring the absorbance of a diluted aliquot at 260 nm.
Specified amounts of the antisense molecules were then tested for their ability to induce exon skipping in an in vitro assay, as described below.
Briefly, normal primary myoblast cultures were prepared from human muscle biopsies obtained after informed consent. The cells were propagated and allowed to differentiate into myotubes using standard culturing techniques. The cells were then transfected with the antisense oligonucleotides by delivery of the oligonucleotides to the cells as cationic lipoplexes, mixtures of antisense molecules or cationic liposome preparations.
The cells were then allowed to grow for another 24 hours, after which total RNA was extracted and molecular analysis commenced. Reverse transcriptase amplification (RT-PCR) was undertaken to study the targeted regions of the dystrophin pre-mRNA or induced exonic re-arrangements.
For example, in the testing of an antisense molecule for inducing exon 19 skipping the RT-PCR test scanned several exons to detect involvement of any adjacent exons. For example, when inducing skipping of exon 19, RT-PCR was carried out with primers that amplified across exons 17 and 21. Amplifications of even larger products in this area (i.e. exons 13-26) were also carried out to ensure that there was minimal amplification bias for the shorter induced skipped transcript. Shorter or exon skipped products tend to be amplified more efficiently and may bias the estimated of the normal and induced transcript.
The sizes of the amplification reaction products were estimated on an agarose gel and compared against appropriate size standards. The final confirmation of identity of these products was carried out by direct DNA sequencing to establish that the correct or expected exon junctions have been maintained.
Once efficient exon skipping had been induced with one antisense molecule, subsequent overlapping antisense molecules may be synthesized and then evaluated in the assay as described above. Our definition of an efficient antisense molecule is one that induces strong and sustained exon skipping at transfection concentrations in the order of 300 nM or less.
Antisense Oligonucleotides Directed at Exon 8
Antisense oligonucleotides directed at exon 8 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 3 shows differing efficiencies of two antisense molecules directed at exon 8 acceptor splice site. H8A(−06+18) [SEQ ID NO:1], which anneals to the last 6 bases of intron 7 and the first 18 bases of exon 8, induces substantial exon 8 and 9 skipping when delivered into cells at a concentration of 20 nM. The shorter antisense molecule, H8A(−06+14) [SEQ ID NO: 4] was only able to induce exon 8 and 9 skipping at 300 nM, a concentration some 15 fold higher than H8A(−06+18), which is the preferred antisense molecule.
This data shows that some particular antisense molecules induce efficient exon skipping while another antisense molecule, which targets a near-by or overlapping region, can be much less efficient. Titration studies show one compound is able to induce targeted exon skipping at 20 nM while the less efficient antisense molecules only induced exon skipping at concentrations of 300 nM and above. Therefore, we have shown that targeting of the antisense molecules to motifs involved in the splicing process plays a crucial role in the overall efficacy of that compound.
Efficacy refers to the ability to induce consistent skipping of a target exon. However, sometimes skipping of the target exons is consistently associated with a flanking exon. That is, we have found that the splicing of some exons is tightly linked. For example, in targeting exon 23 in the mouse model of muscular dystrophy with antisense molecules directed at the donor site of that exon, dystrophin transcripts missing exons 22 and 23 are frequently detected. As another example, when using an antisense molecule directed to exon 8 of the human dystrophin gene, all induced transcripts are missing both exons 8 and 9. Dystrophin transcripts missing only exon 8 are not observed.
Table 2 below discloses antisense molecule sequences that induce exon 8 (and 9) skipping.







TABLE 2







(SEQ ID NOS 1-5, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H8A(−06+18)
5′-GAU AGG UGG UAU
Very



CAA CAU CUG UAA
strong to




20 nM


 


H8A(−03+18)
5′-GAU AGG UGG UAU
Very



CAA CAU CUG
strong




skipping




to 40 nM


 


H8A(−07+18)
5′-GAU AGG UGG UAU
Strong



CAA CAU CUG UAA G
skipping




to 40 nM


 


H8A(−06+14)
5′-GGU GGU AUC AAC
Skipping



AUC UGU AA
to 300 nM


 


H8A(−10+10)
5′-GUA UCA ACA UCU
Patchy/



GUA AGC AC
weak




skipping




to 100 nm









Antisense Oligonucleotides Directed at Exon 7

Antisense oligonucleotides directed at exon 7 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 4 shows the preferred antisense molecule, H7A(+45+67) [SEQ ID NO: 6], and another antisense molecule, H7A(+2+26) [SEQ ID NO: 7], inducing exon 7 skipping. Nested amplification products span exons 3 to 9. Additional products above the induced transcript missing exon 7 arise from amplification from carry-over outer primers from the RT-PCR as well as heteroduplex formation.
Table 3 below discloses antisense molecule sequences for induced exon 7 skipping.







TABLE 3







(SEQ ID NOS 6-9, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H7A(+45+67)
5′-UGC AUG UUC CAG
Strong



UCG UUG UGU GG
skipping




to 20 nM


 


H7A(+02+26)
5′-CAC UAU UCC AGU
Weak



CAA AUA GGU CUG G
skipping




at 100 nM


 


H7D(+15−10)
5′-AUU UAC CAA CCU 
A Weak



UCA GGA UCG AGU A
skipping




to 300 nM


 


H7A(−18+03)
5′-GGC CUA AAA CAC
Weak



AUA CAC AUA
skipping




to 300 nM









Antisense Oligonucleotides Directed at Exon 6

Antisense oligonucleotides directed at exon 6 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 5 shows an example of two non-preferred antisense molecules inducing very low levels of exon 6 skipping in cultured human cells. Targeting this exon for specific removal was first undertaken during a study of the canine model using the oligonucleotides as listed in Table 4, below. Some of the human specific oligonucleotides were also evaluated, as shown in FIG. 5. In this example, both antisense molecules target the donor splice site and only induced low levels of exon 6 skipping. Both H6D(+4−21) [SEQ ID NO: 17] and H6D(+18−4) [SEQ ID NO: 18] would be regarded as non-preferred antisense molecules.
One antisense oligonucleotide that induced very efficient exon 6 skipping in the canine model, C6A(+69+91) [SEQ ID NO: 14], would anneal perfectly to the corresponding region in human dystrophin exon 6. This compound was evaluated, found to be highly efficient at inducing skipping of that target exon, as shown in FIG. 6 and is regarded as the preferred compound for induced exon 6 skipping. Table 4 below discloses antisense molecule sequences for induced exon 6 skipping.







TABLE 4







(SEQ ID NOS 10-18, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





C6A(−10+10)
5′ CAU UUU UGA CCU
No



ACA UGU GG
skipping


 


C6A(−14+06)
5′ UUU GAC CUA CAU
No



GUG GAA AG
skipping


 


C6A(−14+12) 
5′ UAC AUU UUU GAC
No



CUA CAU GUG GAA AG
skipping


 


C6A(−13+09) 
5′ AUU UUU GAC CUA
No



CAU GGG AAA G
skipping


 


CH6A(+69+91)
5′ UAC GAG UUG AUU
Strong



GUC GGA CCC AG
skipping




to 20 nM


 


C6D(+12−13)
5′ GUG GUC UCC UUA
Weak



CCU AUG ACU GUG G
skipping




at 300 nM


 


C6D(+06−11)
5′ GGU CUC CUU ACC
No



UAU GA
skipping


 


H6D(+04−21)
5′ UGU CUC AGU AAU
Weak



CUU CUU ACC UAU
skipping




to 50 nM


 


H6D(+18−04)
5′ UCU UAC CUA UGA 
Very weak



CUA UGG AUG AGA
skipping




to 300 nM









Antisense Oligonucleotides Directed at Exon 4

Antisense oligonucleotides directed at exon 4 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 7 shows an example of a preferred antisense molecule inducing skipping of exon 4 skipping in cultured human cells. In this example, one preferred antisense compound, H4A(+13+32) [SEQ ID NO:19], which targeted a presumed exonic splicing enhancer induced efficient exon skipping at a concentration of 20 nM while other non-preferred antisense oligonucleotides failed to induce even low levels of exon 4 skipping. Another preferred antisense molecule inducing skipping of exon 4 was H4A(+111+40) [SEQ ID NO: 22], which induced efficient exon skipping at a concentration of 20 nM.
Table 5 below discloses antisense molecule sequences for inducing exon 4 skipping.







TABLE 5







(SEQ ID NOS 19, 22, 20, and 21, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H4A(+13+32)
5′ GCA UGA ACU CUU
Skipping



GUG GAU CC
to 20 nM


 


H4A(+11+40)
5′ UGU UCA GGG CAU 
Skipping



GAA CUC UUG UGG AUC
to 20 nM



CUU



 


H4D(+04−16)
5′ CCA GGG UAC UAC
No



UUA CAU UA
skipping


 


H4D(−24−44)
5′ AUC GUG UGU CAC
No



AGC AUC CAG
skipping









Antisense Oligonucleotides Directed at Exon 3

Antisense oligonucleotides directed at exon 3 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H3A(+30+60) [SEQ ID NO:23] induced substantial exon 3 skipping when delivered into cells at a concentration of 20 nM to 600 nM. The antisense molecule, H3A(+35+65) [SEQ ID NO: 24] induced exon skipping at 300 nM.
Table 6 below discloses antisense molecule sequences that induce exon 3 skipping.







TABLE 6







(SEQ ID NOS 23-30, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H3A(+30+60)
UAG GAG GCG CCU CCC
Moderate



AUC CUG UAG GUC ACU
skipping



G
to 20 to




600 nM


 


H3A(+35+65)
AGG UCU AGG AGG CGC
Working



CUC CCA UCC UGU AGG
to 300 nM



U



 


H3A(+30+54)
GCG CCU CCC AUC CUG
Moderate



UAG GUC ACU G
100-600 nM


 


H3D(+46−21)
CUU CGA GGA GGU CUA 
No



GGA GGC GCC UC
skipping


 


H3A(+30+50)
CUC CCA UCC UGU AGG
Moderate



UCA CUG
20-600 nM


 


H3D(+19−03)
UAC CAG UUU UUG CCC
No



UGU CAG G
skipping


 


H3A(−06+20)
UCA AUA UGC UGC UUC
No



CCA AAC UGA AA
skipping


 


H3A(+37+61)
CUA GGA GGC GCC UCC
No



CAU CCU GUA G
skipping









Antisense Oligonucleotides Directed at Exon 5

Antisense oligonucleotides directed at exon 5 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H5A(+20+50) [SEQ ID NO:31] induces substantial exon 5 skipping when delivered into cells at a concentration of 100 nM. Table 7 below shows other antisense molecules tested. The majority of these antisense molecules were not as effective at exon skipping as H5A(+20+50). However, H5A(+15+45) [SEQ ID NO: 40] was able to induce exon 5 skipping at 300 nM.
Table 7 below discloses antisense molecule sequences that induce exon 5 skipping.







TABLE 7







(SEQ ID NOS 31-40, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H5A(+20+50)
UUA UGA UUU CCA UCU
Working



ACG AUG UCA GUA CUU
to 100 nM



C



 


H5D(+25−05)
CUU ACC UGC CAG UGG
No



AGG AUU AUA UUC CAA
skipping



A



 


H5D(+10−15)
CAU CAG GAU UCU UAC
Inconsistent



CUG CCA GUG G
at 300 nM


 


H5A(+10+34)
CGA UGU CAG UAC UUC
Very weak



CAA UAU UCA C



 


H5D(−04−21)
ACC AUU CAU CAG GAU
No



UCU
skipping


 


H5D(+16−02)
ACC UGC CAG UGG AGG
No



AUU
skipping


 


H5A(−07+20)
CCA AUA UUC ACU AAA
No



UCA ACC UGU UAA
skipping


 


H5D(+18−12)
CAG GAU UCU UAC CUG
No



CCA GUG GAG GAU UAU
skipping


 


H5A(+05+35)
ACG AUG UCA GUA CUU
No



CCA AUA UUC ACU AAA
skipping



U



 


H5A(+15+45)
AUU UCC AUC UAC GAU
Working



GUC AGU ACU UCC AAU
to 300 nM



A









Antisense Oligonucleotides Directed at Exon 10

Antisense oligonucleotides directed at exon 10 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H10A(−05+16) [SEQ ID NO:41] induced substantial exon 10 skipping when delivered into cells. Table 8 below shows other antisense molecules tested. The antisense molecules' ability to induce exon skipping was variable. Table 8 below discloses antisense molecule sequences that induce exon 10 skipping.







TABLE 8







(SEQ ID NOS 41-45, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H10A(−05+16)
CAG GAG CUU CCA AAU GCU
Not



GCA
tested


 


H10A(−05+24)
CUU GUC UUC AGG AGC UUC
Not



CAA AUG CUG CA
tested


 


H10A(+98+119)
UCC UCA GCA GAA AGA AGC
Not



CAC G
tested


 


H10A(+130+149)
UUA GAA AUC UCU CCU UGU
No



GC
skipping


 


H10A(−33−14)
UAA AUU GGG UGU UAC ACA
No



AU
skipping









Antisense Oligonucleotides Directed at Exon 11

Antisense oligonucleotides directed at exon 11 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 8B shows an example of H11A(+75+97) [SEQ ID NO:49] antisense molecule inducing exon 11 skipping in cultured human cells. H11A(+75+97) induced substantial exon 11 skipping when delivered into cells at a concentration of 5 nM. Table 9 below shows other antisense molecules tested. The antisense molecules' ability to induce exon skipping was observed at 100 nM.







TABLE 9







(SEQ ID NOS 46-49 and 49, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H11D(+26+49)
CCC UGA GGC AUU CCC AUC
Skipping



UUG AAU
at 100 nM


 


H11D(+11−09)
AGG ACU UAC UUG CUU UGU
Skipping



UU
at 100 nM


 


H11A(+118+140)
CUU GAA UUU AGG AGA UUC
Skipping



AUC UG
at 100 nM


 


H11A(+75+97)
CAU CUU CUG AUA AUU UUC
Skipping



CUG UU
at 100 nM


 


H11A(+75+97)
CAU CUU CUG AUA AUU UUC
Skipping



CUG UU
at 5 nM









Antisense Oligonucleotides Directed at Exon 12

Antisense oligonucleotides directed at exon 12 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H12A(+52+75) [SEQ ID NO:50] induced substantial exon 12 skipping when delivered into cells at a concentration of 5 nM, as shown in FIG. 8A. Table 10 below shows other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. The antisense molecules' ability to induce exon skipping was variable.







TABLE 10







(SEQ ID NOS 50-52, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H12A(+52+75)
UCU UCU GUU UUU GUU AGC
Skipping



CAG UCA
at 5 nM


 


H12A(−10+10)
UCU AUG UAA ACU GAA AAU
Skipping



UU
at 100 nM


 


H12A(+11+30)
UUC UGG AGA UCC AUU AAA
No



AC
skipping









Antisense Oligonucleotides Directed at Exon 13

Antisense oligonucleotides directed at exon 13 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H13A(+77+100) [SEQ ID NO:53] induced substantial exon 13 skipping when delivered into cells at a concentration of 5 nM. Table 11 below includes two other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. These other antisense molecules were unable to induce exon skipping.







TABLE 11







(SEQ ID NOS 53-55, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H13A(+77+100)
CAG CAG UUG CGU GAU CUC
Skipping



CAC UAG
at 5 nM


 


H13A(+55+75)
UUC AUC AAC UAC CAC CAC
No



CAU
skipping


 


H13D(+06−19)
CUA AGC AAA AUA AUC UGA
No



CCU UAA G
skipping









Antisense Oligonucleotides Directed at Exon 14

Antisense oligonucleotides directed at exon 14 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H14A(+37+64) [SEQ ID NO:56] induced weak exon 14 skipping when delivered into cells at a concentration of 100 nM. Table 12 below includes other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. The other antisense molecules were unable to induce exon skipping at any of the concentrations tested.







TABLE 12







(SEQ ID NOS 56-62, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H14A(+37+64)
CUU GUA AAA GAA CCC AGC
Skipping



GGU CUU CUG U
at 100 nM


 


H14A(+14+35)
CAU CUA CAG AUG UUU GCC
No



CAU C
skipping


 


H14A(+51+73)
GAA GGA UGU CUU GUA AAA
No



GAA CC
skipping


 


H14D(−02+18)
ACC UGU UCU UCA GUA AGA
No



CG
skipping


 


H14D(+14−10)
CAU GAC ACA CCU GUU CUU
No



CAG UAA
skipping


 


H14A(+61+80)
CAU UUG AGA AGG AUG UCU
No



UG
skipping


 


H14A(−12+12)
AUC UCC CAA UAC CUG GAG
No



AAG AGA
skipping









Antisense Oligonucleotides Directed at Exon 15

Antisense oligonucleotides directed at exon 15 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H15A(−12+19) [SEQ ID NO:63] and H15A(+48+71) [SEQ ID NO:64] induced substantial exon 15 skipping when delivered into cells at a concentration of 10 Nm, as shown in FIG. 9A. Table 13 below includes other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 Nm. These other antisense molecules were unable to induce exon skipping at any of the concentrations tested.







TABLE 13







(SEQ ID NOS 63-65, 63, and 66, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H15A(−12+19)
GCC AUG CAC UAA AAA GGC
Skipping



ACU GCA AGA CAU U
at 5 nM


 


H15A(+48+71)
UCU UUA AAG CCA GUU GUG
Skipping



UGA AUC
at 5 nM


 


H15A(+08+28)
UUU CUG AAA GCC AUG CAC
No



UAA
skipping


 


H15A(−12+19)
GCC AUG CAC UAA AAA GGC
Skipping



ACU GCA AGA CAU U
at 10 nM


 


H15D(+17−08)
GUA CAU ACG GCC AGU UUU
No



UGA AGA C
skipping









Antisense Oligonucleotides Directed at Exon 16

Antisense oligonucleotides directed at exon 16 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H16A(−12+19) [SEQ ID NO:67] and H16A(−06+25) [SEQ ID NO:68] induced substantial exon 16 skipping when delivered into cells at a concentration of 10 nM, as shown in FIG. 9B. Table 14 below includes other antisense molecules tested. H16A(−06+19) [SEQ ID NO:69] and H16A(+87+109) [SEQ ID NO:70] were tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. These two antisense molecules were able to induce exon skipping at 25 nM and 100 nM, respectively. Additional antisense molecules were tested at 100, 200 and 300 nM and did not result in any exon skipping.







TABLE 14







(SEQ ID NOS 67-78, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H16A(−12+19)
CUA GAU CCG CUU UUA AAA
Skipping



CCU GUU AAA ACA A
at 5 nM


 


H16A(−06+25)
UCU UUU CUA GAU CCG CUU
Skipping



UUA AAA CCU GUU A
at 5 nM


 


H16A(−06+19)
CUA GAU CCG CUU UUA AAA
Skipping



CCU GUU A
at 25 nM


 


H16A(+87+109)
CCG UCU UCU GGG UCA CUG
Skipping



ACU UA
at 100 nM


 


H16A(−07+19)
CUA GAU CCG CUU UUA AAA
No



CCU GUU AA
skipping


 


H16A(−07+13)
CCG CUU UUA AAA CCU GUU
No



AA
skipping


 


H16A(+12+37)
UGG AUU GCU UUU UCU UUU
No



CUA GAU CC
skipping


 


H16A(+92+116)
CAU GCU UCC GUC UUC UGG
No



GUC ACU G
skipping


 


H16A(+45+67)
G AUC UUG UUU GAG UGA
No



AUA CAG U
skipping


 


H16A(+105+126)
GUU AUC CAG CCA UGC UUC
No



CGU C
skipping


 


H16D(+05−20)
UGA UAA UUG GUA UCA CUA
No



ACC UGU G
skipping


 


H16D(+12−11)
GUA UCA CUA ACC UGU GCU
No



GUA C
skipping









Antisense Oligonucleotides Directed at Exon 19

Antisense oligonucleotides directed at exon 19 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H19A(+35+65) [SEQ ID NO:79] induced substantial exon 19 skipping when delivered into cells at a concentration of 10 nM. This antisense molecule also showed very strong exon skipping at concentrations of 25, 50, 100, 300 and 600 nM.
FIG. 10 illustrates exon 19 and 20 skipping using a “cocktail” of antisense oligonucleotides, as tested using gel electrophoresis. It is interesting to note that it was not easy to induce exon 20 skipping using single antisense oligonucleotides H20A(+44+71) [SEQ ID NO:81] or H20A(+149+170) [SEQ ID NO:82], as illustrated in sections 2 and 3 of the gel shown in FIG. 10. Whereas, a “cocktail” of antisense oligonucleotides was more efficient as can be seen in section 4 of FIG. 10 using a “cocktail” of antisense oligonucleotides H20A(+44+71) and H20A(+149+170). When the cocktail was used to target exon 19, skipping was even stronger (see section 5, FIG. 10).
FIG. 11 illustrates gel electrophoresis results of exon 19/20 skipping using “weasels”. The “weasels” were effective in skipping exons 19 and 20 at concentrations of 25, 50, 100, 300 and 600 nM. A further “weasel” sequence is shown in the last row of Table 3C. This compound should give good results.
Antisense Oligonucleotides Directed at Exon 20
Antisense oligonucleotides directed at exon 20 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
None of the antisense oligonucleotides tested induced exon 20 skipping when delivered into cells at a concentration of 10, 25, 50, 300 or 600 nM (see Table 15). Antisense molecules H20A(−11+17) [SEQ ID NO:86] and H20D(+08−20) [SEQ ID NO:87] are yet to be tested.
However, a combination or “cocktail” of H20A(+44+71) [SEQ ID NO: 81] and H20A(+147+168) [SEQ ID NO:82] in a ratio of 1:1, exhibited very strong exon skipping at a concentration of 100 nM and 600 nM. Further, a combination of antisense molecules H19A(+35+65) [SEQ ID NO:80], H20A(+44+71) [SEQ ID NO:81] and H20A(+147+168) [SEQ ID NO:82] in a ratio of 2:1:1, induced very strong exon skipping at a concentration ranging from 10 nM to 600 nM.







TABLE 15







(SEQ ID NOS 81-87, 81-82, and 80-82, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H20A(+44+71)
CUG GCA GAA UUC GAU CCA
No



CCG GCU GUU C
skipping


 


H20A(+147+168)
CAG CAG UAG UUG UCA UCU
No



GCU C
skipping


 


H20A(+185+203)
UGA UGG GGU GGU GGG UUG
No



G
skipping


 


H20A(-08+17)
AUC UGC AUU AAC ACC CUC
No



UAG AAA G
skipping


 


H20A(+30+53)
CCG GCU GUU CAG UUG UUC
No



UGA GGC
skipping


 


H20A(-11+17)
AUC UGC AUU AAC ACC CUC
Not



UAG AAA GAA A
tested yet


 


H20D(+08-20)
GAA GGA GAA GAG AUU CUU
Not



ACC UUA CAA A
tested yet


 


H20A(+44+71) &
CUG GCA GAA UUC GAU CCA
Very



CCG GCU GUU C
strong


H20A(+147+168)
CAG CAG UAG UUG UCA UCU
skipping



GCU C



 


H19A(+35+65)
GCC UGA GCU GAU CUG CUG
Very



GCA UCU UGC AGU U;
strong


H20A(+44+71)
CUG GCA GAA UUC GAU CCA
skipping



CCG GCU GUU C



H20A(+147+168)
CAG CAG UAG UUG UCA UCU




GCU C









Antisense Oligonucleotides Directed at Exon 21

Antisense oligonucleotides directed at exon 21 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H21A(+85+108) [SEQ ID NO:92] and H21A(+85+106) [SEQ ID NO:91] induced exon 21 skipping when delivered into cells at a concentration of 50 nM. Table 16 below includes other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping







TABLE 16







(SEQ ID NOS 90-94, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H21A(−06+16)
GCC GGU UGA CUU CAU CCU
Skips at



GUG C
600 nM


 


H21A(+85+106)
CUG CAU CCA GGA ACA UGG
Skips at



GUC C
50 nM


 


H21A(+85+108)
GUC UGC AUC CAG GAA CAU
Skips at



GGG UC
50 nM


 


H21A(+08+31)
GUU GAA GAU CUG AUA GCC
Skips



GGU UGA
faintly to


 


H21D(+18−07)
UAC UUA CUG UCU GUA GCU
No



CUU UCU
skipping









Antisense Oligonucleotides Directed at Exon 22

Antisense oligonucleotides directed at exon 22 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 12 illustrates differing efficiencies of two antisense molecules directed at exon 22 acceptor splice site. H22A(+125+106) [SEQ ID NO:96] and H22A(+80+101) [SEQ ID NO: 98] induce strong exon 22 skipping from 50 nM to 600 nM concentration.
H22A(+125+146) [SEQ ID NO: 96] and H22A(+80+101) [SEQ ID NO:98] induced exon 22 skipping when delivered into cells at a concentration of 50 nM. Table 17 below shows other antisense molecules tested at a concentration range of 50, 100, 300 and 600 nM. These antisense molecules showed a variable ability to induce exon skipping.







TABLE 17







(SEQ ID NOS 95-99, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H22A(+22+45)
CAC UCA UGG UCU CCU GAU
No



AGC GCA
skipping


 


H22A(+125+146)
CUG CAA UUC CCC GAG UCU
Skipping



CUG C
to 50 nM


 


H22A(+47+69)
ACU GCU GGA CCC AUG UCC
Skipping



UGA UG
to 300 nM


 


H22A(+80+101)
CUA AGU UGA GGU AUG GAG
Skipping



AGU
to 50 nM


 


H22D(+13−11)
UAU UCA CAG ACC UGC AAU
No



UCC CC
skipping









Antisense Oligonucleotides Directed at Exon 23

Antisense oligonucleotides directed at exon 23 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
Table 18 below shows antisense molecules tested at a concentration range of 25, 50, 100, 300 and 600 nM. These antisense molecules showed no ability to induce exon skipping or are yet to be tested.







TABLE 18







(SEQ ID NOS 100-102, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H23A(+34+59)
ACA GUG GUG CUG AGA UAG
No



UAU AGG CC
skipping


 


H23A(+18+39)
UAG GCC ACU UUG UUG CUC
No



UUG C
Skipping


 


H23A(+72+90)
UUC AGA GGG CGC UUU CUU
No



C
Skipping









Antisense Oligonucleotides Directed at Exon 24

Antisense oligonucleotides directed at exon 24 were prepared using similar methods as described above. Table 19 below outlines the antisense oligonucleotides directed at exon 24 that are yet to be tested for their ability to induce exon 24 skipping.







TABLE 19







(SEQ ID NOS 103-104, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H24A(+48+70)
GGG CAG GCC AUU CCU CCU
Needs



UCA GA
testing


 


H24A(−02+22)
UCU UCA GGG UUU GUA UGU
Needs



GAU UCU
testing









Antisense Oligonucleotides Directed at Exon 25

Antisense oligonucleotides directed at exon 25 were prepared using similar methods as described above. Table 20 below shows the antisense oligonucleotides directed at exon 25 that are yet to be tested for their ability to induce exon 25 skipping.







TABLE 20







(SEQ ID NOS 105-107, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H25A(+9+36)
CUG GGC UGA AUU GUC UGA
Needs



AUA UCA CUG
testing


 


H25A(+131+156)
CUG UUG GCA CAU GUG AUC
Needs



CCA CUG AG
testing


 


H25D(+16−08)
GUC UAU ACC UGU UGG CAC
Needs



AUG UGA
testing









Antisense Oligonucleotides Directed at Exon 26

Antisense oligonucleotides directed at exon 26 were prepared using similar methods as described above. Table 21 below outlines the antisense oligonucleotides directed at exon 26 that are yet to be tested for their ability to induce exon 26 skipping.







TABLE 21







(SEQ ID NOS 108-110, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H26A(+132+156)
UGC UUU CUG UAA UUC AUC
Needs



UGG AGU U
testing


 


H26A(-07+19)
CCU CCU UUC UGG CAU AGA
Needs



CCU UCC AC
testing


 


H26A(+68+92)
UGU GUC AUC CAU UCG UGC
Faint



AUC UCU G
skipping




at 600 nM









Antisense Oligonucleotides Directed at Exon 27

Antisense oligonucleotides directed at exon 27 were prepared using similar methods as described above. Table 22 below outlines the antisense oligonucleotides directed at exon 27 that are yet to be tested for their ability to induce exon 27 skipping.







TABLE 22







(SEQ ID NOS 111-113, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H27A(+82+106)
UUA AGG CCU CUU GUG CUA
Needs



CAG GUG G
testing


 


H27A(−4+19)
GGG CCU CUU CUU UAG CUC
Faint



UCU GA
skipping




at 600




and 300 nM


 


H27D(+19−03)
GAC UUC CAA AGU CUU GCA
v. strong



UUU C
skipping




at 600




and 300 nM









Antisense Oligonucleotides Directed at Exon 28

Antisense oligonucleotides directed at exon 28 were prepared using similar methods as described above. Table 23 below outlines the antisense oligonucleotides directed at exon 28 that are yet to be tested for their ability to induce exon 28 skipping.







TABLE 23







(SEQ ID NOS 114-116, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H28A(−05+19) 
GCC AAC AUG CCC AAA CUU
v. strong



CCU AAG
skipping




at 600




and 300 nM


 


H28A(+99+124)
CAG AGA UUU CCU CAG CUC
Needs



CGC CAG GA
testing


 


H28D(+16−05) 
CUU ACA UCU AGC ACC UCA
v. strong



GAG
skipping




at 600




and 300 nM









Antisense Oligonucleotides Directed at Exon 29

Antisense oligonucleotides directed at exon 29 were prepared using similar methods as described above. Table 24 below outlines the antisense oligonucleotides directed at exon 29 that are yet to be tested for their ability to induce exon 29 skipping.







TABLE 24







(SEQ ID NOS 117-119, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H29A(+57+81)
UCC GCC AUC UGU UAG GGU
Needs



CUG UGC C
testing


 


H29A(+18+42)
AUU UGG GUU AUC CUC UGA
v. strong



AUG UCG C
skipping




at 600




and 300 nM


 


H29D(+17-05)
CAU ACC UCU UCA UGU AGU
v. strong



UCC C
skipping




at 600




and 300 nM









Antisense Oligonucleotides Directed at Exon 30

Antisense oligonucleotides directed at exon 30 were prepared using similar methods as described above. Table 25 below outlines the antisense oligonucleotides directed at exon 30 that are yet to be tested for their ability to induce exon 30 skipping.







TABLE 25







(SEQ ID NOS 120-122, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H30A(+122+147)
CAU UUG AGC UGC GUC CAC
Needs



CUU GUC UG
testing


 


H30A(+25+50)
UCC UGG GCA GAC UGG AUG
Very



CUC UGU UC
strong




skipping




at 600 and




300 nM.


 


H30D(+19−04)
UUG CCU GGG CUU CCU GAG
Very



GCA UU
strong




skipping




at 600 and




300 nM.









Antisense Oligonucleotides Directed at Exon 31

Antisense oligonucleotides directed at exon 31 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 13 illustrates differing efficiencies of two antisense molecules directed at exon 31 acceptor splice site and a “cocktail” of exon 31 antisense oligonucleotides at varying concentrations. H31D(+03−22) [SEQ ID NO:124] substantially induced exon 31 skipping when delivered into cells at a concentration of 20 nM. Table 26 below also includes other antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping.







TABLE 26







(SEQ ID NOS 123-126, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H31D(+06−18)
UUC UGA AAU AAC AUA UAC
Skipping to



CUG UGC
300 nM


 


H31D(+03−22)
UAG UUU CUG AAA UAA CAU
Skipping to



AUA CCU G
20 nM


 


H31A(+05+25)
GAC UUG UCA AAU CAG AUU
No



GGA
skipping


 


H31D(+04−20)
GUU UCU GAA AUA ACA UAU
Skipping to



ACC UGU
300 nM









Antisense Oligonucleotides Directed at Exon 32

Antisense oligonucleotides directed at exon 32 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H32D(+04−16) [SEQ ID NO:127] and H32A(+49+73) [SEQ ID NO:130] induced exon 32 skipping when delivered into cells at a concentration of 300 nM. Table 27 below also shows other antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules did not show an ability to induce exon skipping.







TABLE 27







(SEQ ID NOS 127-130, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H32D(+04−16)
CAC CAG AAA UAC AUA CCA
Skipping



CA
to 300 nM


 


H32A(+151+170)
CAA UGA UUU AGC UGU GAC
No



UG
skipping


 


H32A(+10+32)
CGA AAC UUC AUG GAG ACA
No



UCU UG
skipping


 


H32A(+49+73)
CUU GUA GAC GCU GCU CAA
Skipping



AAU UGG C
to 300 nM









Antisense Oligonucleotides Directed at Exon 33

Antisense oligonucleotides directed at exon 33 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 14 shows differing efficiencies of two antisense molecules directed at exon 33 acceptor splice site. H33A(+64+88) [SEQ ID NO:134] substantially induced exon 33 skipping when delivered into cells at a concentration of 10 nM. Table 28 below includes other antisense molecules tested at a concentration of 100, 200 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping.







TABLE 28







(SEQ ID NOS 131-134, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H33D(+09−11)
CAU GCA CAC ACC UUU GCU
No



CC
skipping


 


H33A(+53+76)
UCU GUA CAA UCU GAC GUC
Skipping



CAG UCU
to 200 nM


 


H33A(+30+56)
GUC UUU AUC ACC AUU UCC
Skipping



ACU UCA GAC
to 200 nM


 


H33A(+64+88)
CCG UCU GCU UUU UCU GUA
Skipping



CAA UCU G
to 10 nM









Antisense Oligonucleotides Directed at Exon 34

Antisense oligonucleotides directed at exon 34 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
Table 29 below includes antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping.







TABLE 29







(SEQ ID NOS 135-141, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H34A(+83+104)
UCC AUA UCU GUA GCU GCC
No



AGC C
skipping


 


H34A(+143+165)
CCA GGC AAC UUC AGA AUC
No



CAA AU
skipping


 


H34A(−20+10)
UUU CUG UUA CCU GAA AAG
Not



AAU UAU AAU GAA
tested


 


H34A(+46+70)
CAU UCA UUU CCU UUC GCA
Skipping



UCU UAC G
to 300 nM


 


H34A(+95+120)
UGA UCU CUU UGU CAA UUC
Skipping



CAU AUC UG
to 300 nM


 


H34D(+10−20)
UUC AGU GAU AUA GGU UUU
Not



ACC UUU CCC CAG
tested


 


H34A(+72+96)
CUG UAG CUG CCA GCC AUU
No



CUG UCA AG
skipping









Antisense Oligonucleotides Directed at Exon 35

Antisense oligonucleotides directed at exon 35 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 15 shows differing efficiencies of antisense molecules directed at exon 35 acceptor splice site. H35A(+24+43) [SEQ ID NO:144] substantially induced exon 35 skipping when delivered into cells at a concentration of 20 nM. Table 30 below also includes other antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules showed no ability to induce exon skipping.







TABLE 30







(SEQ ID NOS 142-144, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H35A(+141+161)
UCU UCU GCU CGG GAG GUG
Skipping



ACA
to 20 nM


 


H35A(+116+135)
CCA GUU ACU AUU CAG AAG
No



AC
skipping


 


H35A(+24+43)
UCU UCA GGU GCA CCU UCU
No



GU
skipping









Antisense Oligonucleotides Directed at Exon 36

Antisense oligonucleotides directed at exon 36 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
Antisense molecule H36A(+26+50) [SEQ ID NO:145] induced exon 36 skipping when delivered into cells at a concentration of 300 nM, as shown in FIG. 16.
Antisense Oligonucleotides Directed at Exon 37
Antisense oligonucleotides directed at exon 37 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 17 shows differing efficiencies of two antisense molecules directed at exon 37 acceptor splice site. H37A(+82+105) [SEQ ID NO:148] and H37A(+134+157) [SEQ ID NO:149] substantially induced exon 37 skipping when delivered into cells at a concentration of 10 nM. Table 31 below shows the antisense molecules tested.







TABLE 31







(SEQ ID NOS 147-149, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H37A(+26+50)
CGU GUA GAG UCC ACC UUU
No



GGG CGU A
skipping


 


H37A(+82+105)
UAC UAA UUU CCU GCA GUG
Skipping



GUC ACC
to 10 nM


 


H37A(+134+157)
UUC UGU GUG AAA UGG CUG
Skipping



CAA AUC
to 10 nM









Antisense Oligonucleotides Directed at Exon 38

Antisense oligonucleotides directed at exon 38 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 18 illustrates antisense molecule H38A(+88+112) [SEQ ID NO:152], directed at exon 38 acceptor splice site. H38A(+88+112) substantially induced exon 38 skipping when delivered into cells at a concentration of 10 nM. Table 32 below shows the antisense molecules tested and their ability to induce exon skipping.







TABLE 32







(SEQ ID NOS 150-152, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H38A(−01+19)
CCU UCA AAG GAA UGG AGG
No



CC
skipping


 


H38A(+59+83)
UGC UGA AUU UCA GCC UCC
Skipping



AGU GGU U
to 10 nM


 


H38A(+88+112)
UGA AGU CUU CCU CUU UCA
Skipping



GAU UGA C
to 10 nM









Antisense Oligonucleotides Directed at Exon 39

Antisense oligonucleotides directed at exon 39 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H39A(+62+85) [SEQ ID NO:153] induced exon 39 skipping when delivered into cells at a concentration of 100 nM. Table 33 below shows the antisense molecules tested and their ability to induce exon skipping.







TABLE 33







(SEQ ID NOS 153-156, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H39A(+62+85)
CUG GCU UUC UCU CAU CUG
Skipping



UGA UUC
to 100 nM


 


H39A(+39+58)
GUU GUA AGU UGU CUC CUC
No



UU
skipping


 


H39A(+102+121)
UUG UCU GUA ACA GCU GCU
No



GU
skipping


 


H39D(+10−10)
GCU CUA AUA CCU UGA GAG
Skipping



CA
to 300 nM









Antisense Oligonucleotides Directed at Exon 40

Antisense oligonucleotides directed at exon 40 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 19 illustrates antisense molecule H40A(−05+17) [SEQ ID NO:157] directed at exon 40 acceptor splice site. H40A(−05+17) and H40A(+129+153) [SEQ ID NO:158] both substantially induced exon 40 skipping when delivered into cells at a concentration of 5 nM.
Antisense Oligonucleotides Directed at Exon 42
Antisense oligonucleotides directed at exon 42 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 20 illustrates antisense molecule H42A(−04+23) [SEQ ID NO:159], directed at exon 42 acceptor splice site. H42A(−4+23) and H42D(+19−02) [SEQ ID NO:161] both induced exon 42 skipping when delivered into cells at a concentration of 5 nM. Table 34 below shows the antisense molecules tested and their ability to induce exon 42 skipping.







TABLE 34







(SEQ ID NOS 159-160, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H42A(−4+23)
AUC GUU UCU UCA CGG ACA
Skipping



GUG UGC UGG
to 5 nM


 


H42A(+86+109)
GGG CUU GUG AGA CAU GAG
Skipping



UGA UUU
to 100 nM


 


H42D(+19−02)
A CCU UCA GAG GAC UCC
Skipping



UCU UGC
to 5 nM









Antisense Oligonucleotides Directed at Exon 43

Antisense oligonucleotides directed at exon 43 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H43A(+101+120) [SEQ ID NO:163] induced exon 43 skipping when delivered into cells at a concentration of 25 nM. Table 35 below includes the antisense molecules tested and their ability to induce exon 43 skipping.







TABLE 35







(SEQ ID NOS 162-164, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H43D(+10−15)
UAU GUG UUA CCU ACC CUU
Skipping



GUC GGU C
to 100 nM


 


H43A(+101+120)
GGA GAG AGC UUC CUG UAG
Skipping



CU
to 25 nM


 


H43A(+78+100)
UCA CCC UUU CCA CAG GCG
Skipping



UUG CA
to 200 nM









Antisense Oligonucleotides Directed at Exon 44

Antisense oligonucleotides directed at exon 44 were prepared using similar methods as described above. Testing for the ability of these antisense molecules to induce exon 44 skipping is still in progress. The antisense molecules under review are shown as SEQ ID Nos: 165 to 167 in Table 1A.
Antisense Oligonucleotides Directed at Exon 45
Antisense oligonucleotides directed at exon 45 were prepared using similar methods as described above. Testing for the ability of these antisense molecules to induce exon 45 skipping is still in progress. The antisense molecules under review are shown as SEQ ID Nos: 207 to 211 in Table 1A.
Antisense Oligonucleotides Directed at Exon 46
Antisense oligonucleotides directed at exon 46 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 21 illustrates the efficiency of one antisense molecule directed at exon 46 acceptor splice site. Antisense oligonucleotide H46A(+86+115) [SEQ ID NO:203] showed very strong ability to induce exon 46 skipping. Table 36 below includes antisense molecules tested. These antisense molecules showed varying ability to induce exon 46 skipping.







TABLE 36







(SEQ ID NOS 168-169 and 203-206, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H46D(+16−04)
UUA CCU UGA CUU GCU CAA
No



GC
skipping


 


H46A(+90+109)
UCC AGG UUC AAG UGG GAU
No



AC
skipping


 


H46A(+86+115)
CUC UUU UCC AGG UUC AAG
Good



UGG GAU ACU AGC
skipping




to 100 nM


 


H46A(+107+137)
CAA GCU UUU CUU UUA GUU
Good



GCU GCU CUU UUC C
skipping




to 100 nM


 


H46A(−10+20)
UAU UCU UUU GUU CUU CUA
Weak



GCC UGG AGA AAG
skipping


 


H46A(+50+77)
CUG CUU CCU CCA ACC AUA
Weak



AAA CAA AUU C
skipping









Antisense Oligonucleotides Directed at Exon 47

Antisense oligonucleotides directed at exon 47 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H47A(+76+100) [SEQ ID NO: 170] and H47A(−09+12) [SEQ ID NO:172] both induced exon 47 skipping when delivered into cells at a concentration of 200 nM. H47D(+25−02) [SEQ ID NO: 171] is yet to be prepared and tested.
Antisense Oligonucleotides Directed at Exon 50
Antisense oligonucleotides directed at exon 50 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
Antisense oligonucleotide molecule H50(+02+30) [SEQ ID NO: 173] was a strong inducer of exon skipping. Further, H50A(+07+33) [SEQ ID NO:174] and H50D(+07−18) [SEQ ID NO:175] both induced exon 50 skipping when delivered into cells at a concentration of 100 nM.
Antisense Oligonucleotides Directed at Exon 51
Antisense oligonucleotides directed at exon 51 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 22 illustrates differing efficiencies of two antisense molecules directed at exon 51 acceptor splice site. Antisense oligonucleotide H51A(+66+90) [SEQ ID NO:180] showed the stronger ability to induce exon 51 skipping. Table 37 below includes antisense molecules tested at a concentration range of 25, 50, 100, 300 and 600 nM. These antisense molecules showed varying ability to induce exon 51 skipping. The strongest inducers of exon skipping were antisense oligonucleotide H51A(+61+90) [SEQ ID NO: 179] and H51A(+66+95) [SEQ ID NO: 179].







TABLE 37







(SEQ ID NOS 176-185, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H51A(−01+25)
ACC AGA GUA ACA GUC UGA
Faint



GUA GGA GC
skipping


 


H51D(+16−07)
CUC AUA CCU UCU GCU UGA
Skipping



UGA UC
at




300 nM


 


H51A(+111+134)
UUC UGU CCA AGC CCG GUU
Needs



GAA AUC
re-testing


 


H51A(+61+90)
ACA UCA AGG AAG AUG GCA
Very



UUU CUA GUU
strong




skippin


 


H51A(+66+90)
ACA UCA AGG AAG AUG GCA
skipping



UUU CUA G



 


H51A(+66+95)
CUC CAA CAU CAA GGA AGA
Very



UGG CAU UUC UAG
strong




skipping


 


H51D(+08−17)
AUC AUU UUU UCU CAU ACC
No



UUC UGC U
skipping


 


H51A/D(+08−17)
AUC AUU UUU UCU CAU ACC
No


& (−15+?)
UUC UGC UAG GAG CUA AAA
skipping


 


H51A(+175+195)
CAC CCA CCA UCA CCC UCU
No



GUG
skipping


 


H51A(+199+220)
AUC AUC UCG UUG AUA UCC
No



UCA A
skipping









Antisense Oligonucleotides Directed at Exon 52

Antisense oligonucleotides directed at exon 52 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 22 also shows differing efficiencies of four antisense molecules directed at exon 52 acceptor splice site. The most effective antisense oligonucleotide for inducing exon 52 skipping was H52A(+17+37) [SEQ ID NO:188].
Table 38 below shows antisense molecules tested at a concentration range of 50, 100, 300 and 600 nM. These antisense molecules showed varying ability to induce exon 50 skipping. Antisense molecules H52A(+12+41) [SEQ ID NO:187] and H52A(+17+37) [SEQ ID NO:188] showed the strongest exon 50 skipping at a concentration of 50 nM.







TABLE 38







(SEQ ID NOS 186-190, respectively,


in order of appearance)









Antisense
 
Ability to


Oligonucleotide

induce


name
Sequence
skipping





H52A(−07+14)
UCC UGC AUU GUU GCC UGU
No



AAG
skipping


 


H52A(+12+41)
UCC AAC UGG GGA CGC CUC
Very



UGU UCC AAA UCC
strong




skipping


 


H52A(+17+37)
ACU GGG GAC GCC UCU GUU
Skipping



CCA
to 50 nM


 


H52A(+93+112)
CCG UAA UGA UUG UUC UAG
No



CC
skipping


 


H52D(+05−15)
UGU UAA AAA ACU UAC UUC
No



GA
skipping









Antisense Oligonucleotides Directed at Exon 53

Antisense oligonucleotides directed at exon 53 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
FIG. 22 also shows antisense molecule H53A(+39+69) [SEQ ID NO:193] directed at exon 53 acceptor splice site. This antisense oligonucleotide was able to induce exon 53 skipping at 5, 100, 300 and 600 nM. A “cocktail” of three exon 53 antisense oligonucleotides: H53A(+23+47) [SEQ ID NO:195], H53A(+150+176) [SEQ ID NO:196] and H53D (+14-07) [SEQ ID NO:194], was also tested, as shown in FIG. 20 and exhibited an ability to induce exon skipping.
Table 39 below includes other antisense molecules tested at a concentration range of 50, 100, 300 and 600 nM. These antisense molecules showed varying ability to induce exon 53 skipping. Antisense molecule H53A(+39+69) [SEQ ID NO:193] induced the strongest exon 53 skipping.







TABLE 39







(SEQ ID NOS 191-202, respectively,


in order of appearance)









Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping





H53A(+45+69)
CAU UCA ACU GUU GCC UCC
Faint



GGU UCU G
skipping




at 50 nM


 


H53A(+39+62)
CUG UUG CCU CCG GUU CUG
Faint



AAG GUG
skipping




at 50 nM


 


H53A(+39+69)
CAU UCA ACU GUU GCC UCC
Strong



GGU UCU GAA GGU G
skipping




to 50 nM


 


H53D(+14−07)
UAC UAA CCU UGG UUU CUG
Very faint



UGA
skipping




to 50 nM


 


H53A(+23+47)
CUG AAG GUG UUC UUG UAC
Very faint



UUC AUC C
skipping




to 50 nM


 


H53A(+150+176)
UGU AUA GGG ACC CUC CUU
Very faint



CCA UGA CUC
skipping




to 50 nM


 


H53D(+20−05)
CUA ACC UUG GUU UCU GUG
Not made



AUU UUC U
yet


 


H53D(+09−18)
GGU AUC UUU GAU ACU AAC
Faint at



CUU GGU UUC
600 nM


 


H53A(−12+10)
AUU CUU UCA ACU AGA AUA
No



AAA G
skipping


 


H53A(−07+18)
GAU UCU GAA UUC UUU CAA
No



CUA GAA U
skipping


 


H53A(+07+26)
AUC CCA CUG AUU CUG AAU
No



UC
skipping


 


H53A(+124+145)
UUG GCU CUG GCC UGU CCU
No



AAG A
skipping

## Claims

1. An isolated antisense oligonucleotide of 20 to 50 nucleotides in length comprising at least 17 consecutive nucleotides of SEQ ID NO: 207, wherein the oligonucleotide specifically hybridizes to an exon 45 acceptor splice site of a human dystrophin gene, inducing exon 45 skipping, and wherein the uracil bases are optionally thymine bases.

2. The antisense oligonucleotide of claim 1, wherein the uracil bases are thymine bases.

3. The antisense oligonucleotide of claim 1 comprising SEQ ID NO: 207.

4. The antisense oligonucleotide of claim 1 consisting of SEQ ID NO: 207.

5. The antisense oligonucleotide of claim 1 comprising SEQ ID NO: 207, wherein the uracil bases are thymine bases.

6. The antisense oligonucleotide of claim 1 comprising 25 nucleotides in length.

7. The antisense oligonucleotide of claim 1 comprising 17-30 nucleotides in length.

8. The antisense oligonucleotide of claim 1 comprising 20-31 nucleotides in length.

9. The antisense oligonucleotide of claim 1, wherein the oligonucleotide does not activate RNase H.

10. The antisense oligonucleotide of claim 1, comprising a non-natural backbone.

11. The antisense oligonucleotide of claim 10, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties.

12. The antisense oligonucleotide of claim 11, wherein the non-natural moieties are morpholinos.

13. The antisense oligonucleotide of claim 1, wherein the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

14. The antisense oligonucleotide of claim 13, wherein the non-natural inter-nucleotide linkages are modified phosphates.

15. The antisense oligonucleotide of claim 1, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties and the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

16. The antisense oligonucleotide of claim 15, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.

17. The antisense oligonucleotide of claim 16, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

18. The antisense oligonucleotide of claim 1, wherein the oligonucleotide is a 2′-O-methyl-oligoribonucleotide.

19. The antisense oligonucleotide of claim 1, wherein the oligonucleotide is a peptide nucleic acid.

20. The antisense oligonucleotide of claim 1, wherein the oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.

21. The antisense oligonucleotide of claim 20, wherein the oligonucleotide is conjugated to a polyamine.

22. The antisense oligonucleotide of claim 20, wherein the oligonucleotide is chemically linked to a polyethylene glycol chain.

23. An isolated antisense oligonucleotide of 20 to 50 nucleotides in length comprising at least 17 consecutive nucleotides complementary to an exon 45 target region of a human dystrophin gene designated as annealing site H45A(−06+20), wherein the antisense oligonucleotide specifically hybridizes to the acceptor splice site inducing exon 45 skipping, and wherein uracil bases in the antisense oligonucleotide are optionally thymine bases.

24. The antisense oligonucleotide of claim 23, wherein the uracil bases are thymine bases.

25. The antisense oligonucleotide of claim 23 comprising 25 nucleotides in length.

26. The antisense oligonucleotide of claim 23 comprising 17-30 nucleotides in length.

27. The antisense oligonucleotide of claim 23 comprising 20-31 nucleotides in length.

28. The antisense oligonucleotide of claim 23 comprising at least 17 consecutive nucleotides 100% complementary to the exon 45 target region.

29. The antisense oligonucleotide of claim 23 comprising 20-31 nucleotides in length and at least 17 consecutive nucleotides 100% complementary to the exon 45 target region.

30. The antisense oligonucleotide of claim 23 comprising at least 20 consecutive nucleotides complementary to the exon 45 target region.

31. The antisense oligonucleotide of claim 23 comprising at least 20 consecutive nucleotides 100% complementary to the exon 45 target region.

32. The antisense oligonucleotide of claim 23 comprising 20-31 nucleotides in length and at least 20 consecutive nucleotides complementary to the exon 45 target region.

33. The antisense oligonucleotide of claim 23, wherein the oligonucleotide does not activate RNase H.

34. The antisense oligonucleotide of claim 23, comprising a non-natural backbone.

35. The antisense oligonucleotide of claim 34, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties.

36. The antisense oligonucleotide of claim 35, wherein the non-natural moieties are morpholinos.

37. The antisense oligonucleotide of claim 23, wherein the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

38. The antisense oligonucleotide of claim 37, wherein the non-natural inter-nucleotide linkages are modified phosphates.

39. The antisense oligonucleotide of claim 23, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties and the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

40. The antisense oligonucleotide of claim 39, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.

41. The antisense oligonucleotide of claim 40, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

42. The antisense oligonucleotide of claim 23, wherein the oligonucleotide is a 2′-O-methyl-oligoribonucleotide.

43. The antisense oligonucleotide of claim 23, wherein the oligonucleotide is a peptide nucleic acid.

44. The antisense oligonucleotide of claim 23, wherein the oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.

45. The antisense oligonucleotide of claim 44, wherein the oligonucleotide is conjugated to a polyamine.

46. The antisense oligonucleotide of claim 44, wherein the oligonucleotide is chemically linked to a polyethylene glycol chain.

47. An isolated antisense oligonucleotide of 20 to 50 nucleotides in length comprising at least 20 consecutive nucleotides of SEQ ID NO: 207, wherein the uracil bases are optionally thymine bases.

48. The antisense oligonucleotide of claim 47, wherein the uracil bases are thymine bases.

49. The antisense oligonucleotide of claim 47 comprising 25 nucleotides in length.

50. The antisense oligonucleotide of claim 47 comprising 20-31 nucleotides in length.

51. The antisense oligonucleotide of claim 47, wherein the oligonucleotide does not activate RNase H.

52. The antisense oligonucleotide of claim 47, comprising a non-natural backbone.

53. The antisense oligonucleotide of claim 52, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties.

54. The antisense oligonucleotide of claim 53, wherein the non-natural moieties are morpholinos.

55. The antisense oligonucleotide of claim 47, wherein the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

56. The antisense oligonucleotide of claim 55, wherein the non-natural inter-nucleotide linkages are modified phosphates.

57. The antisense oligonucleotide of claim 47, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties and the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

58. The antisense oligonucleotide of claim 57, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.

59. The antisense oligonucleotide of claim 58, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

60. The antisense oligonucleotide of claim 47, wherein the oligonucleotide is a 2′-O-methyl-oligoribonucleotide.

61. The antisense oligonucleotide of claim 47, wherein the oligonucleotide is a peptide nucleic acid.

62. The antisense oligonucleotide of claim 47, wherein the oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.

63. The antisense oligonucleotide of claim 62, wherein the oligonucleotide is conjugated to a polyamine.

64. The antisense oligonucleotide of claim 62, wherein the oligonucleotide is chemically linked to a polyethylene glycol chain.

65. A pharmaceutical composition comprising an antisense oligonucleotide of claim 1, and a pharmaceutically acceptable carrier.

66. A pharmaceutical composition comprising an antisense oligonucleotide of claim 23, and a pharmaceutically acceptable carrier.

67. A pharmaceutical composition comprising an antisense oligonucleotide of claim 47, and a pharmaceutically acceptable carrier.

68. A method of inducing exon-skipping of dystrophin exon 45, comprising administering a pharmaceutical composition of claim 65.

69. A method of treating Duchenne muscular dystrophy, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition of claim 65.