# 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 issued as U.S. Pat. No. 7,807,816, which application 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; which applications are each 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 120178—411C3_SEQUENCE_LISTING.txt. The text file is 49 KB, was created on Nov. 3, 2009, 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 et al., (1998). No exon skipping, either 23 alone or multiple removal of several flanking exons, could be reproducibly detected using a selection of antisense oligonucleotides directed at the acceptor splice site of intron 22.
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 a antisense oligonucleotide of the present invention, packaged in a suitable container and instructions for its use.
Other aspects and advantages of the invention will become apparent to those skilled in the art from a review of the ensuing description, which proceeds with reference to the following figures.



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 NOS: 213 and 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 molecules [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 (8B) 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) and H15A(−12+19) directed at an exon 15 internal domain; and (9B) strong human exon 16 skipping using antisense molecules H16A(−12+19) and 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) and 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) and H33A(+64+88) directed 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) and H36A(+26+50) directed 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) directed 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    


peptidenucleic 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 CAA 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)
CAG CAG UAG UUG UCA UCU 




GC


 


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(+149+170)
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
CUG CUG GCA UCU UGC AGU 




U


 


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+106)
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)
CTG 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 




UCU 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 



& (−15+)
UUC UGC 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(+149+170)
CAG CAG UAG UUG UCA UCU 




GCU C


 


79
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(+149+170)
CAG CAG UAG UUG UCA UCU 




GCU C


 


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




UGA


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




UUC AUC C


196
H53A(+150+175)
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
NUCELOTIDE SEQUENCE (5′-3′)





 80
H20A(+44+71)-
CUG GCA GAA UUC GAU CCA 




CCG GCU GUU C-


 82
H20A(+149+170)
CAG CAG UAG UUG UCA UCU 




GCU C


 


 81
H19A(+35+53)-
GCC UGA GCU GAU CUG  




CUG GCA UCU UGC AGU U


 88
H20A(+44+63)-
-AUU CGA UCU ACC GGC  




UGU UC-


 89
H20A(+149+168)
AA CUG CUG GCA UCU UGC 




AGU U


 


 80
H19A(+35+53)-
GCC UGA GCU GAU CUG CUG 




GCA UCU UGC AGU U


 88
H20A(+44+63)
-AUU CGA UCU ACC GGC 




UGU UC-


 


 80
H19A(+35+53)-
GCC UGA GCU GAU CUG CUG 




GCA UCU UGC 


 89
H20A(+149+168)
AGU U




-AA 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 



UU-
AUA CCU G- UU-


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




GU


 


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



AA-
UUC AUC C-


196
H53A(+150+
UGU AUA GGG ACC CUC CUU  



175)-AA-
CCA UGA CUC-AA-


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


—
Aimed at exons
UGA CAG CAG UAG UUG UCA



19/20/20
UCU GCU 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 variation 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) are 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 .PHI.m 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).
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 al. (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







Antisense

Ability 



Oligo-

to



nucleotide

induce



name
Sequence
skipping









H8A(−06+18)
5′-GAU AGG UGG  
Very




UAU CAA CAU 
strong




CUG UAA
to 20 nM



 



H8A(−03+18)
5′-GAU AGG UGG  
Very




UAU CAA CAU
strong




CUG
skipping





to 40 nM



 



H8A(−07+18)
5′-GAU AGG    
Strong




UGG UAU CAA 
skipping 




CAU CUG UAA G
to 40 nM



 



H8A(−06+14)
5′-GGU GGU    
Skipping




AUC AAC AUC
to 300 nM




UGU AA




 



H8A(−10+10)
5′-GUA UCA    
Patchy/




ACA UCU GUA 
weak




AGC AC
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





Antisense

Ability


Oligo-

to 


nucleotide

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 
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







Antisense 

Ability 



Oligo

to induce



name
Sequence
skipping









C6A(−10+10)
5′ CAU UUU UGA  
No 




CCU ACA UGU GG
skipping



 



C6A(−14+06)
5′ UUU GAC CUA  
No 




CAU GUG GAA AG
skipping



 



C6A(−14+12)
5′ UAC AUU UUU  
No 




GAC CUA CAU  
skipping




GUG GAA AG




 



C6A(−13+09)
5′ AUU UUU GAC  
No 




CUA CAU GGG  
skipping




AAA G




 



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




AUU GUC GGA  
skipping




CCC AG
to 20 nM



 



C6D(+12−13)
5′ GUG GUC UCC  
Weak  




UUA CCU AUG ACU
skipping




GUG G
at 300 nM



 



C6D(+06−11)
5′ GGU CUC CUU  
No 




ACC UAU GA
skipping



 



H6D(+04−21)
5′ UGU CUC AGU  
Weak  




AAU CUU CUU ACC
skipping




UAU
to 50 nM



 



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




UGA CUA UGG  
skipping




AUG AGA
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







Antisense

Ability 



Oligo-

to



nucleotide

induce



name
Sequence
skipping









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




CUU GUG GAU CC
to 20 nM



 



H4A(+11+40)
5′UGU UCA GGG  
Skipping 




CAU GAA CUC  
to 20 nM




UUG UGG AUC CUU




 



H4D(+04−16)
5′ CCA GGG UAC  
No skipping




UAC UUA CAU UA




 



H4D(−24−44)
5′ AUC GUG UGU  
No skipping




CAC AGC AUC CAG










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









Ability



Antisense

to



Oligonucleotide

induce



name
Sequence
skipping









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




CCC AUC CUG UAG  
skipping




GUC ACU G
to 20 to





600 nM



 



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




CGC CUC CCA UCC  
to




UGU AGG U
300 nM



 



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




CUG UAG GUC ACU 
100-600 nM




G




 



H3D(+46−21)
CUU CGA GGA GGU  
No skipping




CUA GGA GGC GCC 





UC




 



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




AGG UCA CUG
20-600 nM



 



H3D(+19−03)
UAC CAG UUU UUG  
No skipping




CCC UGU CAG G




 



H3A(−06+20)
UCA AUA UGC UGC  
No skipping




UUCCCA AAC UGA 





AA




 



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




UCC CAU CCU GUA 





G










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





Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping







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



ACG AUG UCA GUA CUU
100 nM



C



 


H5D(+25−05)
CUU ACC UGC CAG UGG 
No skipping



AGG AUU AUA UUC CAA




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 skipping



UCU



 


H5D(+16−02)
ACC UGC CAG UGG AGG 
No skipping



AUU



 


H5A(−07+20)
CCA AUA UUC ACU AAA 
No skipping



UCA ACC UGU UAA



 


H5D(+18−12)
CAG GAU UCU UAC CUG 
No skipping



CCA GUG GAG GAU UAU



 


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



CCA AUA UUC ACU AAA




U



 


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



GUC AGU ACU UCC AAU
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





Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping







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



GCU GCA



 


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



UUC CAA AUG CUG CA



 


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



AGC CAC G



 


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



UGU GC



 


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



ACA AU









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





Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping







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



AUC UUG AAU
at 100 nM


 


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



UGU UU
100 nM


 


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



UUC AUC UG
100 nM


 


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



UUC CUG UU
100 nM


 


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



AUC UUG AAU
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





Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping







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



AGC CAG UCA
5 nM


 


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



AAU UU
100 nM


 


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



AAA AC









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





Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping







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



CUC CAC UAG
at 5 nM


 


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



CAC CAU
skipping


 


H13D(+06−19)
CUA AGC AAA AUA AUC
No 



UGA 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





Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping







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



AGC GGU CUU CUG U
at 100 nM


 


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



GCC CAU C
skipping


 


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



AAA GAA CC
skipping


 


H14D(−02+18)
ACC UGU UCU UCA GUA
No



AGA CG
skipping


 


H14D(+14−10)
CAU GAC ACA CCU GUU
No



CUU CAG UAA
skipping


 


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



UCU UG
skipping


 


H14A(−12+12)
AUC UCC CAA UAC
No



CUG GAG 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





Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping







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



GGC ACU GCA AGA CAU
at 5 Nm



U



 


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



GUG UGA AUC
at 5 Nm


 


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



CAC UAA
skipping


 


H15A(−12+19)
GCC AUG CAC UAA AAA
No



GGC ACU GCA AGA CAU 
skipping



U



 


H15D(+17−08)
GUA CAU ACG GCC AGU
No



UUU 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





Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping







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



AAA CCU GUU AAA ACA
at 5 nM



A



 


H16A(−06+25)
UCU UUU CUA GAU
Skipping



CCG CUU UUA AAA
at 5 nM



CCU GUU A



 


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



AAA CCU GUU A
at 25 nM


 


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



CUG ACU UA
at 100 nM


 


H16A(−07+19)
CUA GAU CCG CUU UUA
No 



AAA CCU GUU AA
skipping


 


H16A(−07+13)
CCG CUU UUA AAA CCU
No 



GUU AA
skipping


 


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



UUU CUA GAU CC
skipping


 


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



UGG GUC ACU G
skipping


 


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



UGA AUA CAG U
skipping


 


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



UGC UUC CGU C
skipping


 


H16D(+05−20)
UGA UAA UUG GUA UCA
No 



CUA ACC UGU G
skipping


 


H16D(+12−11)
GUA UCA CUA ACC UGU
No 



GCU 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 H20(+149+170) [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:79], H20A(+44+71) [SEQ ID NO:81] and H20A(+149+170) [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







Antisense

Ability 



Oligo-

to



nucloetide

induce



name
Sequence
skipping









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




CCA CCG GCU GUU C
skipping



 



H20A(+149+170)
CAG CAG UAG UUG UCA
No 




UCU GCU C
skipping



 



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




UUG G
skipping



 



H20A(−08+17)
AUC UGC AUU AAC ACC
No 




CUC UAG AAA G
skipping



 



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




UUC UGA GGC
skipping



 



H20A(−11+17)
AUC UGC AUU AAC ACC
Not 




CUC UAG AAA GAA A
tested





yet



 



H20D(+08−20)
GAA GGA GAA GAG AUU
Not 




CUU ACC UUA CAA A
tested





yet



 



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




CCA CCG GCU GUU C
strong



H20A(+149+170)
CAG CAG UAG UUG UCA
skipping




UCU GCU C




 



H19A(+44+71):
GCC UGA GCU GAU
Very 




CUG CUG GCA UCU
strong




UGC AGU U
skipping



H20A(+44+71);
CUG GCA GAA UUC GAU





CCA CCG GCU GUU C




H20A(+149+170)
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





Antisense

Ability to


Oligonucleotide

induce


name
Sequence
skipping







H21A(−06+16)
GCC GGU UGA CUU
Skips at



CAU CCU GUG C
600 nM


 


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



ACA UGG GUC C
50 nM


 


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



GAA CAU GGG UC
50 nM


 


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



AUA GCC GGU UGA
faintly to


 


H21D(+18−07)
UAC UUA CUG UCU  
No 



GUA GCU 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





Antisense

Ability to


oligonucleotide

induce


name
Sequence
skipping







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



CCU GAU AGC GCA
skipping


 


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



GAG UCU CUG C
to 50 nM


 


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



AUG UCC UGA UG
to 300 nM


 


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



AUG GAG AGU
to 50 nM


 


H22D(+13−11)
UAU UCA CAG ACC  
No 



UGC AAU 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





Antisense

Ability to


oligonucleotide

induce


name
Sequence
skipping







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



UAG UAU AGG CC
skipping


 


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



CUC UUG C
Skipping


 


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



CUU 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





Antisense

Ability to


oligonucleotide

induce


name
Sequence
skipping







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



CCU UCA GA
testing


 


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



UGU 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





Antisense

Ability to


oligonucleotide

induce


name
Sequence
skipping







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



UGA AUA UCA CUG
testing


 


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



AUC CCA CUG AG
testing


 


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



CAC 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







Antisense

Ability



oligo-

to



nucleotide

induce



name
Sequence
skipping









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




UUC AUC UGG AGU
testing




U




 



H26A(−07+19)
CCU CCU UUC UGG   
Needs




CAU AGA CCU UCC
testing




AC




 



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




UCG UGC AUC UCU 
skipping




G
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





Antisense

Ability to


oligonucleotide

induce


name
Sequence
skipping







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



CUA CAG GUG G
testing


 


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



CUC UCU GA
skipping 




at 600 and




300 nM


 


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



GCA 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





Antisense

Ability to


oligonucleotide

induce


name
Sequence
skipping







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



CUU CCU AAG
skipping  




at 600  




and 300 nM


 


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



CUC CGC CAG GA
testing


 


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



UCA 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







Antisense

Ability to



oligonucleotide

induce



name
Sequence
skipping









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




UAG GGU CUG UGC 
testing




C




 



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




CUC UGA AUG UCG 
skipping at




C
600 and 





300 nM



 



H29D(+17−05)
CAU ACC UCU UCA  
v. strong




UGU AGU 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





Antisense




oligonucleo-

Ability to 


tide name
Sequence
induce skipping







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



CAC CUU GUC UG



 


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



AUG CUC UGU UC
skipping at




600 and 300 nM.


 


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



GAG GCA UU
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. H31 D(+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





Antisense

Ability to 


oligonucleo-

induce


tide name
Sequence
skipping







H31D(+06−18)
UUC UGA AAU AAC AUA 
Skipping to 300 



UAC CUG UGC
nM


 


H31D(+03−22)
UAG UUU CUG AAA UAA 
Skipping to 20 



CAU AUA CCU G
nM


 


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



AUU GGA



 


H31D(+04−20)
GUU UCU GAA AUA ACA 
Skipping to 300 



UAU ACC UGU
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





Antisense

Ability to 


oligonucleo-

induce


tide name
Sequence
skipping







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



CCA CA
300 nM


 


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



GAC UG



 


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



ACA UCU UG



 


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



CAA AAU UGG C
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





Antisense

Ability to 


oligonucleo-

induce


tide name
Sequence
skipping







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



GCU CC



 


H33A(+53+76)
UCU GUA CAA UCU GAC 
Skipping to 200 



GUC CAG UCU
nM


 


H33A(+30+56)
GUC UUU AUC ACC AUU 
Skipping to 200 



UCC ACU UCA GAC
nM


 


H33A(+64+88)
CCG UCU GCU UUU UCU 
Skipping to 10 



GUA CAA UCU G
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





Antisense

Ability to 


oligonucleo-

induce


tide name
Sequence
skipping







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



GCC AGC C



 


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



AUC CAA AU



 


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



AAG AAU UAU AAU GAA



 


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



GCA UCU UAC G
300 nM


 


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



UUC CAU AUC UG
300 nM


 


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



UUU ACC UUU CCC CAG



 


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



AUU CUG UCA AG









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





Antisense

Ability to 


oligonucleo-

induce


tide name
Sequence
skipping







H35A(+141+161)
UCU UCU GCU CGG GAG 
Skipping to 20 



GUG ACA
nM


 


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



AAG AC



 


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



UCU GU









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





Antisense

Ability to 


oligonucleo-

induce


tide name
Sequence
skipping







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



UUU GGG CGU A



 


H37A(+82+105)
UAC UAA UUU CCU GCA 
Skipping to 10 



GUG GUC ACC
nM


 


H37A(+134+157)
UUC UGU GUG AAA UGG 
Skipping to 10 



CUG CAA AUC
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





Antisense

Ability to 


oligonucleo-

induce


tide name
Sequence
skipping







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



AGG CC



 


H38A(+59+83)
UGC UGA AUU UCA GCC 
Skipping to 10 



UCC AGU GGU U
nM


 


H38A(+88+112)
UGA AGU CUU CCU CUU 
Skipping to 10 



UCA GAU UCA C
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





Antisense

Ability to 


oligonucleo-

induce


tide name
Sequence
skipping







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



CUG UGA UUC
100 nM


 


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



CUC UU



 


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



GCU GU



 


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



GAG CA
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





Antisense

Ability to 


oligonucleo-

induce


tide name
Sequence
skipping







H42A(−4+23)
AUC GUU UCU UCA CGG 
Skipping to 5 



ACA GUG UGC UGG
nM


 


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



GAG UGA UUU
100 nM


 


H42D(+19−02)
A CCU UCA GAG GAC 
Skipping to 5 



UCC UCU UGC
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





Antisense

Ability to


oligonucleo-

induce


tide name
Sequence
skipping







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



CUU GUC GGU C
100 nM


 


H43A(+101+120)
GGA GAG AGC UUC CUG 
Skipping to 25 



UAG CU
nM


 


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



GCG UUG CA
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





Antisense

Ability to


oligonucleo-

induce


tide name
Sequence
skipping







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



CAA GC



 


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



GAU AC



 


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



AAG UGG GAU ACU AGC
to 100 nM


 


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



GUU GCU GCU CUU UUC 
to 100 nM



C



 


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



CUA GCC UGG AGA AAG



 


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



AUA AAA CAA AUU C









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:181].









TABLE 37





Antisense

Ability to


oligonucleo-

induce


tide name
Sequence
skipping







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



UGA GUA GGA GC
skipping


 


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



UGA UGA UC
300 nM


 


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



GUU GAA AUC
testing


 


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



GCA UUU CUA GUU UGG
skipping


 


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



GCA UUU CUA G



 


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



AGA UGG CAU UUC UAG
skipping


 


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



ACC UUC UGC U



 


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


& (−15+?)
ACC UUC UGC UAG GAG 




CUA AAA



 


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



UCY GUG



 


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



UCC UCA A









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





Antisense

Ability to


oligonucleo-

induce


tide name
Sequence
skipping







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



UGU AAG



 


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



CUC UGU UCC AAA UCC
skipping


 


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



GUU CCA
50 nM


 


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



UAG CC



 


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



UUC GA









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:—H53D(+23+47) [SEQ ID NO:195], H53A(+150+175) [SEQ ID NO:196] and H53A(+14−07) [SEQ ID NO:194], were 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





Antisense

Ability to 


oligonucleo-

induce


tide name
Sequence
skipping







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



UCC GGU UCU G
at 50 nM


 


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



CUG AAG GUG
at 50 nM


 


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



UCC GGU UCU GAA GGU 
skipping



G
to 50 nM


 


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



CUG UGA
skipping to 50 




nM


 


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



UAC UUC AUC C
skipping to 50 




nM


 


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



CUU CCA UGA CUC
skipping to 50 




nM


 


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



GUG AUU UUC U



 


H53D(+09−18)
GGU AUC UUU GAU ACU 
Faint at 600 



AAC CUU GGU UUC
nM


 


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



AUA AAA G



 


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



CAA CUA GAA U



 


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



AAU UC



 


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



CCU AAG A

## Claims

1. An isolated antisense oligonucleotide of 25 to 50 nucleotides in length comprising SEQ ID NO: 180, wherein the uracil bases are optionally thymine bases.

2. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises a non-natural backbone.

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

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

5. The antisense oligonucleotide of claim 2, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties.

6. The antisense oligonucleotide of claim 5, wherein the non-natural moieties are morpholinos.

7. The antisense oligonucleotide of claim 6, wherein the uracil bases are thymine bases.

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

9. The antisense oligonucleotide of claim 8, wherein the non-natural inter-nucleotide linkages are modified phosphates.

10. The antisense oligonucleotide of claim 9, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

11. The antisense oligonucleotide of claim 10, wherein the modified phosphates are phosphoroamidates.

12. The antisense oligonucleotide of claim 10, wherein the modified phosphates are phosphoromorpholidates.

13. 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.

14. The antisense oligonucleotide of claim 13, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.

15. The antisense oligonucleotide of claim 14, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

16. The antisense oligonucleotide of claim 15, wherein the modified phosphates are phosphoroamidates.

17. The antisense oligonucleotide of claim 15, wherein the modified phosphates are phosphoromorpholidates.

18. The antisense oligonucleotide of claim 15, wherein the uracil bases are thymine bases.

19. The antisense oligonucleotide of claim 18, wherein the antisense oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.

20. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide is 25 nucleotides in length.

21. The antisense oligonucleotide of claim 20, wherein the uracil bases are thymine bases.

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

23. The pharmaceutical composition of claim 22, wherein the pharmaceutically acceptable carrier is phosphate-buffered saline.

24. A pharmaceutically acceptable salt of the antisense oligonucleotide of claim 1.