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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is
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 65^th and 85^th 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 internucleoside 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 lipo some 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, intra-peritoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Kits of the Invention

The invention also provides kits for treatment of a patient with a genetic disease which kit comprises at least an antisense molecule, packaged in a suitable container, together with instructions for its use.

In a preferred embodiment, the kits will contain at least one antisense molecule as shown in Table 1A, or a cocktail of antisense molecules as shown in Table 1B or a “weasel” compound as shown in Table 1C. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

Those of ordinary skill in the field should appreciate that applications of the above method has wide application for identifying antisense molecules suitable for use in the treatment of many other diseases.

EXAMPLES

The following Examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these Examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. The references cited herein are expressly incorporated by reference.

Methods of molecular cloning, immunology and protein chemistry, which are not explicitly described in the following examples, are reported in the literature and are known by those skilled in the art. General texts that described conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, included, for example: Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Glover ed., DNA Cloning: A Practical Approach, Volumes I and II, MRL Press, Ltd., Oxford, U.K. (1985); and Ausubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. Current Protocols in Molecular Biology. Greene Publishing Associates/Wiley Intersciences, New York (2002).

Determining Induced Exon Skipping in Human Muscle Cells

Attempts by the inventors to develop a rational approach in antisense molecules design were not completely successful as there did not appear to be a consistent trend that could be applied to all exons. As such, the identification of the most effective and therefore most therapeutic antisense molecules compounds has been the result of empirical studies.

These empirical studies involved the use of computer programs to identify motifs potentially involved in the splicing process. Other computer programs were also used to identify regions of the pre-mRNA which may not have had extensive secondary structure and therefore potential sites for annealing of antisense molecules. Neither of these approaches proved completely reliable in designing antisense oligonucleotides for reliable and efficient induction of exon skipping.

Annealing sites on the human dystrophin pre-mRNA were selected for examination, initially based upon known or predicted motifs or regions involved in splicing. 2OMe antisense oligonucleotides were designed to be complementary to the target sequences under investigation and were synthesised on an Expedite 8909 Nucleic Acid Synthesiser. Upon completion of synthesis, the oligonucleotides were cleaved from the support column and de-protected in ammonium hydroxide before being desalted. The quality of the oligonucleotide synthesis was monitored by the intensity of the trityl signals upon each deprotection step during the synthesis as detected in the synthesis log. The concentration of the antisense oligonucleotide was estimated by measuring the absorbance of a diluted aliquot at 260 nm.

Specified amounts of the antisense molecules were then tested for their ability to induce exon skipping in an in vitro assay, as described below.

Briefly, normal primary myoblast cultures were prepared from human muscle biopsies obtained after informed consent. The cells were propagated and allowed to differentiate into myotubes using standard culturing techniques. The cells were then transfected with the antisense oligonucleotides by delivery of the oligonucleotides to the cells as cationic lipoplexes, mixtures of antisense molecules or cationic liposome preparations.

The cells were then allowed to grow for another 24 hours, after which total RNA was extracted and molecular analysis commenced. Reverse transcriptase amplification (RT-PCR) was undertaken to study the targeted regions of the dystrophin pre-mRNA or induced exonic re-arrangements.

For example, in the testing of an antisense molecule for inducing exon 19 skipping the RT-PCR test scanned several exons to detect involvement of any adjacent exons. For example, when inducing skipping of exon 19, RT-PCR was carried out with primers that amplified across exons 17 and 21. Amplifications of even larger products in this area (i.e. exons 13-26) were also carried out to ensure that there was minimal amplification bias for the shorter induced skipped transcript. Shorter or exon skipped products tend to be amplified more efficiently and may bias the estimated of the normal and induced transcript.

The sizes of the amplification reaction products were estimated on an agarose gel and compared against appropriate size standards. The final confirmation of identity of these products was carried out by direct DNA sequencing to establish that the correct or expected exon junctions have been maintained.

Once efficient exon skipping had been induced with one antisense molecule, subsequent overlapping antisense molecules may be synthesized and then evaluated in the assay as described above. Our definition of an efficient antisense molecule is one that induces strong and sustained exon skipping at transfection concentrations in the order of 300 nM or less.

Antisense Oligonucleotides Directed at Exon 8

Antisense oligonucleotides directed at exon 8 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 3 shows differing efficiencies of two antisense molecules directed at exon 8 acceptor splice site. H8A(−06+18) [SEQ ID NO:1], which anneals to the last 6 bases of intron 7 and the first 18 bases of exon 8, induces substantial exon 8 and 9 skipping when delivered into cells at a concentration of 20 nM. The shorter antisense molecule, H8A(−06+14) [SEQ ID NO: 4] was only able to induce exon 8 and 9 skipping at 300 nM, a concentration some 15 fold higher than H8A(−06+18), which is the preferred antisense molecule.

This data shows that some particular antisense molecules induce efficient exon skipping while another antisense molecule, which targets a near-by or overlapping region, can be much less efficient. Titration studies show one compound is able to induce targeted exon skipping at 20 nM while the less efficient antisense molecules only induced exon skipping at concentrations of 300 nM and above. Therefore, we have shown that targeting of the antisense molecules to motifs involved in the splicing process plays a crucial role in the overall efficacy of that compound.

Efficacy refers to the ability to induce consistent skipping of a target exon. However, sometimes skipping of the target exons is consistently associated with a flanking exon. That is, we have found that the splicing of some exons is tightly linked. For example, in targeting exon 23 in the mouse model of muscular dystrophy with antisense molecules directed at the donor site of that exon, dystrophin transcripts missing exons 22 and 23 are frequently detected. As another example, when using an antisense molecule directed to exon 8 of the human dystrophin gene, all induced transcripts are missing both exons 8 and 9. Dystrophin transcripts missing only exon 8 are not observed.

Table 2 below discloses antisense molecule sequences that induce exon 8 (and 9) skipping.

TABLE 2
(SEQ ID NOS 1-5, respectively, in order of appearance)
Antisense
Oligonucleotide		Ability to induce
name	Sequence	skipping
H8A(−06 + 18)	5′-GAU AGG UGG UAU CAA CAU CUG UAA	Very strong to 20 nM
H8A(−03 + 18)	5′-GAU AGG UGG UAU CAA CAU CUG	Very strong skipping
		to 40n M
H8A(−07 + 18)	5′-GAU AGG UGG UAU CAA CAU CUG UAA G	Strong skipping
		to 40 nM
H8A(−06 + 14)	5′-GGU GGU AUC AAC AUC UGU AA	Skipping to 300 nM
H8A(−10 + 10)	5′-GUA UCA ACA UCU GUA AGC AC	Patchy/weak skipping
		to 100 nm

Antisense Oligonucleotides Directed at Exon 7

Antisense oligonucleotides directed at exon 7 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 4 shows the preferred antisense molecule, H7A(+45+67) [SEQ ID NO: 6], and another antisense molecule, H7A(+2+26) [SEQ ID NO: 7], inducing exon 7 skipping. Nested amplification products span exons 3 to 9. Additional products above the induced transcript missing exon 7 arise from amplification from carry-over outer primers from the RT-PCR as well as heteroduplex formation.

Table 3 below discloses antisense molecule sequences for induced exon 7 skipping.

TABLE 3
(SEQ ID NOS 6-9, respectively, in order of appearance)
Antisense
Oligonucleotide		Ability to induce
name	Sequence	skipping
H7A(+45 + 67)	5′-UGC AUG UUC CAG UCG UUG UGU GG	Strong skipping to
		20 nM
H7A(+02 + 26)	5′-CAC UAU UCC AGU CAA AUA GGU CUG G	Weak skipping at
		100 nM
H7D(+15−10)	5′-AUU UAC CAA CCU UCA GGA UCG AGU A	Weak skipping to
		300 nM
H7A(−18 + 03)	5′-GGC CUA AAA CAC AUA CAC AUA	Weak skipping to
		300 nM

Antisense Oligonucleotides Directed at Exon 6

Antisense oligonucleotides directed at exon 6 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 5 shows an example of two non-preferred antisense molecules inducing very low levels of exon 6 skipping in cultured human cells. Targeting this exon for specific removal was first undertaken during a study of the canine model using the oligonucleotides as listed in Table 4, below. Some of the human specific oligonucleotides were also evaluated, as shown in FIG. 5. In this example, both antisense molecules target the donor splice site and only induced low levels of exon 6 skipping. Both H6D(+4−21) [SEQ ID NO: 17] and H6D(+18-4) [SEQ ID NO: 18] would be regarded as non-preferred antisense molecules.

One antisense oligonucleotide that induced very efficient exon 6 skipping in the canine model, C6A(+69+91) [SEQ ID NO: 14], would anneal perfectly to the corresponding region in human dystrophin exon 6. This compound was evaluated, found to be highly efficient at inducing skipping of that target exon, as shown in FIG. 6 and is regarded as the preferred compound for induced exon 6 skipping. Table 4 below discloses antisense molecule sequences for induced exon 6 skipping.

TABLE 4
(SEQ ID NOS 10-18, respectively, in order of appearance)
Antisense
Oligo		Ability to induce
name	Sequence	skipping
C6A(−10 + 10)	5' CAU UUU UGA CCU ACA UGU GG	No skipping
C6A(−14 + 06)	5' UUU GAC CUA CAU GUG GAA AG	No skipping
C6A(−14 + 12)	5' UAC AUU UUU GAC CUA CAU GUG GAA AG	No skipping
C6A(−13 + 09)	5' AUU UUU GAC CUA CAU GGG AAA G	No skipping
CH6A(+69 + 91)	5' UAC GAG UUG AUU GUC GGA CCC AG	Strong skipping to
		20 nM
C6D(+12−13)	5' GUG GUC UCC UUA CCU AUG ACU GUG G	Weak skipping at
		300 nM
C6D(+06−11)	5' GGU CUC CUU ACC UAU GA	No skipping
H6D(+04−21)	5' UGU CUC AGU AAU CUU CUU ACC UAU	Weak skipping to
		50 nM
H6D(+18−04)	5' UCU UAC CUA UGA CUA UGG AUG AGA	Very weak skipping
		to 300 nM

Antisense Oligonucleotides Directed at Exon 4

Antisense oligonucleotides directed at exon 4 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 7 shows an example of a preferred antisense molecule inducing skipping of exon 4 skipping in cultured human cells. In this example, one preferred antisense compound, H4A(+13+32) [SEQ ID NO:19], which targeted a presumed exonic splicing enhancer induced efficient exon skipping at a concentration of 20 nM while other non-preferred antisense oligonucleotides failed to induce even low levels of exon 4 skipping. Another preferred antisense molecule inducing skipping of exon 4 was H4A(+111+40) [SEQ ID NO:22], which induced efficient exon skipping at a concentration of 20 nM.

Table 5 below discloses antisense molecule sequences for inducing exon 4 skipping.

TABLE 5
(SEQ ID NOS 19, 22, 20, and 21, respectively, in order of appearance)
Antisense
Oligonucleotide		Ability to induce
name	Sequence	skipping
H4A(+13 + 32)	5′ GCA UGA ACU CUU GUG GAU CC	Skipping to 20 nM
H4A(+11 + 40)	5′ UGU UCA GGG CAU GAA CUC UUG UGG AUC	Skipping to 20 nM
	CUU
H4D(+04−16)	5′ CCA GGG UAC UAC UUA CAU UA	No skipping
H4D(−24−44)	5′ AUC GUG UGU CAC AGC AUC CAG	No skipping

Antisense Oligonucleotides Directed at Exon 3

Antisense oligonucleotides directed at exon 3 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H3A(+30+60) [SEQ ID NO:23] induced substantial exon 3 skipping when delivered into cells at a concentration of 20 nM to 600 nM. The antisense molecule, H3A(+35+65) [SEQ ID NO: 24] induced exon skipping at 300 nM.

Table 6 below discloses antisense molecule sequences that induce exon 3 skipping.

TABLE 6
(SEQ ID NOS 23-30, respectively, in order of appearance)
Antisense
Oligonucleotide		Ability to induce
name	Sequence	skipping
H3A(+30 + 60)	UAG GAG GCG CCU CCC AUC CUG UAG GUC	Moderate skipping
	ACU G	to 20 to 600 nM
H3A(+35 + 65)	AGG UCU AGG AGG CGC CUC CCA UCC UGU	Working to 300 nM
	AGG U
H3A(+30 + 54)	GCG CCU CCC AUC CUG UAG GUC ACU G	Moderate 100 - 600 nM
H3D(+46−21)	CUU CGA GGA GGU CUA GGA GGC GCC UC	No skipping
H3A(+30 + 50)	CUC CCA UCC UGU AGG UCA CUG	Moderate 20 - 600 nM
H3D(+19−03)	UAC CAG UUU UUG CCC UGU CAG G	No skipping
H3A(−06 + 20)	UCA AUA UGC UGC UUCCCA AAC UGA AA	No skipping
H3A(+37 + 61)	CUA GGA GGC GCC UCC CAU CCU GUA G	No skipping

Antisense Oligonucleotides Directed at Exon 5

Antisense oligonucleotides directed at exon 5 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H5A(+20+50) [SEQ ID NO:31] induces substantial exon 5 skipping when delivered into cells at a concentration of 100 nM. Table 7 below shows other antisense molecules tested. The majority of these antisense molecules were not as effective at exon skipping as H5A(+20+50). However, H5A(+15+45) [SEQ ID NO: 40] was able to induce exon 5 skipping at 300 nM.

Table 7 below discloses antisense molecule sequences that induce exon 5 skipping.

TABLE 7
(SEQ ID NOS 31-40, respectively, in order of appearance)
Antisense		Ability
Oligonucleotide		to induce
name	Sequence	skipping
H5A(+20 + 50)	UUA UGA UUU CCA UCU ACG AUG UCA GUA	Working to
	CUU C	100 nM
H5D(+25−05)	CUU ACC UGC CAG UGG AGG AUU AUA UUC	No skipping
	CAA A
H5D(+10−15)	CAU CAG GAU UCU UAC CUG CCA GUG G	Inconsistent at
		300 nM
H5A(+10 + 34)	CGA UGU CAG UAC UUC CAA UAU UCA C	Very weak
H5D(−04−21)	ACC AUU CAU CAG GAU UCU	No skipping
H5D(+16−02)	ACC UGC CAG UGG AGG AUU	No skipping
H5A(−07 + 20)	CCA AUA UUC ACU AAA UCA ACC UGU UAA	No skipping
H5D(+18−12)	CAG GAU UCU UAC CUG CCA GUG GAG GAU	No skipping
	UAU
H5A(+05 + 35)	ACG AUG UCA GUA CUU CCA AUA UUC ACU	No skipping
	AAA U
H5A(+15 + 45)	AUU UCC AUC UAC GAU GUC AGU ACU UCC	Working to
	AAU A	300 nM

Antisense Oligonucleotides Directed at Exon 10

Antisense oligonucleotides directed at exon 10 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H10A(−05+16) [SEQ ID NO:41] induced substantial exon 10 skipping when delivered into cells. Table 8 below shows other antisense molecules tested. The antisense molecules ability to induce exon skipping was variable. Table 8 below discloses antisense molecule sequences that induce exon 10 skipping.

TABLE 8
(SEQ ID NOS 41-45, respectively, in order of appearance)
Antisense		Ability
Oligonucleotide		to induce
name	Sequence	skipping
H10A(−05 + 16)	CAG GAG CUU CCA AAU GCU GCA	Not tested
H10A(−05 + 24)	CUU GUC UUC AGG AGC UUC CAA AUG CUG	Not tested
	CA
H10A(+98 + 119)	UCC UCA GCA GAA AGA AGC CAC G	Not tested
H10A(+130 + 149)	UUA GAA AUC UCU CCU UGU GC	No skipping
H10A(−33−14)	UAA AUU GGG UGU UAC ACA AU	No skipping

Antisense Oligonucleotides Directed at Exon 11

Antisense oligonucleotides directed at exon 11 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 8B shows an example of H11A(+75+97) [SEQ ID NO:49] antisense molecule inducing exon 11 skipping in cultured human cells. H11A(+75+97) induced substantial exon 11 skipping when delivered into cells at a concentration of 5 nM. Table 9 below shows other antisense molecules tested. The antisense molecules ability to induce exon skipping was observed at 100 nM.

TABLE 9
(SEQ ID NOS 46-49 and 49, respectively,
in order of appearance)
Antisense		Ability
Oligo -		to
nucleotide		induce
name	Sequence	skipping
H11D	CCC UGA GGC AUU CCC AUC	Skipping
(+26 + 49)	UUG AAU	at
		100 nM
H11D	AGG ACU UAC UUG CUU UGU UU	Skipping
(+11−09)		at
		100 nM
H11A	CUU GAA UUU AGG AGA UUC	Skipping
(+118 + 140)	AUC UG	at
		100 nM
H11A	CAU CUU CUG AUA AUU UUC	Skipping
(+75 + 97)	CUG UU	at
		100 nM
H11A	CAU CUU CUG AUA AUU	Skipping
(+75 + 97)	UUC CUG UU	at 5 nM

Antisense Oligonucleotides Directed at Exon 12

Antisense oligonucleotides directed at exon 12 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H12A(+52+75) [SEQ ID NO:50] induced substantial exon 12 skipping when delivered into cells at a concentration of 5 nM, as shown in FIG. 8A. Table 10 below shows other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. The antisense molecules ability to induce exon skipping was variable.

TABLE 10
(SEQ ID NOS 50-52, respectively,
in order of appearance)
Antisense		Ability to
Oligonucleotide		induce
name	Sequence	skipping
H12A(+52 + 75)	UCU UCU GUU UUU GUU AGC	Skipping
	CAG UCA	at 5 nM
H12A(−10 + 10)	UCU AUG UAA ACU GAA AAU	Skipping
	UU	at 100 nM
H12A(+11 + 30)	UUC UGG AGA UCC AUU AAA	No
	AC	skipping

Antisense Oligonucleotides Directed at Exon 13

Antisense oligonucleotides directed at exon 13 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H13A(+77+100) [SEQ ID NO:53] induced substantial exon 13 skipping when delivered into cells at a concentration of 5 nM. Table 11 below includes two other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. These other antisense molecules were unable to induce exon skipping.

TABLE 11
(SEQ ID NOS 53-55, respectively,
in order of appearance)
Antisense		Ability to
Oligonucleotide		induce
name	Sequence	skipping
H13A(+77 + 100)	CAG CAG UUG CGU GAU CUC	Skipping at
	CAC UAG	5 nM
H13A(+55 + 75)	UUC AUC AAC UAC CAC CAC	No skipping
	CAU
H13D(+06 − 19)	CUA AGC AAA AUA AUC UGA	No skipping
	CCU UAA G

Antisense Oligonucleotides Directed at Exon 14

Antisense oligonucleotides directed at exon 14 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H14A(+37+64) [SEQ ID NO:56] induced weak exon 14 skipping when delivered into cells at a concentration of 100 nM. Table 12 below includes other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. The other antisense molecules were unable to induce exon skipping at any of the concentrations tested.

TABLE 12
(SEQ ID NOS 56-62, respectively,
in order of appearance)
Antisense		Ability to
Oligonucleotide		induce
name	Sequence	skipping
H14A(+37 + 64)	CUU GUA AAA GAA CCC AGC	Skipping at
	GGU CUU CUG U	100 nM
H14A(+14 + 35)	CAU CUA CAG AUG UUU GCC	No skipping
	CAU C
H14A(+51 + 73)	GAA GGA UGU CUU GUA AAA	No skipping
	GAA CC
H14D(-02 + 18)	ACC UGU UCU UCA GUA AGA	No skipping
	CG
H14D(+14 − 10)	CAU GAC ACA CCU GUU CUU	No skipping
	CAG UAA
H14A(+61 + 80)	CAU UUG AGA AGG AUG UCU	No skipping
	UG
H14A(−12 + 12)	AUC UCC CAA UAC CUG GAG	No skipping
	AAG AGA

Antisense Oligonucleotides Directed at Exon 15

Antisense oligonucleotides directed at exon 15 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

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

TABLE 13
(SEQ ID NOS 63-65, 63, and 66, respectively,
in order of appearance)
Antisense		Ability
Oligo-		to
nucleotide		induce
name	Sequence	skipping
H15A(−12 + 19)	GCC AUG CAC UAA AAA GGC	Skipping
	ACU GCA AGA CAU U	at 5 nM
H15A(+48 + 71)	UCU UUA AAG CCA GUU GUG	Skipping
	UGA AUC	at 5 nM
H15A(+08 + 28)	UUU CUG AAA GCC AUG CAC	No
	UAA	skipping
H15A(−12 + 19)	GCC AUG CAC UAA AAA GGC	Skipping
	ACU GCA AGA CAU U	at 10 nM
H15D(+17 − 08)	GUA CAU ACG GCC AGU UUU	No
	UGA AGA C	skipping

Antisense Oligonucleotides Directed at Exon 16

Antisense oligonucleotides directed at exon 16 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

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

TABLE 14
(SEQ ID NOS 67-78, respectively,
in order of appearance)
Antisense		Ability to
Oligonucleotide		induce
name	Sequence	skipping
H16A(−12 + 19)	CUA GAU CCG CUU UUA AAA	Skipping at
	CCU GUU AAA ACA A	5 nM
H16A(−06 + 25)	UCU UUU CUA GAU CCG CUU	Skipping at
	UUA AAA CCU GUU A	5 nM
H16A(−06 + 19)	CUA GAU CCG CUU UUA AAA	Skipping at
	CCU GUU A	25 nM
H16A(+87 + 109)	CCG UCU UCU GGG UCA CUG	Skipping at
	ACU UA	100 nM
H16A(−07 + 19)	CUA GAU CCG CUU UUA AAA	No skipping
	CCU GUU AA
H16A(−07 + 13)	CCG CUU UUA AAA CCU GUU	No skipping
	AA
H16A(+12 + 37)	UGG AUU GCU UUU UCU UUU	No skipping
	CUA GAU CC
H16A(+92 + 116)	CAU GCU UCC GUC UUC UGG	No skipping
	GUC ACU G
H16A(+45 + 67)	G AUC UUG UUU GAG UGA	No skipping
	AUA CAG U
H16A(+105 + 126)	GUU AUC CAG CCA UGC UUC	No skipping
	CGU C
H16D(+05 − 20)	UGA UAA UUG GUA UCA CUA	No skipping
	ACC UGU G
H16D(+12 − 11)	GUA UCA CUA ACC UGU GCU	No skipping
	GUA C

Antisense Oligonucleotides Directed at Exon 19

Antisense oligonucleotides directed at exon 19 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H19A(+35+65) [SEQ ID NO:79] induced substantial exon 19 skipping when delivered into cells at a concentration of 10 nM. This antisense molecule also showed very strong exon skipping at concentrations of 25, 50, 100, 300 and 600 nM.

FIG. 10 illustrates exon 19 and 20 skipping using a “cocktail” of antisense oligonucleotides, as tested using gel electrophoresis. It is interesting to note that it was not easy to induce exon 20 skipping using single antisense oligonucleotides H20A(+44+71) [SEQ ID NO:81] or H20A(+149+170) [SEQ ID NO:82], as illustrated in sections 2 and 3 of the gel shown in FIG. 10. Whereas, a “cocktail” of antisense oligonucleotides was more efficient as can be seen in section 4 of FIG. 10 using a “cocktail” of antisense oligonucleotides H20A(+44+71) and H20A(+149+170). When the cocktail was used to target exon 19, skipping was even stronger (see section 5, FIG. 10).

FIG. 11 illustrates gel electrophoresis results of exon 19/20 skipping using “weasels” The “weasels” were effective in skipping exons 19 and 20 at concentrations of 25, 50, 100, 300 and 600 nM. A further “weasel” sequence is shown in the last row of Table 3C. This compound should give good results.

Antisense Oligonucleotides Directed at Exon 20

Antisense oligonucleotides directed at exon 20 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

None of the antisense oligonucleotides tested induced exon 20 skipping when delivered into cells at a concentration of 10, 25, 50, 300 or 600 nM (see Table 15). Antisense molecules H20A(−11+17) [SEQ ID NO:86] and H20D(+08-20) [SEQ ID NO:87] are yet to be tested.

However, a combination or “cocktail” of H20A(+44+71) [SEQ ID NO: 81] and H20A(+147+168) [SEQ ID NO:82] in a ratio of 1:1, exhibited very strong exon skipping at a concentration of 100 nM and 600 nM. Further, a combination of antisense molecules H19A(+35+65) [SEQ ID NO:80], H20A(+44+71) [SEQ ID NO:81] and H20A(+147+168) [SEQ ID NO:82] in a ratio of 2:1:1, induced very strong exon skipping at a concentration ranging from 10 nM to 600 nM.

TABLE 15
(SEQ ID NOS 81-87, 81-82, and 80-82, respectively,
in order of appearance)
Antisense		Ability to
Oligonucleotide		induce
name	Sequence	skipping
H20A(+44 + 71)	CUG GCA GAA UUC GAU CCA CCG GCU GUU C	No skipping
H20A(+147 + 168)	CAG CAG UAG UUG UCA UCU GCU C	No skipping
H20A(+185 + 203)	UGA UGG GGU GGU GGG UUG G	No skipping
H20A(−08 + 17)	AUC UGC AUU AAC ACC CUC UAG AAA G	No skipping
H20A(+30 + 53)	CCG GCU GUU CAG UUG UUC UGA GGC	No skipping
H20A(−11 + 17)	AUC UGC AUU AAC ACC CUC UAG AAA GAA	Not tested
	A	yet
H20D(+08 − 20)	GAA GGA GAA GAG AUU CUU ACC UUA	Not tested
	CAA A	yet
H20A(+44 + 71) &	CUG GCA GAA UUC GAU CCA CCG GCU GUU	Very strong
	C	skipping
H20A(+147 + 168)	CAG CAG UAG UUG UCA UCU GCU C
H19A(+35 + 65);	GCC UGA GCU GAU CUG CUG GCA UCU UGC	Very strong
	AGU U;	skipping
H20A(+44 + 71);	CUG GCA GAA UUC GAU CCA CCG GCU GUU
	C;
H20A(+147 + 168)	CAG CAG UAG UUG UCA UCU GCU C

Antisense Oligonucleotides Directed at Exon 21

Antisense oligonucleotides directed at exon 21 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H21A(+85+108) [SEQ ID NO:92] and H21A(+85+106) [SEQ ID NO:91] induced exon 21 skipping when delivered into cells at a concentration of 50 nM. Table 16 below includes other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping

TABLE 16
(SEQ ID NOS 90-94, respectively,
in order of appearance)
Antisense		Ability to
Oligonucleotide		induce
name	Sequence	skipping
H21A(−06 + 16)	GCC GGU UGA CUU CAU CCU	Skips at
	GUG C	600 nM
H21A(+85 + 106)	CUG CAU CCA GGA ACA UGG	Skips at 50
	GUC C	nM
H21A(+85 + 108)	GUC UGC AUC CAG GAA CAU	Skips at 50
	GGG UC	nM
H21A(+08 + 31)	GUU GAA GAU CUG AUA GCC	Skips
	GGU UGA	faintly to
H21D(+18 − 07)	UAC UUA CUG UCU GUA GCU	No skipping
	CUU UCU

Antisense Oligonucleotides Directed at Exon 22

Antisense oligonucleotides directed at exon 22 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 12 illustrates differing efficiencies of two antisense molecules directed at exon 22 acceptor splice site. H22A(+125+106) [SEQ ID NO:96] and H22A(+80+101) [SEQ ID NO: 98] induce strong exon 22 skipping from 50 nM to 600 nM concentration.

H22A(+125+146) [SEQ ID NO:96] and H22A(+80+101) [SEQ ID NO:98] induced exon 22 skipping when delivered into cells at a concentration of 50 nM. Table 17 below shows other antisense molecules tested at a concentration range of 50, 100, 300 and 600 nM. These antisense molecules showed a variable ability to induce exon skipping.

TABLE 17
(SEQ ID NOS 95-99, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H22A(+22 + 45)	CAC UCA UGG UCU CCU GAU	No skipping
	AGC GCA
H22A(+125 + 146)	CUG CAA UUC CCC GAG UCU	Skipping to
	CUG C	50 nM
H22A(+47 + 69)	ACU GCU GGA CCC AUG UCC	Skipping to
	UGA UG	300 nM
H22A(+80 + 101)	CUA AGU UGA GGU AUG GAG	Skipping to
	AGU	50 nM
H22D(+13 - 11)	UAU UCA CAG ACC UGC AAU	No skipping
	UCC CC

Antisense Oligonucleotides Directed at Exon 23

Antisense oligonucleotides directed at exon 23 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

Table 18 below shows antisense molecules tested at a concentration range of 25, 50, 100, 300 and 600 nM. These antisense molecules showed no ability to induce exon skipping or are yet to be tested.

TABLE 18
(SEQ ID NOS 100-102, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H23A(+34 + 59)	ACA GUG GUG CUG AGA UAG	No skipping
	UAU AGG CC
H23A(+18 + 39)	UAG GCC ACU UUG UUG CUC	No Skipping
	UUG C
H23A(+72 + 90)	UUC AGA GGG CGC UUU CUU	No Skipping
	C

Antisense Oligonucleotides Directed at Exon 24

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

TABLE 19
(SEQ ID NOS 103-104, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H24A(+48 + 70)	GGG CAG GCC AUU CCU CCU	Needs
	UCA GA	testing
H24A(−02 + 22)	UCU UCA GGG UUU GUA UGU	Needs
	GAU UCU	testing

Antisense Oligonucleotides Directed at Exon 25

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

TABLE 20
(SEQ ID NOS 105-107, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		skipping
name	Sequence	induce
H25A(+9 + 36)	CUG GGC UGA AUU GUC UGA	Needs
	AUA UCA CUG	testing
H25A(+131 + 156)	CUG UUG GCA CAU GUG AUC	Needs
	CCA CUG AG	testing
H25D(+16 − 08)	GUC UAU ACC UGU UGG CAC	Needs
	AUG UGA	testing

Antisense Oligonucleotides Directed at Exon 26

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

TABLE 21
(SEQ ID NOS 108-110, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H26A(+132 + 156)	UGC UUU CUG UAA UUC AUC	Needs
	UGG AGU U	testing
H26A(−07 + 19)	CCU CCU UUC UGG CAU AGA	Needs
	CCU UCC AC	testing
H26A(+68 + 92)	UGU GUC AUC CAU UCG UGC	Faint
	AUC UCU G	skipping
		at 600 nM

Antisense Oligonucleotides Directed at Exon 27

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

TABLE 22
(SEQ ID NOS 111-113, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H27A(+82 + 106)	UUA AGG CCU CUU GUG CUA	Needs
	CAG GUG G	testing
H27A(−4 + 19)	GGG CCU CUU CUU UAG CUC	Faint
	UCU GA	skipping
		at 600 and
		300 nM
H27D(+19 − 03)	GAC UUC CAA AGU CUU GCA	v. strong
	UUU C	skipping
		at 600 and
		300 nM

Antisense Oligonucleotides Directed at Exon 28

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

TABLE 23
(SEQ ID NOS 114-116, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H28A(−05 + 19)	GCC AAC AUG CCC AAA CUU	v. strong
	CCU AAG	skipping at
		600 and
		300 nM
H28A(+99 − 124)	CAG AGA UUU CCU CAG CUC	Needs
	CGC CAG GA	testing
H28D(+16 − 05)	CUU ACA UCU AGC ACC UCA	v. strong
	GAG	skipping at
		600 and
		300 nM

Antisense Oligonucleotides Directed at Exon 29

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

TABLE 24
(SEQ ID NOS 117-119, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H29A(+57 + 81)	UCC GCC AUC UGU UAG GGU	Needs
	CUG UGC C	testing
H29A(+18 + 42)	AUU UGG GUU AUC CUC UGA	v. strong
	AUG UCG C	skipping at
		600 and
		300 nM
H29D(+17 − 05)	CAU ACC UCU UCA UGU AGU	v. strong
	UCC C	skipping at
		600 and
		300 nM

Antisense Oligonucleotides Directed at Exon 30

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

TABLE 25
(SEQ ID NOS 120-122, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H30A(+122+147)	CAU UUG AGC UGC	Needs testing
	GUC CAC CUU GUC UG
H30A(+25+50)	UCC UGG GCA GAC	Very strong
	UGG AUG CUC UGU UC	skipping at
		600 and 300
		nM.
H30D(+19−04)	UUG CCU GGG CUU	Very strong
	CCU 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
(SEQ ID NOS 123-126, respectively,
in order of appearance)
	Antisense		Ability to
	oligonucleotide		induce
	name	Sequence	skipping
	H31D(+06−18)	UUC UGA AAU AAC	Skipping
		AUA UAC CUG UGC	to 300 nM
	H31D(+03−22)	UAG UUU CUG AAA	Skipping
		UAA CAU AUA CCU G	to 20 nM
	H31A(+05+25)	GAC UUG UCA AAU	No skipping
		CAG AUU GGA
	H31D(+04−20)	GUU UCU GAA AUA	Skipping
		ACA UAU ACC UGU	to 300 nM

Antisense Oligonucleotides Directed at Exon 32

Antisense oligonucleotides directed at exon 32 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H32D(+04−16) [SEQ ID NO:127] and H32A(+49+73) [SEQ ID NO:130] induced exon 32 skipping when delivered into cells at a concentration of 300 nM. Table 27 below also shows other antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules did not show an ability to induce exon skipping.

TABLE 27
(SEQ ID NOS 127-130, respectively,
in order of appearance)
	Antisense		Ability to
	oligonucleotide		induce
	name	Sequence	skipping
	H32D(+04−16)	CAC CAG AAA UAC	Skipping to
		AUA CCA CA	300 nM
	H32A(+151+170)	CAA UGA UUU AGC	No skipping
		UGU GAC UG
	H32A(+10+32)	CGA AAC UUC AUG	No skipping
		GAG ACA UCU UG
	H32A(+49+73)	CUU GUA GAC GCU	Skipping to
		GCU 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
(SEQ ID NOS 131-134,
respectively, in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H33D(+09−11)	CAU GCA CAC ACC	No skipping
	UUU GCU CC
H33A(+53+76)	UCU GUA CAA UCU	Skipping to
	GAC GUC CAG UCU	200 nM
H33A(+30+56)	GUC UUU AUC ACC	Skipping to
	AUU UCC ACU UCA GAC	200 nM
H33A(+64+88)	CCG UCU GCU UUU	Skipping to
	UCU GUA CAA UCU G	10 nM

Antisense Oligonucleotides Directed at Exon 34

Antisense oligonucleotides directed at exon 34 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

Table 29 below includes antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping.

TABLE 29
(SEQ ID NOS 135-141, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H34A(+83+104)	UCC AUA UCU GUA	No skipping
	GCU GCC AGC C
H34A(+143+165)	CCA GGC AAC UUC	No skipping
	AGA 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	Skipping to
	UUC GCA UCU UAC G	300 nM
H34A(+95+120)	UGA UCU CUU UGU	Skipping to
	CAA 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
(SEQ ID NOS 142-144, respectively,
in order of appearance)
	Antisense		Ability to
	oligonucleotide		induce
	name	Sequence	skipping
	H35A(+141+161)	UCU UCU GCU CGG	Skipping to
		GAG GUG ACA	20 nM
	H35A(+116+135)	CCA GUU ACU AUU	No skipping
		CAG AAG AC
	H35A(+24+43)	UCU UCA GGU GCA	No skipping
		CCU 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
(SEQ ID NOS 147-149, respectively,
in order of appearance)
	Antisense		Ability to
	oligonucleotide		induce
	name	Sequence	skipping
	H37A(+26+50)	CGU GUA GAG UCC	No skipping
		ACC UUU GGG CGU A
	H37A(+82+105)	UAC UAA UUU CCU	Skipping to
		GCA GUG GUC ACC	10 nM
	H37A(+134+157)	UUC UGU GUG AAA	Skipping to
		UGG CUG CAA AUC	10 nM

Antisense Oligonucleotides Directed at Exon 38

Antisense oligonucleotides directed at exon 38 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 18 illustrates antisense molecule H38A(+88+112) [SEQ ID NO:152], directed at exon 38 acceptor splice site. H38A(+88+112) substantially induced exon 38 skipping when delivered into cells at a concentration of 10 nM. Table 32 below shows the antisense molecules tested and their ability to induce exon skipping.

TABLE 32
(SEQ ID NOS 150-152, respectively,
in order of appearance)
	Antisense		Ability to
	oligonucleotide		induce
	name	Sequence	skipping
	H38A(−01+19)	CCU UCA AAG GAA	No skipping
		UGG AGG CC
	H38A(+59+83)	UGC UGA AUU UCA	Skipping to
		GCC UCC AGU GGU U	10 nM
	H38A(+88+112)	UGA AGU CUU CCU	Skipping to
		CUU UCA GAU UCA C	10 nM

Antisense Oligonucleotides Directed at Exon 39

Antisense oligonucleotides directed at exon 39 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H39A(+62+85) [SEQ ID NO:153] induced exon 39 skipping when delivered into cells at a concentration of 100 nM. Table 33 below shows the antisense molecules tested and their ability to induce exon skipping.

TABLE 33
(SEQ ID NOS 153-156, respectively,
in order of appearance)
	Antisense		Ability to
	oligonucleotide		induce
	name	Sequence	skipping
	H39A(+62+85)	CUG GCU UUC UCU	Skipping to
		CAU CUG UGA UUC	100 nM
	H39A(+39+58)	GUU GUA AGU UGU	No skipping
		CUC CUC UU
	H39A(+102+121)	UUG UCU GUA ACA	No skipping
		GCU GCU GU
	H39D(+10−10)	GCU CUA AUA CCU	Skipping to
		UGA 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
(SEQ ID NOS 159-160,
respectively, in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H42A(−4+23)	AUC GUU UCU UCA CGG	Skipping to
	ACA GUG UGC UGG	5 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
	UCC UCU UGC	5 nM

Antisense Oligonucleotides Directed at Exon 43

Antisense oligonucleotides directed at exon 43 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H43A(+101+120) [SEQ ID NO:163] induced exon 43 skipping when delivered into cells at a concentration of 25 nM. Table 35 below includes the antisense molecules tested and their ability to induce exon 43 skipping.

TABLE 35
(SEQ ID NOS 162-164, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H43D(+10−15)	UAU GUG UUA CCU ACC	Skipping
	CUU GUC GGU C	to 100 nM
H43A(+101+120)	GGA GAG AGC UUC CUG	Skipping
	UAG CU	to 25 nM
H43A(+78+100)	UCA CCC UUU CCA CAG	Skipping
	GCG UUG CA	to 200 nM

Antisense Oligonucleotides Directed at Exon 44

Antisense oligonucleotides directed at exon 44 were prepared using similar methods as described above. Testing for the ability of these antisense molecules to induce exon 44 skipping is still in progress. The antisense molecules under review are shown as SEQ ID Nos: 165 to 167 in Table 1A.

Antisense Oligonucleotides Directed at Exon 45

Antisense oligonucleotides directed at exon 45 were prepared using similar methods as described above. Testing for the ability of these antisense molecules to induce exon 45 skipping is still in progress. The antisense molecules under review are shown as SEQ ID Nos: 207 to 211 in Table 1A.

Antisense Oligonucleotides Directed at Exon 46

Antisense oligonucleotides directed at exon 46 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 21 illustrates the efficiency of one antisense molecule directed at exon 46 acceptor splice site. Antisense oligonucleotide H46A(+86+115) [SEQ ID NO:203] showed very strong ability to induce exon 46 skipping. Table 36 below includes antisense molecules tested. These antisense molecules showed varying ability to induce exon 46 skipping.

TABLE 36
(SEQ ID NOS 168-169 and 203-206, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H46D(+16−04)	UUA CCU UGA CUU	No skipping
	GCU CAA GC
\
H46A(+90+109)	UCC AGG UUC AAG	No skipping
	UGG GAU AC
H46A(+86+115)	CUC UUU UCC AGG	Good skipping
	UUC AAG UGG GAU	to 100 nM
	ACU AGC
H46A(+107+137)	CAA GCU UUU CUU	Good skipping
	UUA GUU GCU GCU	to 100 nM
	CUU UUC C
H46A(−10+20)	UAU UCU UUU GUU	Weak skipping
	CUU CUA GCC UGG
	AGA AAG
H46A(+50+77)	CUG CUU CCU CCA	Weak skipping
	ACC 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 skipping. The strongest inducers of exon skipping were antisense oligonucleotide H51A(+61+90) [SEQ ID NO: 179] and H51A(+66+95) [SEQ ID NO: 179].

TABLE 37
(SEQ ID NOS 176-185, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H51A(−01 +25)	ACC AGA GUA ACA GUC	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
	ACC UUC UGC U	skipping
H51A/D(+08 −17) &	AUC AUU UUU UCU CAU	No
(−15 +?)	ACC UUC UGC UAG GAG	skipping
	CUA AAA
H51A(+175 +195)	CAC CCA CCA UCA GCC	No
	UCU GUG	skipping
H51A(+199 +220)	AUC AUC UCG UUG AUA	No
	UCC UCA A	skipping

Antisense Oligonucleotides Directed at Exon 52

Antisense oligonucleotides directed at exon 52 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 22 also shows differing efficiencies of four antisense molecules directed at exon 52 acceptor splice site. The most effective antisense oligonucleotide for inducing exon 52 skipping was H52A(+17+37) [SEQ ID NO:188].

Table 38 below shows antisense molecules tested at a concentration range of 50, 100, 300 and 600 nM. These antisense molecules showed varying ability to induce exon 50 skipping. Antisense molecules H52A(+12+41) [SEQ ID NO:187] and H52A(+17+37) [SEQ ID NO:188] showed the strongest exon 50 skipping at a concentration of 50 nM.

TABLE 38
(SEQ ID NOS 186-190, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H52A(−07+14)	UCC UGC AUU GUU GCC	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: H53A(+23+47) [SEQ ID NO:195], H53A(+150+176) [SEQ ID NO:196] and H53D (+14-07) [SEQ ID NO:194], was also tested, as shown in FIG. 20 and exhibited an ability to induce exon skipping.

Table 39 below includes other antisense molecules tested at a concentration range of 50, 100, 300 and 600 nM. These antisense molecules showed varying ability to induce exon 53 skipping. Antisense molecule H53A(+39+69) [SEQ ID NO:193] induced the strongest exon 53 skipping.

TABLE 39
(SEQ ID NOS 191-202, respectively,
in order of appearance)
Antisense		Ability to
oligonucleotide		induce
name	Sequence	skipping
H53A(+45+69)	CAU UCA ACU GUU	Faint
	GCC UCC GGU UCU G	skipping
		at 50 nM
H53A(+39+62)	CUG UUG CCU CCG	Faint
	GUU CUG AAG GUG	skipping
		at 50 nM
H53A(+39+69)	CAU UCA ACU GUU	Strong
	GCC UCC GGU UCU	skipping
	GAA GGU G	to 50 nM
H53D(+14−07)	UAC UAA CCU UGG	Very faint
	UUU CUG UGA	skipping
		to 50 nM
H53A(+23+47)	CUG AAG GUG UUC	Very faint
	UUG UAC UUC AUC C	skipping
		to 50 nM
H53A(+150+176)	UGU AUA GGG ACC	Very faint
	CUC CUU CCA UGA CUC	skipping
		to 50 nM
H53D(+20−05)	CUA ACC UUG GUU	Not made
	UCU GUG AUU UUC U	yet
H53D(+09−18)	GGU AUC UUU GAU	Faint
	ACU AAC CUU GGU UUC	at 600 nM
H53A(−12+10)	AUU CUU UCA ACU	No skipping
	AGA AUA AAA G
H53A(−07+18)	GAU UCU GAA UUC	No skipping
	UUU CAA CUA GAA U
H53A(+07+26)	AUC CCA CUG AUU	No skipping
	CUG AAU UC
H53A(+124+145)	UUG GCU CUG GCC	No skipping
	UGU CCU AAG A

Claims

1. A method of inducing skipping of exon 51 in a dystrophin gene in a subject comprising administering a pharmaceutical composition comprising an antisense oligonucleotide of 30 to 50 nucleotides in length comprising SEQ ID NO: 181, wherein the uracil bases are optionally thymine bases, and a pharmaceutically acceptable carrier.

2. The method of claim 1, wherein the antisense oligonucleotide comprises SEQ ID NO:181.

3. The method of claim 1, wherein the antisense oligonucleotide consists of SEQ ID NO:181 and wherein the uracil bases are thymine bases.

4. The method of claim 1, wherein the subject is a human.

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

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

7. The method of claim 1, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties.

8. The method of claim 7, wherein the non-natural moieties are morpholinos.

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

10. The method of claim 9, wherein the non-natural inter-nucleotide linkages are modified phosphates.

11. The method 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.

12. The method of claim 11, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.

13. The method of claim 12, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

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

15. The method of claim 1, wherein the oligonucleotide is a peptide nucleic acid.

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

17. The method of claim 1, wherein the oligonucleotide is conjugated to a polyamine.

18. The method of claim 1, wherein the oligonucleotide is chemically linked to a polyethylene glycol chain.

19. A method of correcting a defective gene for dystrophin in a subject comprising administering a pharmaceutical composition comprising an antisense oligonucleotide of 30 to 50 nucleotides in length comprising SEQ ID NO: 181, wherein the uracil bases are optionally thymine bases, and a pharmaceutically acceptable carrier.

20. The method of claim 19 wherein the antisense oligonucleotide comprises SEQ ID NO:181.

21. The method of claim 19, wherein the antisense oligonucleotide consists of SEQ ID NO:181, and wherein the uracil bases are thymine bases.

22. A method of restoring or increasing functional dystrophin protein production in a subject comprising administering a pharmaceutical composition comprising an antisense oligonucleotide of 30 to 50 nucleotides in length comprising SEQ ID NO: 181, wherein the uracil bases are optionally thymine bases, and a pharmaceutically acceptable carrier.

23. The method of claim 22, wherein the antisense oligonucleotide comprises SEQ ID NO:181.

24. The method of claim 22, wherein the antisense oligonucleotide consists of SEQ ID NO:181, and wherein the uracil bases are thymine bases.

25. A method of treating muscular dystrophy associated with a defective gene for dystrophin in a subject comprising administering a pharmaceutical composition comprising an antisense oligonucleotide of 30 to 50 nucleotides in length comprising SEQ ID NO: 181, wherein the uracil bases are optionally thymine bases, and a pharmaceutically acceptable carrier.

26. The method of claim 25, wherein the antisense oligonucleotide comprises SEQ ID NO:181.

27. The method of claim 25, wherein the antisense oligonucleotide consists of SEQ ID NO:181, and wherein the uracil bases are thymine bases.

28. The method of claim 25, wherein the subject is a human and the muscular dystrophy is Becker muscular dystrophy.

29. The method of claim 25, wherein the subject is a human and the muscular dystrophy is duchenne muscular dystrophy.

30. The method of claim 1, wherein the pharmaceutical carrier is phosphate buffered saline.

31. The method of claim 1, further comprising administering a steroid to the subject.

32. A method of inducing skipping of exon 51 in a dystrophin gene in a human patient comprising administering an injectable solution comprising:

an antisense oligonucleotide of 30 nucleotides in length comprising the base sequence 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 181), in which the uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain;

and phosphate-buffered saline,

wherein the injectable solution is formulated for intravenous administration.

33. A method of correcting a defective gene for dystrophin in a human patient comprising administering an injectable solution comprising:

an antisense oligonucleotide of 30 nucleotides in length comprising the base sequence 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 181), in which the uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain;

and phosphate-buffered saline,

wherein the injectable solution is formulated for intravenous administration.

34. A method of restoring or increasing functional dystrophin protein production in a human patient comprising administering an injectable solution comprising:

an antisense oligonucleotide of 30 nucleotides in length comprising the base sequence 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 181), in which the uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain;

and phosphate-buffered saline,

wherein the injectable solution is formulated for intravenous administration.

35. A method of treating duchenne muscular dystrophy in a human patient comprising administering an injectable solution comprising:

an antisense oligonucleotide of 30 nucleotides in length comprising the base sequence 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 181), in which the uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain;

and phosphate-buffered saline,

wherein the injectable solution is formulated for intravenous administration.