antisense molecules capable of binding to a selected target site in the dystrophin gene to induce exon skipping are described.
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0. 27. 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 parenteral administration.
0. 24. 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.
0. 33. 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 subcutaneous administration.
0. 30. 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 intramuscular administration.
0. 25. 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
a pharmaceutically acceptable carrier or diluent;
wherein the injectable solution is formulated for parenteral administration.
0. 22. 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
a pharmaceutically acceptable carrier or diluent;
wherein the injectable solution is formulated for intravenous administration.
0. 31. 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
a pharmaceutically acceptable carrier or diluent;
wherein the injectable solution is formulated for subcutaneous administration.
0. 28. 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
a pharmaceutically acceptable carrier or diluent;
wherein the injectable solution is formulated for intramuscular administration.
0. 1. An isolated antisense oligonucleotide of 30 to 50 nucleotides in length comprising SEQ ID NO: 181, wherein the uracil bases are optionally thymine bases.
0. 2. The antisense oligonucleotide of
0. 3. The antisense oligonucleotide of
0. 4. The antisense oligonucleotide of
0. 5. The antisense oligonucleotide of
0. 6. The antisense oligonucleotide of
0. 7. The antisense oligonucleotide of
0. 8. The antisense oligonucleotide of
0. 9. The antisense oligonucleotide of
0. 10. The antisense oligonucleotide of
0. 11. The antisense oligonucleotide of
0. 12. The antisense oligonucleotide of
0. 13. The antisense oligonucleotide of
0. 14. The antisense oligonucleotide of
0. 15. The antisense oligonucleotide of
0. 16. The antisense oligonucleotide of
0. 17. The antisense oligonucleotide of
0. 18. The antisense oligonucleotide of
0. 19. The antisense oligonucleotide of
0. 20. The antisense oligonucleotide of
0. 21. The antisense oligonucleotide of
0. 23. The injectable solution of claim 22, wherein the pharmaceutically acceptable carrier or diluent comprises an isotonic saline solution.
0. 26. The injectable solution of claim 25, wherein the pharmaceutically acceptable carrier or diluent comprises an isotonic saline solution.
0. 29. The injectable solution of claim 28, wherein the pharmaceutically acceptable carrier or diluent comprises an isotonic saline solution.
0. 32. The injectable solution of claim 31, wherein the pharmaceutically acceptable carrier or diluent comprises an isotonic saline solution.
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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.
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
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-5-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 US patent 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, 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.
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.
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
Anti-
sense
Oligo-
Ability
nucle-
to
otide
induce
name
Sequence
skipping
H8A
5′-GAU AGG UGG UAU CAA CAU CUG UAA
Very
(−06 +
strong
18)
to 20 nM
H8A
5′-GAU AGG UGG UAU CAA CAU CUG
Very
(−03 +
strong
18)
skipping
to 40 nM
H8A
5′-GAU AGG UGG UAU CAA CAU CUG UAA G
Strong
(−07 +
skipping
18)
to 40 nM
H8A
5′-GGU GGU AUC AAC AUC UGU AA
Skipping
(−06 +
to 300
14)
nM
H8A
5′-GUA UCA ACA UCU GUA AGC AC
Patchy/
(−10 +
weak
10)
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.
Table 3 below discloses antisense molecule sequences for induced exon 7 skipping.
TABLE 3
Anti-
sense
Abili-
Oligo-
ty to
nucle-
induce
otide
skip-
name
Sequence
ping
H7A
5′-UGC AUG UUC CAG UCG UUG UGU GG
Strong
(+45 +
skip-
67)
ping
to 20
nM
H7A
5′-CAC UAU UCC AGU CAA AUA GGU
Weak
(+02 +
CUG G
skip-
26)
ping
at 100
nM
H7D
5′-AUU UAC CAA CCU UCA GGA UCG
Weak
(+15 −
AGU A
skip-
10)
ping
to 300
nM
H7A
5′-GGC CUA AAA CAC AUA CAC AUA
Weak
(−18 +
skip-
03)
ping
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.
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
TABLE 4
Anti-
sense
Ability to
Oligo
induce
name
Sequence
skipping
C6A
5′ CAU UUU UGA CCU ACA UGU GG
No skipping
(−10 +
10)
C6A
5′ UUU GAC CUA CAU GUG GAA AG
No skipping
(−14 +
06)
C6A
5′ UAC AUU UUU GAC CUA CAU GUG
No skipping
(−14 +
GAA AG
12)
C6A
5′ AUU UUU GAC CUA CAU GGG
No skipping
(−13 +
AAA G
09)
CH6A
5′ UAC GAG UUG AUU GUC GGA CCC
Strong
(+69 +
AG
skipping to
91)
20 nM
C6D
5′ GUG GUC UCC UUA CCU AUG ACU
Weak skip-
(+12 −
GUG G
ping at
13)
300 nM
C6D
5′ GGU CUC CUU ACC UAU GA
No skipping
(+06 −
11)
H6D
5′ UGU CUC AGU AAU CUU CUU ACC
Weak skip-
(+04 −
UAU
ping to
21)
50 nM
H6D
5′ UCU UAC CUA UGA CUA UGG AUG
Very weak
(+18 −
AGA
skipping
04)
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.
Table 5 below discloses antisense molecule sequences for inducing exon 4 skipping.
TABLE 5
Anti-
sense
Oligo-
Ability
nucle-
to
otide
induce
name
Sequence
skipping
H4A
5′ GCA UGA ACU CUU GUG GAU CC
Skipping
(+13 +
to 20
32)
nM
H4A
5′UGU UCA GGG CAU GAA CUC UUG
Skipping
(+11 +
UGG AUC CUU
to 20
40)
nM
H4D
5′ CCA GGG UAC UAC UUA CAU UA
No
(+04 −
skipping
16)
H4D
5′ AUC GUG UGU CAC AGC AUC CAG
No
(−24 −
skipping
44)
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
Anti-
sense
Oligo-
Ability
nucleo-
to
tide
induce
name
Sequence
skipping
H3A
UAG GAG GCG CCU CCC AUC CUG UAG
Moderate
(+30 +
GUC ACU G
skipping
60)
to 20 to
600 nM
H3A
AGG UCU AGG AGG CGC CUC CCA UCC
Working
(+35 +
UGU AGG U
to 300
65)
nM
H3A
GCG CCU CCC AUC CUG UAG GUC ACU G
Moderate
(+30 +
100-600
54)
nM
H3D
CUU CGA GGA GGU CUA GGA GGC GCC UC
No
(+46 −
skipping
21)
H3A
CUC CCA UCC UGU AGG UCA CUG
Moderate
(+30 +
20-600
50)
nM
H3D
UAC CAG UUU UUG CCC UGU CAG G
No
(+19 −
skipping
03)
H3A
UCA AUA UGC UGC UUCCCA AAC UGA AA
No
(−06 +
skipping
20)
H3A
CUA GGA GGC GCC UCC CAU CCU GUA G
No
(+37 +
skipping
61)
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
Anti-
sense
Oligo-
Ability
nucle-
to
otide
induce
name
Sequence
skipping
H5A
UUA UGA UUU CCA UCU ACG AUG UCA
Working
(+20 +
GUA CUU C
to 100
50)
nM
H5D
CUU ACC UGC CAG UGG AGG AUU AUA
No
(+25 −
UUC CAA A
skipping
05)
H5D
CAU CAG GAU UCU UAC CUG CCA GUG G
Incon-
(+10 −
sistent
15)
at 300
nM
H5A
CGA UGU CAG UAC UUC CAA UAU UCA C
Very
(+10 +
weak
34)
H5D
ACC AUU CAU CAG GAU UCU
No
(−04 −
skipping
21)
H5D
ACC UGC CAG UGG AGG AUU
No
(+16 −
skipping
02)
H5A
CCA AUA UUC ACU AAA UCA ACC UGU
No
(−07 +
UAA
skipping
20)
H5D
CAG GAU UCU UAC CUG CCA GUG GAG
No
(+18 −
GAU UAU
skipping
12)
H5A
ACG AUG UCA GUA CUU CCA AUA UUC
No
(+05 +
ACU AAA U
skipping
35)
H5A
AUU UCC AUC UAC GAU GUC AGU ACU
Working
(+15 +
UCC AAU A
to 300
45)
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
Anti-
sense
Oligo-
Ability
nucle-
to
otide
induce
name
Sequence
skipping
H10A
CAG GAG CUU CCA AAU GCU GCA
Not
(−05 +
tested
16)
H10A
CUU GUC UUC AGG AGC UUC CAA AUG
Not
(−05 +
CUG CA
tested
24)
H10A
UCC UCA GCA GAA AGA AGC CAC G
Not
(+98 +
tested
119)
H10A
UUA GAA AUC UCU CCU UGU GC
No
(+130 +
skipping
149)
H10A
UAA AUU GGG UGU UAC ACA AU
No
(−33 −
skipping
14)
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.
TABLE 9
Anti-
sense
Oligonu-
Ability to
cleotide
induce
name
Sequence
skipping
H11D
CCC UGA GGC AUU CCC AUC UUG AAU
Skipping at
(+26 +
100 nM
49)
H11D
AGG ACU UAC UUG CUU UGU UU
Skipping at
(+11 −
100 nM
09)
H11A
CUU GAA UUU AGG AGA UUC AUG UG
Skipping at
(+118 +
100 nM
140)
H11A
CAU CUU CUG AUA AUU UUC CUG UU
Skipping at
(+75 +
100 nM
97)
H11D
CCC UGA GGC AUU CCC AUC UUG AAU
Skipping at
(+26 +
5 nM
49)
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
TABLE 10
Anti-
sense
Oligonu-
Ability to
cleotide
induce
name
Sequence
skipping
H12A
UCU UCU GUU UUU GUU AGC CAG UCA
Skipping at
(+52 +
5 nM
75)
H12A
UCU AUG UAA ACU GAA AAU UU
Skipping at
(−10 +
100 nM
10)
H12
UUC UGG AGA UCC AUU AAA AC
No skipping
(+11 +
30)
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
Anti-
Abili-
sense
ty to
Oligonu-
induce
cleotide
skip-
name
Sequence
ping
H13A
CAG CAG UUG CGU GAU CUC CAC UAG
Skip-
(+77 +
ping
100)
at 5
nM
H13A
UUC AUC AAC UAC CAC CAC CAU
No
(+55 +
skip-
75)
ping
H13D
CUA AGC AAA AUA AUC UGA CCU UAA G
No
(+06 −
skip-
19)
ping
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
Anti-
sense
Abili-
Oligo-
ty to
nucle-
induce
otide
skip-
name
Sequence
ping
H14A
CUU GUA AAA GAA CCC AGC GGU CUU CUG U
Skip-
(+37 +
ping
64)
at 100
nM
H14A
CAU CUA CAG AUG UUU GCC CAU C
No
(+14 +
skip-
35)
ping
H14A
GAA GGA UGU CUU GUA AAA GAA CC
No
(+51 +
skip-
73)
ping
H14D
ACC UGU UCU UCA GUA AGA CG
No
(−02 +
skip-
18)
ping
H14D
CAU GAC ACA CCU GUU CUU CAG UAA
No
(+14 −
skip-
10)
ping
H14A
CAU UUG AGA AGG AUG UCU UG
No
(+61 +
skip-
80)
ping
H14A
AUC UCC CAA UAC CUG GAG AAG AGA
No
(−12 +
skip-
12)
ping
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
TABLE 13
Anti-
sense
Oligo-
Ability
nucle-
to in-
otide
duce
name
Sequence
skipping
H15A
GCC AUG CAC UAA AAA GGC ACU GCA AGA
Skipping
(−12 +
CAU U
at 5 Nm
19)
H15A
UCU UUA AAG CCA GUU GUG UGA AUC
Skipping
(+48 +
at 5 Nm
71)
H15A
UUU CUG AAA GCC AUG CAC UAA
No
(+08 +
skipping
28)
H15A
GCC AUG CAC UAA AAA GGC ACU GCA AGA
No
(−12 +
CAU U
skipping
19)
H15D
GUA CAU ACG GCC AGU UUU UGA AGA C
No
(+17 −
skipping
08)
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
TABLE 14
Anti-
Abili-
sense
ty to
Oligonu-
induce
cleotide
skip-
name
Sequence
ping
H16A
CUA GAU CCG CUU UUA AAA CCU GUU AAA
Skip-
(−12 +
ACA A
ping
19)
at 5
nM
H16A
UCU UUU CUA GAU CCG CUU UUA AAA CCU
Skip-
(−06 +
GUU A
ping
25)
at 5
nM
H16A
CUA GAU CCG CUU UUA AAA CCU GUU A
Skip-
(−06 +
ping
19)
at 25
nM
H16A
CCG UCU UCU GGG UGA CUG ACU UA
Skip-
(+87 +
ping
109)
at 100
nM
H16A
CUA GAU CCG CUU UUA AAA CCU GUU AA
No
(−07 +
skip-
19)
ping
H16A
CCG CUU UUA AAA CCU GUU AA
No
(−07 +
skip-
13)
ping
H16A
UGG AUU GCU UUU UCU UUU CUA GAU CC
No
(+12 +
skip-
37)
ping
H16A
CAU GCU UCC GUC UUC UGG GUC ACU G
No
(+92 +
skip-
116)
ping
H16A
G AUC UUG UUU GAG UGA AUA CAG U
No
(+45 +
skip-
67)
ping
H16A
GUU AUC CAG CCA UGC UUC CGU C
No
(+105 +
skip-
126)
ping
H16D
UGA UAA UUG GUA UCA CUA ACC UGU G
No
(+05 −
skip-
20)
ping
H16D
GUA UCA CUA ACC UGU GCU GUA C
No
(+12 −
skip-
11)
ping
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.
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
Anti-
Abili-
sense
ty to
Oligonu-
induce
cloetide
skip-
name
Sequence
ping
H20A
CUG GCA GAA UUC GAU CCA CCG GCU GUU
No
(+44 +
C
skip-
71)
ping
H20A
CAG CAG UAG UUG UCA UCU GCU C
No
(+149 +
skip-
170)
ping
H20A
UGA UGG GGU GGU GGG UUG G
No
(+185 +
skip-
203)
ping
H20A
AUC UGC AUU AAC ACC CUC UAG AAA G
No
(−08 +
skip-
17)
ping
H20A
CCG GCU GUU CAG UUG UUC UGA GGC
No
(+30 +
skip-
53)
ping
H20A
AUC UGC AUU AAC ACC CUC UAG AAA GAA
Not
(−11 +
A
tested
17)
yet
H20D
GAA GGA GAA GAG AUU CUU ACC UUA CAA
Not
(+08 −
A
tested
20)
yet
H20A
CUG GCA GAA UUC GAU CCA CCG GCU GUU
Very
(+44 +
C
strong
71) &
skip-
H20A
CAG CAG UAG UUG UCA UCU GCU C
ping
(+149 +
170)
H19A
GCC UGA GCU GAU CUG CUG GCA UCU UGC
Very
(+44 +
AGU U
strong
71):
skip-
H20A
CUG GCA GAA UUC GAU CCA CCG GCU GUU
ping
(+44 +
C
71);
H20A
CAG CAG UAG UUG UCA UCU GCU C
(+149 +
170)
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
Oligonucle-
to in-
otide
duce
name
Sequence
skipping
H21A
GCC GGU UGA CUU CAU CCU GUG C
Skips at
(−06 + 16)
600 nM
H21A
CUG CAU CCA GGA ACA UGG GUC C
Skips at
(+85 + 106)
50 nM
H21A
GUC UGC AUC CAG GAA CAU GGG UC
Skips at
(+85 + 108)
50 nM
H21A
GUU GAA GAU CUG AUA GCC GGU UGA
Skips
(+08 + 31)
faintly
to
H21D
UAC UUA CUG UCU GUA GCU CUU UCU
No
(+18 − 07)
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.
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
Abili-
Antisense
ty to
oligonucleo-
induce
tide
skip-
name
Sequence
ping
H22A
CAC UCA UGG UCU CCU GAU AGC GCA
No
(+22 + 45)
skip-
ping
H22A
CUG CAA UUC CCC GAG UCU CUG C
Skip-
(+125 + 146)
ping
to 50
nM
H22A
ACU GCU GGA CCC AUG UCC UGA UG
Skip-
(+47 + 69)
ping
to 300
nM
H22A
CUA AGU UGA GGU AUG GAG AGU
Skip-
(+80 + 101)
ping
to 50
nM
H22D
UAU UCA CAG ACC UGC AAU UCC CC
No
(+13 − 11)
skip-
ping
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
Anti-
sense
Ability
oligonu-
to in-
cleotide
duce
name
Sequence
skipping
H23A
ACA GUG GUG CUG AGA UAG UAU AGG CC
No
(+34 +
skipping
59)
H23A
UAG GCC ACU UUG UUG CUC UUG C
No
(+18 +
Skipping
39)
H23A
UUC AGA GGG CGC UUU CUU C
No
(+72 +
Skipping
90)
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
oligonucle-
to in-
otide
duce
name
Sequence
skipping
H24A
GGG CAG GCC AUU CCU CCU UCA GA
Needs
(+48 + 70)
testing
H24A
UCU UCA GGG UUU GUA UGU GAU UCU
Needs
(-02 + 22)
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
Anti-
Abili-
sense
ty to
oligonu-
induce
cleotide
skip-
name
Sequence
ping
H25A
CUG GGC UGA AUU GUC UGA AUA UCA CUG
Needs
(+9 +
test-
36)
ing
H25A
CUG UUG GCA CAU GUG AUC CCA CUG AG
Needs
(+131 +
test-
156)
ing
H25D
GUC UAU ACC UGU UGG CAC AUG UGA
Needs
(+16 −
test-
08)
ing
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
Anti-
sense
Ability
oligonu-
to in-
cleotide
duce
name
Sequence
skipping
H26A
UGC UUU CUG UAA UUC AUC UGG AGU U
Needs
(+132 +
testing
156)
H26A
CCU CCU UUC UGG CAU AGA CCU UCC AC
Needs
(−07 +
testing
19)
H26A
UGU GUC AUC CAU UCG UGC AUC UCU G
Faint
(+68 +
skipping
92)
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
Anti-
sense
oligo-
Ability
nucle-
to
otide
induce
name
Sequence
skipping
H27A
UUA AGG CCU CUU GUG CUA CAG GUG G
Needs
(+82 +
testing
106)
H27A
GGG CCU CUU CUU UAG CUC UCU GA
Faint
(−4 +
skipping
19)
at 600
and 300
nM
H27D
GAC UUC CAA AGU CUU GCA UUU C
v. strong
(+19 −
skipping
03)
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
Anti-
sense
oligo-
nucle-
Ability
otide
to induce
name
Sequence
skipping
H28A
GCC AAC AUG CCC AAA CUU CCU AAG
v. strong
(−05 +
skipping
19)
at 600
and 300
nM
H28A
CAG AGA UUU CCU CAG CUC CGC CAG GA
Needs
(+99 +
testing
124)
H28D
CUU ACA UCU AGC ACC UCA GAG
v. strong
(+16 −
skipping
05)
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
Anti-
sense
oligonu-
Ability to
cleotide
induce
name
Sequence
skipping
H29A
UCC GCC AUC UGU UAG GGU CUG
Needs testing
(+57 +
UGC C
81)
H29A
AUU UGG GUU AUC CUC UGA AUG
v. strong
(+18 +
UCG C
skipping at
42)
600 and 300
nM
H29D
CAU ACC UCU UCA UGU AGU UCC C
v. strong
(+17 −
skipping at
05)
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
Anti-
sense
oligonu-
cleotide
Ability to in-
name
Sequence
duce skipping
H30A
CAU UUG AGC UGC GUC CAC CUU
Needs testing
(+122 +
GUC UG
147)
H30A
UCC UGG GCA GAC UGG AUG CUC
Very strong
(+25 +
UGU UC
skipping at
50)
600 and 300
nM.
H30D
UUG CCU GGG CUU CCU GAG GCA
Very strong
(+19 −
UU
skipping at
04)
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.
TABLE 26
Anti-
sense
oligo-
Ability
nucleo-
to in-
tide
duce
name
Sequence
skipping
H31D
UUC UGA AAU AAC AUA UAC CUG UGC
Skipping
(+06 −
to 300
18)
nM
H31D
UAG UUU CUG AAA UAA CAU AUA CCU G
Skipping
(+03 −
to 20 nM
22)
H31A
GAC UUG UCA AAU CAG AUU GGA
No
(+05 +
skipping
25)
H31D
GUU UCU GAA AUA ACA UAU ACC UGU
Skipping
(+04 −
to 300
20)
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
Abili-
ty to
Antisense
induce
oligonucleo-
skip-
tide name
Sequence
ping
H32D
CAC CAG AAA UAC AUA CCA CA
Skip-
(+04 − 16)
ping
to 300
nM
H32A
CAA UGA UUU AGC UGU GAC UG
No
(+151 + 170)
skip-
ping
H32A
CGA AAC UUC AUG GAG ACA UCU UG
No
(+10 + 32)
skip-
ping
H32A
CUU GUA GAC GCU GCU CAA AAU
Skip-
(+49 + 73)
UGG C
ping
to 300
nM
Antisense Oligonucleotides Directed at Exon 33
Antisense oligonucleotides directed at exon 33 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
TABLE 28
Antisense
Ability
oligonucle-
to
otide
induce
name
Sequence
skipping
H33D
CAU GCA CAC ACC UUU GCU CC
No
(+09 − 11)
skipping
H33A
UCU GUA CAA UCU GAC GUC CAG UCU
Skipping
(+53 + 76)
to 200
nM
H33A
GUC UUU AUC ACC AUU UCC ACU UCA
Skipping
(+30 + 56)
GAC
to 200
nM
H33A
CCG UCU GCU UUU UCU GUA CAA UCU
Skipping
(+64 + 88)
G
to 10 nM
Antisense Oligonucleotides Directed at Exon 34
Antisense oligonucleotides directed at exon 34 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
Table 29 below includes antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping.
TABLE 29
Anti-
Abili-
sense
ty to
oligonu-
induce
cleotide
skip-
name
Sequence
ping
H34A
UCC AUA UCU GUA GCU GCC AGC C
No
(+83 +
skip-
104)
ping
H34A
CCA GGC AAC UUC AGA AUC CAA AU
No
(+143 +
skip-
165)
ping
H34A
UUU CUG UUA CCU GAA AAG AAU UAU AAU
Not
(−20 +
GAA
tested
10)
H34A
CAU UCA UUU CCU UUC GCA UCU UAC G
Skip-
(+46 +
ping
70)
to 300
nM
H34A
UGA UCU CUU UGU CAA UUC CAU AUC UG
Skip-
(+95 +
ping
120)
to 300
nM
H34D
UUC AGU GAU AUA GGU UUU ACC UUU
Not
(+10 −
CCC CAG
tested
20)
H34A
CUG UAG CUG CCA GCC AUU CUG UCA AG
No
(+72 +
skip-
96)
ping
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.
TABLE 30
Antisense
oligonucleo-
Ability
tide
to induce
name
Sequence
skipping
H35A
UCU UCU GCU CGG GAG GUG ACA
Skipping
(+141 + 161)
to 20 nM
H35A
CCA GUU ACU AUU CAG AAG AC
No
(+116 + 135)
skipping
H35A
UCU UCA GGU GCA CCU UCU GU
No
(+24 + 43)
skipping
Antisense Oligonucleotides Directed at Exon 36
Antisense oligonucleotides directed at exon 36 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
Antisense molecule H36A(+26+50) [SEQ ID NO:145] induced exon 36 skipping when delivered into cells at a concentration of 300 nM, as shown in
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.
TABLE 31
Anti-
sense
Ability
oligonu-
to
cleotide
induce
name
Sequence
skipping
H37A
CGU GUA GAG UCC ACC UUU GGG CGU A
No
(+26 +5
skipping
0)
H37A
UAC UAA UUU CCU GCA GUG GUC ACC
Skipping
(+82 +
to 10 nM
105)
H37A
UUC UGU GUG AAA UGG CUG CAA AUC
Skipping
(+134 +
to 10 nM
157)
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.
TABLE 32
Anti-
sense
Ability
oligonu-
to
cleotide
induce
name
Sequence
skipping
H38A
CCU UCA AAG GAA UGG AGG CC
No
(−01 +
skipping
19)
H38A
UGC UGA AUU UCA GCC UCC AGU GGU
Skipping
(+59 +
U
to 10 nM
83)
H38A
UGA AGU CUU CCU CUU UCA GAU UCA C
Skipping
(+88 +
to 10 nM
112)
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
Abili-
ty to
Antisense
induce
oligonucleo-
skip-
tide name
Sequence
ping
H39A
CUG GCU UUC UCU CAU CUG UGA UUC
Skip-
(+62 + 85)
ping
to 100
nM
H39A
GUU GUA AGU UGU CUC CUC UU
No
(+39 + 58)
skip-
ping
H39A
UUG UCU GUA ACA GCU GCU GU
No
(+102 + 121)
skip-
ping
H39D
GCU CUA AUA CCU UGA GAG CA
Skip-
(+10 − 10)
ping
to 300
nM
Antisense Oligonucleotides Directed at Exon 40
Antisense oligonucleotides directed at exon 40 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
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.
TABLE 34
Antisense
Ability
oligonucle-
to
otide
induce
name
Sequence
skipping
H42A
AUC GUU UCU UCA CGG ACA GUG UGC
Skipping
(−4 + 23)
UGG
to 5 nM
H42A
GGG CUU GUG AGA CAU GAG UGA UUU
Skipping
(+86 + 109)
to 100
nM
H42D
A CCU UCA GAG GAC UCC UCU UGC
Skipping
(+19 − 02)
to 5 nM
Antisense Oligonucleotides Directed at Exon 43
Antisense oligonucleotides directed at exon 43 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H43A(+101+120) [SEQ ID NO:163] induced exon 43 skipping when delivered into cells at a concentration of 25 nM. Table 35 below includes the antisense molecules tested and their ability to induce exon 43 skipping.
TABLE 35
Anti-
sense
Ability
oligonu-
to
cleotide
induce
name
Sequence
skipping
H43D
UAU GUG UUA CCU ACC CUU GUC GGU C
Skipping
(+10 −
to 100
15)
nM
H43A
GGA GAG AGC UUC CUG UAG CU
Skipping
(+101 +
to 25 nM
120)
H43A
UCA CCC UUU CCA CAG GCG UUG CA
Skipping
(+78 +
to 200
100)
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.
TABLE 36
Antisense
Ability
oligonucle-
to
otide
induce
name
Sequence
skipping
H46D
UUA CCU UGA CUU GCU CAA GC
No
(+16 − 04)
skipping
H46A
UCC AGG UUC AAG UGG GAU AC
No
(+90 + 109)
skipping
H46A
CUC UUU UCC AGG UUC AAG UGG GAU
Good
(+86 + 115)
ACU AGC
skipping
to 100
nM
H46A
CAA GCU UUU CUU UUA GUU GCU GCU
Good
(+107 +
CUU UUC C
skipping
137)
to 100
nM
H46A
UAU UCU UUU GUU CUU CUA GCC UGG
Weak
(−10 + 20)
AGA AAG
skipping
H46A
CUG CUU CCU CCA ACC AUA AAA CAA
Weak
(+50 + 77)
AUU C
skipping
Antisense Oligonucleotides Directed at Exon 47
Antisense oligonucleotides directed at exon 47 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
H47A(+76+100) [SEQ ID NO:170] and H47A(−09+12) [SEQ ID NO:172] both induced exon 47 skipping when delivered into cells at a concentration of 200 nM. H47D(+25−02) [SEQ ID NO: 171] is yet to be prepared and tested.
Antisense Oligonucleotides Directed at Exon 50
Antisense oligonucleotides directed at exon 50 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
Antisense oligonucleotide molecule H50(+02+30) [SEQ ID NO: 173] was a strong inducer of exon skipping. Further, H50A(+07+33) [SEQ ID NO:174] and H50D(+07−18) [SEQ ID NO:175] both induced exon 50 skipping when delivered into cells at a concentration of 100 nM.
Antisense Oligonucleotides Directed at Exon 51
Antisense oligonucleotides directed at exon 51 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.
TABLE 37
Anti-
Abili-
sense
ty to
oligonu-
induce
cleotide
skip-
name
Sequence
ping
H51A
ACC AGA GUA ACA GUC UGA GUA GGA GC
Faint
(−01 +
skip-
25)
ping
H51D
CUC AUA CCU UCU GCU UGA UGA UC
Skip-
(+16 −
ping
07)
at 300
nM
H51A
UUC UGU CCA AGC CCG GUU GAA AUC
Needs
(+111 +
re-
134)
test-
ing
H51A
ACA UCA AGG AAG AUG GCA UUU CUA GUU
Very
(+61 +
UGG
strong
90)
skip-
ping
H51A
ACA UCA AGG AAG AUG GCA UUU CUA G
skip-
(+66 +
ping
90)
H51A
CUC CAA CAU CAA GGA AGA UGG CAU UUC
Very
(+66 +
UAG
strong
95)
skip-
ping
H51D
AUC AUU UUU UCU CAU ACC UUC UGC U
No
(+08 −
skip-
17)
ping
H51A/D
AUC AUU UUU UCU CAU ACC UUC UGC UAG
No
(+08 −
GAG CUA AAA
skip-
17) &
ping
(−15 −
?)
H51A
CAC CCA CCA UCA CCC UCY GUG
No
(+175 +
skip-
195)
ping
H51A
AUC AUC UCG UUG AUA UCC UCA A
No
(+199 +
skip-
220)
ping
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.
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
Anti-
Abili-
sense
ty to
oligonu-
induce
cleotide
skip-
name
Sequence
ping
H52A
UCC UGC AUU GUU GCC UGU AAG
No
(−07 +
skip-
14)
ping
H52A
UCC AAC UGG GGA CGC CUC UGU UCC AAA
Very
(+12 +
UCC
strong
41)
skip-
ping
H52A
ACU GGG GAC GCC UCU GUU CCA
Skip-
(+17 +
ping
37)
to 50
nM
H52A
CCG UAA UGA UUG UUC UAG CC
No
(+93 +
skip-
112)
ping
H52D
UGU UAA AAA ACU UAC UUC GA
No
(+05 −
skip-
15)
ping
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.
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
Anti-
sense
Ability
oligonu-
to
cleotide
induce
name
Sequence
skipping
H53A
CAU UCA ACU GUU GCC UCC GGU UCU G
Faint
(+45 +
skipping
69)
at 50 nM
H53A
CUG UUG CCU CCG GUU CUG AAG GUG
Faint
(+39 +
skipping
62)
at 50 nM
H53A
CAU UCA ACU GUU GCC UCC GGU UCU
Strong
(+39 +
GAA GGU G
skipping
69)
to 50 nM
H53D
UAC UAA CCU UGG UUU CUG UGA
Very
(+14 −
faint
07)
skipping
to 50 nM
H53A
CUG AAG GUG UUC UUG UAC UUC AUC C
Very
(+23 +
faint
47)
skipping
to 50 nM
H53A
UGU AUA GGG ACC CUC CUU CCA UGA
Very
(+150 +
CUC
faint
176)
skipping
to 50 nM
H53D
CUA ACC UUG GUU UCU GUG AUU UUC U
Not made
(+20 −
yet
05)
H53D
GGU AUC UUU GAU ACU AAC CUU GGU
Faint at
(+09 −
UUC
600 nM
18)
H53A
AUU CUU UCA ACU AGA AUA AAA G
No
(−12 +
skipping
10)
H53A
GAU UCU GAA UUC UUU CAA CUA GAA U
No
(−07 +
skipping
18)
H53A
AUC CCA CUG AUU CUG AAU UC
No
(+07 +
skipping
26)
H53A
UUG GCU CUG GCC UGU CCU AAG A
No
(+124 +
skipping
145)
Wilton, Stephen Donald, Fletcher, Sue, McClorey, Graham
| Patent | Priority | Assignee | Title |
| 10875880, | May 24 2016 | Sarepta Therapeutics, Inc | Processes for preparing oligomers |
| 10947533, | May 24 2016 | Sarepta Therapeutics, Inc | Processes for preparing phosphorodiamidate morpholino oligomers |
| 10961262, | May 24 2016 | BIOPHARMA CREDIT PLC | Processes for preparing phosphorodiamidate morpholino oligomers |
| 11168141, | Aug 02 2018 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
| 11248056, | Aug 02 2018 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
| 11369689, | Aug 02 2018 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
| 11384105, | May 24 2016 | Sarepta Therapeutics, Inc. | Processes for preparing oligomers |
| 11472824, | May 24 2016 | BIOPHARMA CREDIT PLC | Processes for preparing phosphorodiamidate morpholino oligomers |
| 11497815, | Aug 02 2018 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
| 11629197, | Aug 02 2018 | Dyne Therapeutics, Inc. | Methods of using muscle targeting complexes to deliver an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy or a disease associated with muscle weakness |
| 11633496, | Aug 02 2018 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
| 11679161, | Jul 09 2021 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy |
| 11771776, | Jul 09 2021 | DYNE THERAPEUTICS, INC | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
| 11787869, | Aug 02 2018 | Dyne Therapeutics, Inc. | Methods of using muscle targeting complexes to deliver an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy or a disease associated with muscle weakness |
| 11795233, | Aug 02 2018 | Dyne Therapeutics, Inc. | Muscle-targeting complex comprising an anti-transferrin receptor antibody linked to an oligonucleotide |
| 11795234, | Aug 02 2018 | Dyne Therapeutics, Inc. | Methods of producing muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide |
| 11833217, | Aug 02 2018 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
| 11844843, | Jul 09 2021 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy |
| Patent | Priority | Assignee | Title |
| 4458066, | Feb 29 1980 | UNIVERSITY PATENTS, INC | Process for preparing polynucleotides |
| 5034506, | Mar 15 1985 | ANTIVIRALS, INC | Uncharged morpholino-based polymers having achiral intersubunit linkages |
| 5138045, | Jul 27 1990 | Isis Pharmaceuticals | Polyamine conjugated oligonucleotides |
| 5142047, | Mar 15 1985 | Anti-Gene Development Group; ANTIVIRALS, INC | Uncharged polynucleotide-binding polymers |
| 5149797, | Feb 15 1990 | WORCESTER FOUNDATION FOR BIOMEDICAL RESEARCH, INC | Method of site-specific alteration of RNA and production of encoded polypeptides |
| 5166315, | Dec 20 1989 | ANTIVIRALS, INC | Sequence-specific binding polymers for duplex nucleic acids |
| 5185444, | Mar 15 1985 | ANTIVIRALS, INC | Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages |
| 5190931, | Oct 20 1983 | Research Foundation of State University of New York, The | Regulation of gene expression by employing translational inhibition of MRNA utilizing interfering complementary MRNA |
| 5217866, | Mar 15 1985 | ANTIVIRALS, INC | Polynucleotide assay reagent and method |
| 5506337, | Mar 15 1985 | ANTIVIRALS INC | Morpholino-subunit combinatorial library and method |
| 5521063, | Mar 15 1985 | ANTIVIRALS INC AN OREGON CORPORATION | Polynucleotide reagent containing chiral subunits and methods of use |
| 5627274, | Jan 26 1995 | The University of North Carolina at Chapel Hill | Antisense oligonucleotides which combat aberrant splicing and methods of using the same |
| 5665593, | May 11 1993 | University of North Carolina | Antisense oligonucleotides which combat aberrant splicing and methods of using the same |
| 5698685, | Mar 15 1985 | Antivirals Inc. | Morpholino-subunit combinatorial library and method |
| 5801154, | Apr 16 1996 | Isis Pharmaceuticals, Inc | Antisense oligonucleotide modulation of multidrug resistance-associated protein |
| 5869252, | Mar 31 1992 | Abbott Laboratories | Method of multiplex ligase chain reaction |
| 5892023, | May 26 1995 | Societe de Conseils de Recherches et d'Applications Scientifiques | Anti sense oligonucleotides for blocking IgE receptor synthesis |
| 5916808, | May 11 1993 | The University of North Carolina at Chapel Hill | Antisense oligonucleotides which combat aberrant splicing and methods of using the same |
| 5976879, | May 11 1993 | The University of North Carolina at Chapel Hill | Antisense oligonucleotides which combat aberrant splicing and methods of using the same |
| 6153436, | Jan 10 1997 | BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS, THE | Method of gene delivery using wildtype adeno associated viral (AAV) vectors with insertions |
| 6210892, | Oct 07 1998 | Ionis Pharmaceuticals, Inc | Alteration of cellular behavior by antisense modulation of mRNA processing |
| 6312900, | Apr 14 1997 | ISIS PHARMACEUTICALS INC | Antisense oligonucleotide compositions and methods for the modulation of activating protein 1 |
| 6391636, | May 31 1994 | ISIS Pharmaceuticals, Inc. | Antisense oligonucleotide modulation of raf gene expression |
| 6451991, | Feb 14 1996 | ISIS PHARMACEUTICALS INC ; Novartis AG; Isis Pharmaceuticals, Inc | Sugar-modified gapped oligonucleotides |
| 6653466, | May 21 1999 | JCR PHARMACEUTICALS CO., LTD.; Masafumi, Matsuo | Pharmaceutical composition for treatment of duchenne muscular dystrophy |
| 6653467, | Apr 26 2000 | JCR Pharmaceutical Co., Ltd.; Masafumi, Matsuo | Medicament for treatment of Duchenne muscular dystrophy |
| 6656732, | May 18 2001 | Ionis Pharmaceuticals, Inc | Antisense inhibition of src-c expression |
| 6727355, | Aug 25 2000 | JCR PHARMACEUTICALS CO , LTD ; Matsuo, Masafumi | Pharmaceutical composition for treatment of Duchenne muscular dystrophy |
| 6784291, | May 04 2000 | AVI BIOPHARMA, INC | Splice-region antisense composition and method |
| 6806084, | Jun 04 1992 | The Regents of the University of California | Methods for compositions for in vivo gene delivery |
| 7001761, | Apr 28 2000 | ASKLEPIOS BIOPHARMACEUTICAL, INC | DNA sequences comprising dystrophin minigenes and methods of use thereof |
| 7070807, | Dec 29 1999 | Branched histidine copolymers and methods for using same | |
| 7163695, | Dec 29 1999 | Histidine copolymer and methods for using same | |
| 7250289, | Nov 20 2002 | Affymetrix, Inc | Methods of genetic analysis of mouse |
| 7314750, | Nov 20 2002 | Affymetrix, Inc | Addressable oligonucleotide array of the rat genome |
| 7468418, | Apr 29 2003 | Sarepta Therapeutics, Inc | Compositions for enhancing transport of molecules into cells |
| 7534879, | Mar 21 2003 | Academisch Ziekenhuis Leiden | Modulation of exon recognition in pre-mRNA by interfering with the secondary RNA structure |
| 7655785, | Nov 14 2002 | Rosetta Genomics Ltd. | Bioinformatically detectable group of novel regulatory oligonucleotides and uses thereof |
| 7655788, | Nov 14 2002 | Thermo Fisher Scientific Inc | siRNA targeting DNA-damage-inducible transcript 4 (DDIT4) |
| 7807816, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 7902160, | Nov 25 2002 | DAIICHI SANKYO COMPANY, LIMITED | ENA nucleic acid drugs modifying splicing in mRNA precursor |
| 7960541, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 7973015, | Sep 21 2000 | LEIDEN, ACADEMISCH ZIEKENHUIS | Induction of exon skipping in eukaryotic cells |
| 8084601, | Sep 11 2008 | Royal Holloway and Bedford New College Royal Holloway, University of London | Oligomers |
| 8232384, | Jun 28 2005 | University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 8324371, | Sep 11 2008 | Royal Holloway and Bedford New College, University of London | Oligomers |
| 8361979, | May 19 2006 | Academisch Ziekenhuis Leiden | Means and method for inducing exon-skipping |
| 8436163, | May 04 2000 | AVI BioPharma, Inc. | Splice-region antisense composition and method |
| 8450474, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 8455634, | Jun 28 2005 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 8455635, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 8455636, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 8461325, | Sep 11 2008 | Royal Holloway, University of London | Oligomers |
| 8476423, | Jun 28 2004 | The University of Western Austrailia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 8486907, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 8501703, | Aug 30 2005 | Ionis Pharmaceuticals, Inc | Chimeric oligomeric compounds for modulation of splicing |
| 8501704, | Nov 08 2005 | Sarepta Therapeutics, Inc | Immunosuppression compound and treatment method |
| 8524676, | Sep 08 2005 | Sarepta Therapeutics, Inc | Method for treating enterovirus or rhinovirus infection using antisense antiviral compounds |
| 8524880, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 8536147, | Jul 13 2005 | Sarepta Therapeutics, Inc | Antibacterial antisense oligonucleotide and method |
| 8552172, | Sep 11 2008 | Royal Holloway and Bedford New College Royal Holloway, University of London | Oligomers |
| 8592386, | Dec 17 2008 | Sarepta Therapeutics, Inc | Antisense compositions and methods for modulating contact hypersensitivity or contact dermatitis |
| 8618270, | Aug 05 2003 | Sarepta Therapeutics, Inc | Oligonucleotide analog and method for treating flavivirus infections |
| 8624019, | Nov 25 2002 | DAIICHI SANKYO COMPANY, LIMITED | ENA nucleic acid drugs modifying splicing in mRNA precursor |
| 8637483, | Nov 12 2009 | The University of Western Australia | Antisense molecules and methods for treating pathologies |
| 8697858, | Nov 13 2009 | Sarepta Therapeutics, Inc | Antisense antiviral compound and method for treating influenza viral infection |
| 8741863, | Jun 29 2007 | Sarepta Therapeutics, Inc. | Compound and method for treating myotonic dystrophy |
| 8759307, | Dec 24 2003 | Sarepta Therapeutics, Inc | Oligonucleotide compound and method for treating nidovirus infections |
| 8759507, | Mar 21 2003 | Academisch Ziekenhuis Leiden | Modulation of exon recognition in pre-mRNA by interfering RNA structure |
| 8779128, | May 28 2010 | Sarepta Therapeutics, Inc | Oligonucleotide analogues having modified intersubunit linkages and/or terminal groups |
| 8785407, | May 10 2006 | Sarepta Therapeutics, Inc | Antisense antiviral agent and method for treating ssRNA viral infection |
| 8785410, | Feb 09 2005 | Sarepta Therapeutics, Inc | Antisense composition and method for treating muscle atrophy |
| 8835402, | Jun 26 2009 | Sarepta Therapeutics, Inc. | Compound and method for treating myotonic dystrophy |
| 8865883, | Oct 24 2008 | Sarepta Therapeutics, Inc | Multiple exon skipping compositions for DMD |
| 8871918, | Oct 24 2008 | Sarepta Therapeutics, Inc | Multiple exon skipping compositions for DMD |
| 8877725, | May 24 2004 | Sarepta Therapeutics, Inc. | Peptide conjugated, inosine-substituted antisense oligomer compound and method |
| 8895722, | May 04 2000 | Sarepta Therapeutics, Inc. | Splice-region antisense composition and method |
| 8906872, | Sep 16 2004 | Sarepta Therapeutics, Inc | Antisense antiviral compound and method for treating ssRNA viral infection |
| 9018368, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 9024007, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 9035040, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 9175286, | Jun 28 2005 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 9217148, | Mar 14 2013 | Sarepta Therapeutics, Inc. | Exon skipping compositions for treating muscular dystrophy |
| 9228187, | Nov 12 2009 | The University of Western Australia | Antisense molecules and methods for treating pathologies |
| 9234198, | Oct 24 2008 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
| 9249416, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 9416361, | May 04 2000 | BIOPHARMA CREDIT PLC | Splice-region antisense composition and method |
| 9422555, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 9434948, | Oct 24 2008 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
| 9441229, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 9447415, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 9447416, | Oct 24 2008 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
| 9447417, | Oct 24 2008 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
| 9453225, | Oct 24 2008 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
| 9506058, | Mar 15 2013 | BIOPHARMA CREDIT PLC | Compositions for treating muscular dystrophy |
| 9605262, | Jun 28 2004 | The University of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
| 9758783, | Nov 12 2009 | The University of Western Australia | Antisense molecules and methods for treating pathologies |
| 20010056077, | |||
| 20020049173, | |||
| 20020055481, | |||
| 20020110819, | |||
| 20020156235, | |||
| 20030166588, | |||
| 20030224353, | |||
| 20030235845, | |||
| 20040248833, | |||
| 20040254137, | |||
| 20040266720, | |||
| 20050026164, | |||
| 20050048495, | |||
| 20050153935, | |||
| 20060099616, | |||
| 20060147952, | |||
| 20060148740, | |||
| 20060287268, | |||
| 20070037165, | |||
| 20070082861, | |||
| 20070265215, | |||
| 20080194463, | |||
| 20080200409, | |||
| 20080209581, | |||
| 20090076246, | |||
| 20090082547, | |||
| 20090088562, | |||
| 20090099066, | |||
| 20090228998, | |||
| 20090269755, | |||
| 20090312532, | |||
| 20100016215, | |||
| 20100130591, | |||
| 20100168212, | |||
| 20110015253, | |||
| 20110015258, | |||
| 20110046203, | |||
| 20110046360, | |||
| 20110110960, | |||
| 20110263682, | |||
| 20110263686, | |||
| 20110281787, | |||
| 20110294753, | |||
| 20110312086, | |||
| 20120022134, | |||
| 20120022144, | |||
| 20120022145, | |||
| 20120029057, | |||
| 20120029058, | |||
| 20120029059, | |||
| 20120029060, | |||
| 20120041050, | |||
| 20120046342, | |||
| 20120053228, | |||
| 20120059042, | |||
| 20120065169, | |||
| 20120065244, | |||
| 20120108652, | |||
| 20120108653, | |||
| 20120115150, | |||
| 20120122801, | |||
| 20120149756, | |||
| 20120172415, | |||
| 20120202752, | |||
| 20120289457, | |||
| 20130072671, | |||
| 20130090465, | |||
| 20130116310, | |||
| 20130190390, | |||
| 20130197220, | |||
| 20130211062, | |||
| 20130217755, | |||
| 20130253033, | |||
| 20130253180, | |||
| 20130274313, | |||
| 20130289096, | |||
| 20130302806, | |||
| 20130331438, | |||
| 20140045916, | |||
| 20140057964, | |||
| 20140080896, | |||
| 20140080898, | |||
| 20140094500, | |||
| 20140113955, | |||
| 20140128592, | |||
| 20140155587, | |||
| 20140213635, | |||
| 20140221458, | |||
| 20140243515, | |||
| 20140243516, | |||
| 20140275212, | |||
| 20140296323, | |||
| 20140315862, | |||
| 20140315977, | |||
| 20140316123, | |||
| 20140323544, | |||
| 20140329762, | |||
| 20140329881, | |||
| 20140343266, | |||
| 20140350067, | |||
| 20140350076, | |||
| 20140357698, | |||
| 20140357855, | |||
| 20150045413, | |||
| 20150057330, | |||
| 20150152415, | |||
| 20150232839, | |||
| 20150353931, | |||
| 20150361428, | |||
| 20150376615, | |||
| 20150376616, | |||
| 20150376617, | |||
| 20150376618, | |||
| 20160002631, | |||
| 20160002632, | |||
| 20160002633, | |||
| 20160002634, | |||
| 20160002635, | |||
| 20160002637, | |||
| 20160040162, | |||
| 20160177301, | |||
| 20160298111, | |||
| 20170009233, | |||
| 20170009234, | |||
| 20170283799, | |||
| 20170292125, | |||
| 20170369875, | |||
| 20170369876, | |||
| 20180002689, | |||
| 20180002697, | |||
| 20180016574, | |||
| AU2003284638, | |||
| AU780517, | |||
| CA2507125, | |||
| EP1054058, | |||
| EP1160318, | |||
| EP1191097, | |||
| EP1191098, | |||
| EP1495769, | |||
| EP1544297, | |||
| EP1568769, | |||
| EP1606407, | |||
| EP1619249, | |||
| EP1766010, | |||
| EP1857548, | |||
| EP2119783, | |||
| EP2135948, | |||
| EP2206781, | |||
| EP2258863, | |||
| EP2284264, | |||
| EP2374885, | |||
| EP2386636, | |||
| EP2392660, | |||
| EP2435582, | |||
| EP2435583, | |||
| EP2488165, | |||
| EP2500430, | |||
| EP2530153, | |||
| EP2530154, | |||
| EP2530155, | |||
| EP2530156, | |||
| EP2581448, | |||
| EP2594640, | |||
| EP2594641, | |||
| EP2594642, | |||
| EP2602322, | |||
| EP2607484, | |||
| EP2612917, | |||
| EP2614827, | |||
| EP2623507, | |||
| EP2636740, | |||
| EP2636741, | |||
| EP2636742, | |||
| EP2799548, | |||
| EP2801618, | |||
| JP2000325085, | |||
| JP2002010790, | |||
| JP2002325582, | |||
| JP2002340857, | |||
| JP2002529499, | |||
| JP2004509622, | |||
| JP2010268815, | |||
| JP2011101655, | |||
| JP2011200235, | |||
| JP2014054250, | |||
| JP2014111638, | |||
| JP2014138589, | |||
| JP4777777, | |||
| JP4846965, | |||
| JP5138722, | |||
| JP5378423, | |||
| WO15780, | |||
| WO44897, | |||
| WO78341, | |||
| WO149775, | |||
| WO172765, | |||
| WO183503, | |||
| WO183740, | |||
| WO218656, | |||
| WO224906, | |||
| WO229406, | |||
| WO3053341, | |||
| WO4048570, | |||
| WO4083432, | |||
| WO4083446, | |||
| WO2005115479, | |||
| WO2006000057, | |||
| WO2006021724, | |||
| WO2006112705, | |||
| WO2007058894, | |||
| WO2007133812, | |||
| WO2007135105, | |||
| WO2008036127, | |||
| WO2009054725, | |||
| WO2009101399, | |||
| WO2009139630, | |||
| WO2010048586, | |||
| WO2010050801, | |||
| WO2010050802, | |||
| WO2010115993, | |||
| WO2010123369, | |||
| WO2010136415, | |||
| WO2010136417, | |||
| WO2010150231, | |||
| WO2011024077, | |||
| WO2011045747, | |||
| WO2011057350, | |||
| WO2011143008, | |||
| WO2012001941, | |||
| WO2012029986, | |||
| WO2012043730, | |||
| WO2012109296, | |||
| WO2012150960, | |||
| WO2013033407, | |||
| WO2013053928, | |||
| WO2013100190, | |||
| WO2013112053, | |||
| WO2013142087, | |||
| WO2014007620, | |||
| WO2014100714, | |||
| WO2014144978, | |||
| WO2014153220, | |||
| WO2014153240, | |||
| WO2014172669, | |||
| WO2017059131, | |||
| WO9320227, | |||
| WO9402595, | |||
| WO9426887, | |||
| WO9610391, | |||
| WO9610392, | |||
| WO9730067, | |||
| WO9734638, | |||
| WO2024906, |
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