Multiple exon skipping compositions for DMD

Abstract

Provision of multiple exon skipping compositions for DMD. [Solution] Antisense molecules capable of binding to selected target sites in the human dystrophin gene to induce exon skipping and methods of using the antisense molecules to treat muscular dystrophy are provided. Embodiments of the present invention generally relate to antisense compounds capable of binding to selected targets to induce exon skipping and methods of using the antisense compounds to induce exon skipping. In certain embodiments, two or more antisense oligonucleotides of the present invention can be combined together to induce single or multiple exon skipping.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61 / 108,416, filed October 24, 2008, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing References The sequence listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference. The name of the text containing the sequence listing is 120178_410PC_SEQUENCE_LISTING.txt. The text file is 156 KB, was created on October 23, 2009, and has been submitted electronically via EFS-Web.

[0003] FIELD OF THE INVENTION The present invention relates to novel antisense compounds and compositions suitable for facilitating exon skipping in the human dystrophin gene. The present invention also provides methods for inducing exon skipping using antisense compositions adapted for use in the methods of the invention. [Background technology]

[0004] Background of the Invention Antisense technology has been developed using a range of chemistries to affect gene expression at various levels (transcription, splicing, stability, and translation). Much of the research has focused on the use of antisense compounds to correct or compensate for aberrant or disease-related genes across a wide range of indications. Antisense molecules can specifically inhibit gene expression, and for this reason, much research effort related to oligonucleotides as regulators of gene expression has focused on inhibiting the expression of targeted genes or the function of cis-acting elements. Antisense oligonucleotides are typically directed against either the sense strand (e.g., mRNA) or the minus strand of RNA, in the case of certain viral RNA targets. To achieve the desired effect of specific gene downregulation, oligonucleotides generally either promote the decay of the targeted mRNA, block mRNA translation, or block the function of cis-acting RNA elements, thereby effectively disrupting either the de novo synthesis of the target protein or viral RNA replication.

[0005] However, such techniques are not useful when the goal is to upregulate the production of native proteins or to compensate for mutations that induce premature termination of translation, such as nonsense or frameshift mutations. In these cases, the defective gene transcript should not be subject to targeted degradation or steric inhibition, and therefore, the antisense oligonucleotide chemistry should not promote target mRNA decay or block translation.

[0006] In various genetic diseases, the effect of mutations on the final expression of a gene can be regulated through the process of targeted exon skipping during the splicing process. The splicing process is directed by a complex, multi-component mechanism that brings adjacent exon-intron junctions in pre-mRNA into close proximity and performs phosphodiester bond cleavage at the end of the intron, with their subsequent reformation between exons spliced ​​together. This complex and highly precise process is mediated by sequence motifs in pre-mRNA, a relatively short, semi-conserved RNA segment, to which various nuclear splicing factors bind, and these factors then participate in the splicing reaction. By altering the way the splicing machinery reads or recognizes motifs involved in pre-mRNA processing, it is possible to create differentially spliced ​​mRNA molecules. It is now recognized that the majority of human genes are alternatively spliced ​​during normal gene expression, but the mechanisms involved have not been identified.

[0007] In cases where a normally functioning protein is prematurely terminated due to a mutation within it, it has been shown that a means to restore functional protein production through antisense technology is possible through intervention in the splicing process, and if the exon associated with a disease-causing mutation can be specifically removed from a gene, a truncated protein product can sometimes be produced that has similar biological properties to the native protein or has sufficient biological activity to ameliorate the disease caused by the mutation associated with the exon (Sierakowska, Sambade et al., 1996; Wilton, Lloyd et al., 1999; van Deutekom, Bremmer-Bout et al., 2001; Lu, Mann et al., 2003; Aartsma-Rus, Janson et al., 2004). Kole et al. (Patent Document 1; Patent Document 2; Patent Document 3; and Patent Document 4) disclose a method of combating aberrant splicing using modified antisense oligonucleotide analogs that do not promote the decay of the targeted pre-mRNA. Bennett et al. (US Pat. No. 6,210,892) describe antisense modulation of wild-type cellular mRNA processing using antisense oligonucleotide analogs that also do not induce RNAse H-mediated cleavage of the target RNA.

[0008] The process of targeted exon skipping appears to be particularly useful in long genes, where there are many exons and introns, where the gene organization of exons is redundant, or where proteins can function without one or more specific exons.Attempts to redirect gene processing for the treatment of genetic diseases associated with shortening caused by mutations in various genes have focused on the use of antisense oligonucleotides that either: (1) completely or partially overlap with the element involved in the splicing process; or (2) bind to pre-mRNA at a position close enough to the element to disrupt the binding and function of the splicing factors that normally mediate the specific splicing reaction that occurs at that element.

[0009] Duchenne muscular dystrophy (DMD) is caused by the lack of expression of the protein dystrophin.The gene encoding this protein contains 79 exons, spanning over 2 million nucleotides of DNA.Any exon mutation, characterized by changing the reading frame of exon, introducing a stop codon, or removing the entire reading frame of exon(s) or one or more exon duplications, has the potential to disrupt the production of functional dystrophin and cause DMD.

[0010] Becker muscular dystrophy (BMD), a milder form of muscular dystrophy, has been found to occur when mutations, typically deletions of one or more exons, result in the correct reading frame along the entire dystrophin transcript, such that translation of mRNA into protein does not terminate prematurely. If the combination of upstream and downstream exons in the processing of mutated dystrophin pre-mRNA maintains the correct reading frame of the gene, the result is an mRNA that encodes a protein with a short internal deletion that retains some activity, resulting in the Becker phenotype.

[0011] Deletion of an exon (or exons) that does not change the reading frame of the dystrophin protein results in a BMD phenotype, whereas exon deletions that cause a frameshift result in DMD (Monaco, Bertelson et al., 1988). Generally, dystrophin mutations, including point mutations and exon deletions that change the reading frame and thus disrupt proper protein translation, result in DMD. It should also be noted that some BMD and DMD patients have exon deletions that encompass multiple exons.

[0012] Antisense molecules may provide a tool in the treatment of Duchenne muscular dystrophy (DMD), but attempts to induce exon skipping using antisense molecules have had mixed success. Successful skipping of dystrophin exon 19 from dystrophin pre-mRNA was achieved using various antisense molecules directed at adjacent splice sites or motifs within the exon included in the definition of the exon, as described by Errington et al. (Errington, Mann et al., 2003).

[0013] The first example of specific and reproducible exon skipping in the mdx mouse model was reported by Wilton et al. (Wilton, Lloyd et al., 1999). By directing antisense molecules to the donor splice site, exon 23 skipping was induced in dystrophin mRNA within 6 hours of treatment of cultured cells. Wilton et al. also described using longer antisense oligonucleotides to target the acceptor region of mouse dystrophin pre-mRNA. While the first antisense oligonucleotides directed at the intron 23 donor splice site induced exon skipping in primary myoblast cultures, this compound was found to be much less effective in immortalized cell cultures expressing higher levels of dystrophin. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] U.S. Patent No. 5,627,274 [Patent Document 2] U.S. Patent No. 5,916,808 [Patent Document 3] U.S. Patent No. 5,976,879 [Patent Document 4] U.S. Patent No. 5,665,593 Summary of the Invention [Problem to be solved by the invention]

[0015] Despite these attempts, there remains a need for improved antisense oligomers targeted to multiple dystrophin exons, and improved muscle delivery compositions and methods for therapeutic application in DMD. [Means for solving the problem]

[0016] Summary of the Invention Embodiments of the present invention generally relate to antisense compounds capable of binding to a selected target to induce exon skipping, and methods of using the antisense compounds to induce exon skipping. In certain embodiments, two or more antisense oligonucleotides of the present invention can be combined together to induce single or multiple exon skipping.

[0017] In certain embodiments, it is possible to improve exon skipping of single or multiple exons by covalently linking two or more antisense oligonucleotide molecules together (see, e.g., Aartsma-Rus, Janson et al., 2004).

[0018] In certain embodiments, the antisense compounds of the invention induce exon skipping in the human dystrophin gene, thereby enabling muscle cells to produce functional dystrophin protein.

[0019] The antisense oligonucleotide compounds (also referred to herein as oligomers) of the invention typically: (i) comprise morpholino subunits and phosphorus-containing intersubunit linkages connecting the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit; (ii) contain between 10 and 40, preferably between 20 and 35, nucleotide bases; and (iii) comprise a base sequence effective to hybridize to at least 12 consecutive bases of a target sequence in a dystrophin pre-mRNA and induce exon skipping.

[0020] In certain embodiments, the antisense compounds herein may comprise a phosphorus-containing intersubunit linkage connecting the morpholino nitrogen of one subunit to the 5′ exocyclic carbon of an adjacent subunit according to the following structure (I):

[0021] [ka] where: Y1 is -O-, -S-, -NH-, or -CH2-; Z is O or S; Pj is a purine or pyrimidine base-pairing moiety that is effective to bind to a base in a polynucleotide by base-specific hydrogen bonding; and X is fluoro, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, amino, optionally substituted alkylamino, or optionally substituted heterocyclyl.

[0022] In certain embodiments, the above-described uncharged intersubunit linkages may be dispersed with linkages that are positively charged at physiological pH, where the total number of positively charged linkages is between 2 and no more than half of the total number of linkages. For example, the positively charged linkage may have the structure above, where X is an optionally substituted 1-piperazinyl. In other embodiments, the positively charged linkage may have the structure above, where X is a substituted 1-piperazinyl, where the 1-piperazinyl is substituted at the 4-position with an optionally substituted alkylguanidinyl moiety.

[0023] When the administered antisense compound is effective for targeting a splice site of preprocessed human dystrophin, it may have a base sequence complementary to a target region comprising at least 12 consecutive bases in the preprocessed messenger RNA (mRNA) human dystrophin transcript. Exemplary antisense sequences include those identified by SEQ ID NOS: 1-569 and 612-633.

[0024] In certain embodiments, the antisense sequences of the invention are included in: (a) any of the sequences identified by SEQ ID NOs: 1-20, preferably SEQ ID NOs: 4, 8, 11, and 12, and more preferably SEQ ID NO: 12, for use in causing skipping of exon 44 in the processing of preprocessed mRNA of human dystrophin; (b) any of the sequences identified by SEQ ID NOs: 21-76 and 612-624, preferably SEQ ID NOs: 27, 29, 34, and 39, and more preferably SEQ ID NO: 34, for use in causing skipping of exon 45 in the processing of preprocessed mRNA of human dystrophin; (c) any of the sequences identified by SEQ ID NOs: 77-125, preferably SEQ ID NOs: 21-53, and more preferably SEQ ID NOs: 82, 84-87, 90, 96, 98, and 101, for use in causing skipping of exon 46 in the processing of preprocessed mRNA of human dystrophin; (d) any of the sequences identified by SEQ ID NOs: 126-169, preferably SEQ ID NOs: 126-149, and more preferably SEQ ID NOs: 126, 128-130, 132, 144, and 146-149, for use in causing skipping of exon 47 in the processing of preprocessed mRNA of human dystrophin; (e) any of the sequences identified by SEQ ID NOs: 170-224 and 634, preferably SEQ ID NOs: 170-201 and 634, and more preferably SEQ ID NOs: 176, 178, 181-183, 194, and 198-201, for use in causing skipping of exon 48 in the processing of preprocessed mRNA of human dystrophin; (f) any of the sequences identified by SEQ ID NOs: 225-266, preferably SEQ ID NOs: 225-248, and more preferably SEQ ID NOs: 227, 229, 234, 236, 237, and 244-248, for use in causing exon 49 skipping in the processing of human dystrophin preprocessed mRNA; (g) any of the sequences identified by SEQ ID NOs: 267-308, preferably SEQ ID NOs: 277, 287, and 290, and more preferably SEQ ID NO: 287, for use in causing skipping of exon 50 in the processing of preprocessed mRNA of human dystrophin; (h) any of the sequences identified by SEQ ID NOs: 309-371, preferably SEQ ID NOs: 324, 326, and 327, and more preferably SEQ ID NO: 327, for use in causing exon 51 skipping in the processing of human dystrophin preprocessed mRNA; (i) any of the sequences identified by SEQ ID NOs: 372 to 415, preferably SEQ ID NOs: 372 to 397, and more preferably SEQ ID NOs: 379 to 382, ​​384, 390, and 392 to 395, for use in causing skipping of exon 52 in the processing of preprocessed mRNA of human dystrophin; (j) any of the sequences identified by SEQ ID NOs: 416-475 and 625-633, preferably SEQ ID NOs: 428, 429, and 431, and more preferably SEQ ID NO: 429, for use in causing exon 53 skipping in the processing of human dystrophin preprocessed mRNA; (k) any of the sequences identified by SEQ ID NOs: 476-519, preferably SEQ ID NOs: 476-499, and more preferably SEQ ID NOs: 479-482, 484, 489, and 491-493, for use in causing skipping of exon 54 in the processing of preprocessed mRNA of human dystrophin; (l) Any of the sequences identified by SEQ ID NOs: 520-569 and 635, preferably SEQ ID NOs: 520-546 and 635, and more preferably SEQ ID NOs: 524-528, 537, 539, 540, 542, and 544, for use in causing skipping of exon 55 in the processing of preprocessed mRNA of human dystrophin.

[0025] In certain embodiments, the compound may be conjugated to an arginine-rich polypeptide effective to facilitate cellular uptake of the compound. Exemplary peptides include those identified by SEQ ID NOS: 570-578, among others, described herein.

[0026] In one exemplary embodiment, the arginine-rich peptide is covalently attached at its N- or C-terminus to the 3' or 5' end of the antisense compound, and in one exemplary embodiment, the antisense compound is composed of morpholino subunits and a phosphorus-containing intersubunit linkage connecting the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit.

[0027] Generally, the peptide-oligomer conjugate may further comprise a homing peptide that is selective for a selected mammalian tissue, i.e., the same tissue targeted by the cell-penetrating peptide. This conjugate may be a cell-penetrating peptide-homing peptide-antisense oligomer type, or more preferably, a homing peptide-cell-penetrating peptide-antisense oligomer type. For example, the peptide conjugate compound for use in treating Duchenne muscular dystrophy as described above may further comprise a homing peptide that is selective for muscle tissue, such as the peptide having the sequence identified as SEQ ID NO: 579, conjugated to a cell-penetrating peptide. Exemplary conjugates of this type include those referred to herein as CP06062-MSP-PMO (cell-penetrating peptide-homing peptide-antisense oligomer) and MSP-CP06062-PMO (homing peptide-cell-penetrating peptide-antisense oligomer) (see SEQ ID NOs: 580-583).

[0028] In some embodiments, the peptide is conjugated to the oligomer via a linker moiety. In certain embodiments, the linker moiety may comprise an optionally substituted piperazinyl moiety. In other embodiments, the linker moiety may further comprise β-alanine and / or 6-aminohexanoic acid subunits. In still other embodiments, the peptide is directly conjugated to the oligomer without a linker moiety.

[0029] The peptide may be conjugated to the oligomer at any moiety suitable for forming a covalent bond between the peptide and the oligomer, or between the linker moiety and the oligomer. For example, in some embodiments, the peptide may be conjugated to the 3' end of the oligomer. In other embodiments, the peptide may be conjugated to the oligomer at the 5' end of the oligomer. In still other embodiments, the peptide may be conjugated to the oligomer through any of the intersubunit linkages.

[0030] In some embodiments, the peptide is conjugated to the oligomer at the 5' end of the oligomer. In embodiments involving phosphorus-containing intersubunit linkages, the peptide may be conjugated to the oligomer via a covalent bond to the phosphorus of the terminal linking group. Conjugation in this manner may be with or without a linker moiety as described above.

[0031] In yet other embodiments, the peptide may be conjugated to the oligomer at the 3'-terminus of the oligomer. In certain further embodiments, the peptide may be conjugated to the nitrogen atom of the 3'-terminal morpholino group of the oligomer. In this regard, the peptide may be conjugated to the oligomer either directly or via a linker moiety as described above.

[0032] In some embodiments, the oligomer may be conjugated to a moiety that enhances the solubility of the oligomer in aqueous media. In some embodiments, the moiety that enhances the solubility of the oligomer in aqueous media is polyethylene glycol. In yet further embodiments, the moiety that enhances the solubility of the oligomer in aqueous media is triethylene glycol. For example, in some embodiments, the moiety that enhances the solubility of the oligomer in aqueous media may be conjugated to the oligomer at the 5' end of the oligomer. The moiety that enhances the solubility of the oligomer in aqueous media may be conjugated to the oligomer either directly or through a linker moiety as described above.

[0033] Certain embodiments of the present invention provide antisense molecules selected and / or adapted to assist in the prophylactic or therapeutic treatment of genetic disorders, comprising at least an antisense molecule in a form suitable for delivery to a patient.

[0034] Certain embodiments of the present invention provide methods for treating patients suffering from genetic diseases in which there is a mutation in a gene encoding a particular protein and the effect of the mutation can be neutralized by exon skipping, comprising: (a) selecting an antisense molecule according to the methods described herein; and (b) administering the molecule to a patient in need of such treatment. The present invention also includes the use of the purified and isolated antisense oligonucleotides of the present invention for the manufacture of a medicament for the treatment of a genetic disease.

[0035] Certain embodiments provide methods for treating muscular dystrophy, such as conditions characterized by Duchenne muscular dystrophy, comprising administering to the patient an effective amount of a suitably designed antisense oligonucleotide as described herein, which is associated with a specific genetic lesion in the patient in need of treatment. Additionally, certain embodiments provide methods for prophylactically treating a patient to prevent or at least minimize muscular dystrophy, including Duchenne muscular dystrophy, comprising administering to the patient an effective amount of an antisense oligonucleotide or a pharmaceutical composition comprising one or more of these biological molecules.

[0036] Certain embodiments relate to methods of treating muscular dystrophy in a subject, comprising administering to the subject an effective amount of a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 1-569 and 612-635 and is capable of forming a heteroduplex structure with a complementary mRNA sequence in a dystrophin gene exon, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C, wherein the exon is selected from the group consisting of exons 44-55.

[0037] In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD). In certain embodiments, the muscular dystrophy is Becker muscular dystrophy (BMD).

[0038] In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 1-20, and the exon is exon 44. In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 21-76 and 612-624, and the exon is exon 45.

[0039] In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 77-125 and the exon is exon 46. In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 126-169 and the exon is exon 47.

[0040] In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 170-224 and 634, and the exon is exon 48. In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 225-266, and the exon is exon 49.

[0041] In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 267-308 and the exon is exon 50. In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 309-371 and the exon is exon 51.

[0042] In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 372-415, and the exon is exon 52. In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 416-475 and 625-633, and the exon is exon 53. In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 476-519, and the exon is exon 54. In certain embodiments, the sequence is selected from the group consisting of SEQ ID NOs: 520-569, and 635, and the exon is exon 55. In certain embodiments, the sequence comprises or consists essentially of SEQ ID NO: 287.

[0043] Certain embodiments provide kits for treating genetic diseases, the kit comprising at least one antisense oligonucleotide of the invention packaged in a suitable container and instructions for its use. In preferred embodiments of the present invention, for example, the following is provided: (Item 1) 1. A composition for use in causing exon 44 skipping in the processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 20 and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 44 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 2) 2. The composition of claim 1, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 8, 11, and 12. (Item 3) 2. The composition of claim 1, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 4) 1. A composition for use in causing skipping of exon 45 in processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 21-76 and 612-624, and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 45 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 5) 5. The composition of claim 4, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 27, 29, 34, and 39. (Item 6) 6. The composition of claim 5, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 29 and 34. (Item 7) 5. The composition according to item 4, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 8) 1. A composition for use in causing skipping of exon 46 in processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 77 to 125 and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 46 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 9) Item 9. The composition according to item 8, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 77 to 105. (Item 10) 10. The composition of claim 9, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 82, 84-87, 90, 96, 98, 99, and 101. (Item 11) 9. The composition of claim 8, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 12) 1. A composition for use in causing skipping of exon 47 in processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 126 to 169 and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 47 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 13) Item 13. The composition according to item 12, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 126 to 149. (Item 14) Item 14. The composition according to item 13, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 126, 128-130, 132, 144, and 146-149. (Item 15) Item 13. The composition of item 12, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 16) 1. A composition for use in causing skipping of exon 48 in processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 170-224, and 634, and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 48 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 17) Item 17. The composition according to item 16, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 170 to 201, and 634. (Item 18) Item 18. The composition of item 17, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 176, 178, 181-183, 194, and 198-201. (Item 19) Item 17. The composition of item 16, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 20) 1. A composition for use in causing exon 49 skipping in the processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 225 to 266 and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 49 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 21) 21. The composition according to item 20, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 225 to 248. (Item 22) 22. The composition of claim 21, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 227, 229, 234, 236, 237, and 244-248. (Item 23) 21. The composition of claim 20, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 24) 1. A composition for use in causing skipping of exon 50 in processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 267-308 and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 50 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 25) 25. The composition of claim 24, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 277, 287, 290, and 291. (Item 26) 26. The composition of claim 25, wherein the compound comprises a sequence consisting of SEQ ID NO: 287. (Item 27) 25. The composition of claim 24, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 28) 1. A composition for use in causing skipping of exon 51 in processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 309 to 371 and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 51 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 29) 29. The composition of claim 28, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 324, 326, and 327. (Item 30) 30. The composition of claim 29, wherein the compound comprises a sequence consisting of SEQ ID NO: 327. (Item 31) 29. The composition of item 28, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 32) 1. A composition for use in causing skipping of exon 52 in processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 372 to 415 and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 52 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 33) 33. The composition according to item 32, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 372 to 397. (Item 34) 34. The composition according to item 33, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 379 to 382, ​​384, 390, and 392 to 395. (Item 35) 33. The composition of claim 32, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 36) 1. A composition for use in causing exon 53 skipping in the processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 416-475 and 625-633, and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 53 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 37) 37. The composition of claim 36, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 428, 429, and 431. (Item 38) 38. The composition of claim 37, wherein the compound contains a sequence consisting of SEQ ID NO: 429. (Item 39) 37. The composition of claim 36, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 40) 1. A composition for use in causing skipping of exon 54 in processing of preprocessed mRNA of human dystrophin, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 476 to 519 and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 54 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 41) 41. The composition according to item 40, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 476 to 499. (Item 42) 42. The composition according to item 41, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 479-482, 484, 489, and 491-493. (Item 43) 41. The composition of claim 40, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 44) 1. A composition for use in causing skipping of exon 55 in processing of a preprocessed mRNA transcript of the human dystrophin gene, the composition comprising: 1. A composition comprising a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 520-569 and 635, and is capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 55 of the dystrophin gene, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C. (Item 45) 45. The composition according to item 44, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 520 to 546, and 635. (Item 46) 46. ​​The composition of item 45, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOs: 524-528, 537, 539, 540, 542, and 544. (Item 47) 45. The composition of claim 44, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOs: 570 to 578. (Item 48) A method for treating muscular dystrophy in a subject, the method comprising the step of administering to the subject an effective amount of a substantially uncharged antisense compound containing 20 to 35 morpholino subunits linked by phosphorus-containing intersubunit linkages that connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the compound comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 569 and 612 to 635 and is capable of forming a heteroduplex structure with a complementary mRNA sequence in a dystrophin gene exon, wherein the heteroduplex structure between the compound and the mRNA has a Tm of at least 45°C, and the exon is selected from the group consisting of exons 44 to 55. (Item 49) Item 49. The method of item 48, wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD). (Item 50) Item 49. The method of item 48, wherein the muscular dystrophy is Becker muscular dystrophy (BMD). (Item 51) Item 49. The method according to Item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 1 to 20, and the exon is exon 44. (Item 52) Item 49. The method according to item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 21 to 76 and 612 to 624, and the exon is exon 45. (Item 53) Item 49. The method according to Item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 77 to 125, and the exon is exon 46. (Item 54) Item 49. The method according to Item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 126 to 169, and the exon is exon 47. (Item 55) Item 49. The method of item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 170 to 224 and 634, and the exon is exon 48. (Item 56) Item 49. The method according to Item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 225 to 266, and the exon is exon 49. (Item 57) Item 49. The method of item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 267 to 308, and the exon is exon 50. (Item 58) Item 49. The method of item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 309 to 371, and the exon is exon 51. (Item 59) Item 49. The method of item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 372 to 415, and the exon is exon 52. (Item 60) Item 49. The method according to item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 416 to 475 and 625 to 633, and the exon is exon 53. (Item 61) Item 49. The method according to Item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 476 to 519, and the exon is exon 54. (Item 62) Item 49. The method of item 48, wherein the sequence is selected from the group consisting of SEQ ID NOs: 520 to 569 and 635, and the exon is exon 55. (Item 63) 49. The method of item 48, wherein the sequence comprises SEQ ID NO: 287. (Item 64) Item 65. The method of item 48, wherein the compound is conjugated to an arginine-rich peptide. Item 65. The method according to Item 64, wherein the arginine-rich peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 570 to 578.

[0044] These and other objects and features will be more fully understood when the following detailed description of the invention is read in conjunction with the drawings. [Brief explanation of the drawings]

[0045] [Figure 1A] FIG. 1A shows an exemplary morpholino oligomer structure having phosphorodiamidate linkages. [Figure 1B] FIG. 1B shows a conjugate of an arginine-rich peptide and an antisense oligomer, according to an embodiment of the present invention. [Figure 1C] FIG. 1C shows a conjugate as in FIG. 1B in which the backbone linkages contain one or more positively charged groups. [Figure 1D] 1D-G show exemplary morpholino oligonucleotide repeat subunit segments designated D through G. [Figure 1E] 1D-G show exemplary morpholino oligonucleotide repeat subunit segments designated D through G. [Figure 1F] 1D-G show exemplary morpholino oligonucleotide repeat subunit segments designated D through G. [Figure 1G]1D-G show exemplary morpholino oligonucleotide repeat subunit segments designated D through G. [Figure 2A] FIG. 2A shows the relative location and results of antisense oligomers designed to induce human dystrophin exon 51 skipping. [Figure 2B] Figures 2B-C show the relative activity in cultured human rhabdomyosarcoma (RD) cells and human primary skeletal muscle cells of the three best oligomers selected from the exon 51 scan (SEQ ID NOs: 324, 326, and 327) compared to sequences effective in inducing exon 51 skipping (AVI-5658; SEQ ID NO: 588 and h51AON1; SEQ ID NO: 594). [Figure 2C] Figures 2B-C show the relative activity in cultured human rhabdomyosarcoma (RD) cells and human primary skeletal muscle cells of the three best oligomers selected from the exon 51 scan (SEQ ID NOs: 324, 326, and 327) compared to sequences effective in inducing exon 51 skipping (AVI-5658; SEQ ID NO: 588 and h51AON1; SEQ ID NO: 594). [Figure 2D] FIG. 2D shows the relative locations within exon 51 of three selected oligomers compared to the specified sequence. [Figure 3A] FIG. 3A shows the relative locations and results of an antisense oligomer exon 50 scan designed to induce skipping of human dystrophin exon 50 compared to other sequences that induce exon 50 skipping. [Figure 3B] FIG. 3B shows the relative positions and results of antisense sequences selected from the exon 50 scan (SEQ ID NOs: 277, 287, 290, and 291) compared to other sequences (SEQ ID NOs: 584 and 585). [Figure 4A] FIG. 4A shows the relative locations and results of an antisense oligomer exon 53 scan designed to induce skipping of human dystrophin exon 53. [Figure 4B]Figure 4B shows the relative positions of specific sequences used to compare exon skipping activity to oligomers selected to be most active in the exon 53 scan. [Figure 4C] Figures 4C-F show the results of the dose-ranging study summarized in Figure 4G, using oligomers selected as most effective in the exon 53 scan (SEQ ID NOS: 422, 428, 429, and 431). [Figure 4D] Figures 4C-F show the results of the dose-ranging study summarized in Figure 4G, using oligomers selected as most effective in the exon 53 scan (SEQ ID NOS: 422, 428, 429, and 431). [Figure 4E] Figures 4C-F show the results of the dose-ranging study summarized in Figure 4G, using oligomers selected as most effective in the exon 53 scan (SEQ ID NOS: 422, 428, 429, and 431). [Figure 4F] Figures 4C-F show the results of the dose-ranging study summarized in Figure 4G, using oligomers selected as most effective in the exon 53 scan (SEQ ID NOS: 422, 428, 429, and 431). [Figure 4G] Figures 4C-F show the results of the dose-ranging study summarized in Figure 4G, using oligomers selected as most effective in the exon 53 scan (SEQ ID NOS: 422, 428, 429, and 431). [Figure 4H] Figures 4H and 4I show the relative activity of specific sequences (SEQ ID NOs: 608-611) compared to the activity of the most active exon 53 skipping oligomer (SEQ ID NO: 429) in both RD cells and human primary skeletal muscle cells. [Figure 4I] Figures 4H and 4I show the relative activity of specific sequences (SEQ ID NOs: 608-611) compared to the activity of the most active exon 53 skipping oligomer (SEQ ID NO: 429) in both RD cells and human primary skeletal muscle cells. [Figure 5A]FIG. 5A shows the relative locations and results of an antisense oligomer exon 44 scan designed to induce skipping of human dystrophin exon 44. [Figure 5B] FIG. 5B shows the relative location within exon 44 of specific sequences used to compare exon skipping activity against oligomers selected to be most active in the exon 44 scan. [Figure 5C] Figures 5C-G show the results of the dose-ranging study summarized in Figure 5H using oligomers selected as most effective in the exon 44 scan (SEQ ID NOS: 4, 8, 11, 12, and 13). [Figure 5D] Figures 5C-G show the results of the dose-ranging study summarized in Figure 5H using oligomers selected as most effective in the exon 44 scan (SEQ ID NOS: 4, 8, 11, 12, and 13). [Figure 5E] Figures 5C-G show the results of the dose-ranging study summarized in Figure 5H using oligomers selected as most effective in the exon 44 scan (SEQ ID NOS: 4, 8, 11, 12, and 13). [Figure 5F] Figures 5C-G show the results of the dose-ranging study summarized in Figure 5H using oligomers selected as most effective in the exon 44 scan (SEQ ID NOS: 4, 8, 11, 12, and 13). [Figure 5G] Figures 5C-G show the results of the dose-ranging study summarized in Figure 5H using oligomers selected as most effective in the exon 44 scan (SEQ ID NOS: 4, 8, 11, 12, and 13). [Figure 5H] Figures 5C-G show the results of the dose-ranging study summarized in Figure 5H using oligomers selected as most effective in the exon 44 scan (SEQ ID NOS: 4, 8, 11, 12, and 13). [Figure 5I]Figures 5I and 5J show the relative activity of specific sequences (SEQ ID NOs: 600-603) compared to the activity of the most active exon 53 skipping oligomer (SEQ ID NO: 12) in both RD cells and human primary skeletal muscle cells. [Figure 5J] Figures 5I and 5J show the relative activity of specific sequences (SEQ ID NOs: 600-603) compared to the activity of the most active exon 53 skipping oligomer (SEQ ID NO: 12) in both RD cells and human primary skeletal muscle cells. [Figure 6A] FIG. 6A shows the relative locations and results of an antisense oligomer exon 45 scan designed to induce skipping of human dystrophin exon 45. [Figure 6B] FIG. 6B shows the relative location within exon 45 of specific sequences used to compare exon skipping activity against oligomers selected to be most active in the exon 45 scan. [Figure 6C] Figures 6C-F show the results of the dose-ranging study summarized in Figure 6H using oligomers selected as most effective in the exon 45 scan (SEQ ID NOs: 27, 29, 34, and 39). Figure 6G uses a relatively inactive oligomer (SEQ ID NO: 49) as a negative control. [Figure 6D] Figures 6C-F show the results of the dose-ranging study summarized in Figure 6H using oligomers selected as most effective in the exon 45 scan (SEQ ID NOs: 27, 29, 34, and 39). Figure 6G uses a relatively inactive oligomer (SEQ ID NO: 49) as a negative control. [Figure 6E] Figures 6C-F show the results of the dose-ranging study summarized in Figure 6H using oligomers selected as most effective in the exon 45 scan (SEQ ID NOs: 27, 29, 34, and 39). Figure 6G uses a relatively inactive oligomer (SEQ ID NO: 49) as a negative control. [Figure 6F]Figures 6C-F show the results of the dose-ranging study summarized in Figure 6H using oligomers selected as most effective in the exon 45 scan (SEQ ID NOs: 27, 29, 34, and 39). Figure 6G uses a relatively inactive oligomer (SEQ ID NO: 49) as a negative control. [Figure 6G] Figures 6C-F show the results of the dose-ranging study summarized in Figure 6H using oligomers selected as most effective in the exon 45 scan (SEQ ID NOs: 27, 29, 34, and 39). Figure 6G uses a relatively inactive oligomer (SEQ ID NO: 49) as a negative control. [Figure 6H] Figures 6C-F show the results of the dose-ranging study summarized in Figure 6H using oligomers selected as most effective in the exon 45 scan (SEQ ID NOs: 27, 29, 34, and 39). Figure 6G uses a relatively inactive oligomer (SEQ ID NO: 49) as a negative control. [Figure 6I] Figures 6I and 6J show the relative activity of specific sequences (SEQ ID NOs: 604-607) compared to the activity of the most active exon 53 skipping oligomer (SEQ ID NO: 34) in both RD cells and human primary skeletal muscle cells. [Figure 6J] Figures 6I and 6J show the relative activity of specific sequences (SEQ ID NOs: 604-607) compared to the activity of the most active exon 53 skipping oligomer (SEQ ID NO: 34) in both RD cells and human primary skeletal muscle cells. DETAILED DESCRIPTION OF THE INVENTION

[0046] The embodiments of the present invention generally relate to the improvement of antisense compounds and their use methods, which are specifically designed to induce exon skipping in dystrophin gene.Dystrophin plays a vital role in muscle function, and various muscle-related diseases are characterized by the mutation of this gene.Therefore, in certain embodiments, the improvement of antisense compounds described herein induces exon skipping in mutant human dystrophin gene, such as the mutant dystrophin gene found in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

[0047] Due to the abnormal mRNA splicing events caused by mutations, these mutant human dystrophin genes either express defective dystrophin proteins or do not express any measurable dystrophin, which leads to various types of muscular dystrophy.To treat this condition, the antisense compounds of the present invention typically hybridize to selected regions of the pre-processed RNA of mutant human dystrophin genes, and induce exon skipping and differential splicing in otherwise abnormally spliced ​​dystrophin mRNA, thereby allowing muscle cells to produce mRNA transcripts encoding functional dystrophin proteins.In certain embodiments, the resulting dystrophin protein is not necessarily "wild-type" dystrophin, but rather a truncated, yet functional or semi-functional form of dystrophin.

[0048] By increasing the levels of functional dystrophin protein in muscle cells, these and related embodiments may be useful in the prevention and treatment of forms of muscular dystrophies, such as DMD and BMD, which are characterized by the expression of defective dystrophin protein due to aberrant mRNA splicing. Certain oligomers described herein further provide improved dystrophin-exon-specific targeting over other oligomers in use, thereby offering significant practical advantages over alternative methods of treating related forms of muscular dystrophies.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0050] definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0051] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the referenced quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0052] "Coding sequence" means any nucleic acid sequence that contributes to the coding of the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not contribute to the coding of the polypeptide product of a gene.

[0053] Throughout this specification, unless the context requires to the contrary, the words "comprise", "comprises" and "comprising" will be understood to mean the inclusion of the stated step or component or group of steps or components, but not to the exclusion of any other step or component or group of steps or components.

[0054] "Consisting of" is meant to include and be limited to whatever comes before the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed components are required or essential, and that other components may not be present. "Consisting essentially of" is meant to include any components listed before the phrase, and is limited to other components that do not interfere with or contribute to the activity or action identified in this disclosure for the listed components. Thus, the phrase "consisting essentially of" indicates that the listed components are required or essential, but that other components are optional and may or may not be present depending on whether they materially affect the activity or action of the listed components.

[0055] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., sequences of nucleotides) related by the base-pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA." Complementarity can be "partial," in which only some of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. While complete complementarity is often desired, certain embodiments can include one or more, but preferably 6, 5, 4, 3, 2, or 1, mismatches with respect to the target RNA. Variations at any position within the oligomer are included. In certain embodiments, sequence variations near the ends of the oligomer are generally preferred, with internal variations being preferred, and, if present, are typically within about 6, 5, 4, 3, 2, or 1 nucleotide of the 5' and / or 3' ends.

[0056] The terms "cell penetrating peptide" or "CPP" are used interchangeably and refer to cationic cell penetrating peptides, also known as transport peptides, carrier peptides, or peptide transduction domains. These peptides, as provided herein, have the potential to induce cell penetration in 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells of a given cell culture population, including all integers therebetween, and enable macromolecular transport in multiple tissues in vivo upon systemic administration.

[0057] The terms "antisense oligomer" or "antisense compound" are used interchangeably and refer to an array of cyclic subunits, each having a base-pairing moiety, joined by intersubunit linkages that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically RNA) by Watson-Crick base pairing, forming a nucleic acid:oligomer heteroduplex in the target sequence. The cyclic subunits are based on ribose or another pentose sugar, or, in preferred embodiments, morpholino groups (see description of morpholino oligomers below).

[0058] Such antisense oligomers can be designed to block or inhibit translation of mRNA or to inhibit natural pre-mRNA splicing processing and may be said to be "directed" or "targeted" to the target sequence to which they hybridize. In certain embodiments, the target sequence includes the AUG start codon of the mRNA, the 3' or 5' splice site of the preprocessed mRNA, or a region containing a branch point. The target sequence may be within an exon or an intron. Target sequences for splice sites may include mRNA sequences having 1 to about 25 base pairs of their 5' end downstream of the normal splice acceptor junction in the preprocessed mRNA. Preferred target sequences for splicing are any region of the preprocessed mRNA that contains a splice site, whether entirely contained within exon coding sequence or spanning a splice acceptor or donor site. Oligomers are more commonly said to "target" a biologically relevant target, such as a protein, virus, or bacterium, when they are targeted to the target nucleic acid in the manner described above. Included are antisense oligomers that comprise, consist essentially of, or consist of one or more of SEQ ID NOS: 1-569 and 612-635. Also included are variants of these antisense oligomers, including those with 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity or homology (including all integers therebetween) to any one of SEQ ID NOS: 1-569 and 612-635, and / or variants that differ from these sequences by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, preferably those that induce exon skipping of one or more selected human dystrophin exons.Also included are oligomers of any one or more of SEQ ID NOs: 584-611 and 634-635, which contain an appropriate number of charged linkages as described herein, e.g., up to about 1 for every 2-5 uncharged linkages, such as about 4-5 for every 10 uncharged linkages, and / or contain an Arg-rich peptide attached thereto, also as described herein.

[0059] "Morpholino oligomer" or "PMO" (phosphoramidate or phosphorodiamidate morpholino oligomer) refers to an oligonucleotide analog composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus-containing linkages that are 1 to 3 atoms long, preferably 2 atoms long, and preferably uncharged or cationic, connecting the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring has a purine or pyrimidine base-pairing moiety that is effective for binding to a base in a polynucleotide by base-specific hydrogen bonding. See, for example, the structure in Figure 1A, which shows a preferred phosphorodiamidate linkage. Variations can be made to this linkage as long as they do not interfere with binding or activity. For example, the oxygen attached to the phosphorus may be substituted with sulfur (thiophosphorodiamidate). The 5' oxygen may be substituted with amino or lower alkyl-substituted amino. The pendant nitrogen attached to the phosphorus may be unsubstituted, monosubstituted, or disubstituted with an (optionally substituted) lower alkyl. See also the discussion of cationic linkage below. The synthesis, structure, and binding properties of morpholino oligomers are detailed in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, as well as PCT Application No. PCT / US07 / 11435 (cationic linkage), all of which are incorporated herein by reference.

[0060] Purine or pyrimidine base pairing moieties typically also include, for example, the following bases: adenine, cytosine, guanine, uracil, thymine, or inosine. pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethyloxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), or 6-azapyrimidine or 6-alkylpyrimidine (e.g., 6-methyluridine), propyne, quesocine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, β-D-gas Lactosilcoeosin, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, β-D-mannosilcoeosin, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). In this regard, "modified base" means a nucleotide base other than adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), as exemplified above; such bases can be used at any position in the antisense molecule. Those skilled in the art will understand that T and U are interchangeable, depending on the use of the oligomer.For example, with other antisense chemistries such as 2'-O-methyl antisense oligonucleotides, which are more RNA-like, T bases may be represented as U (see, eg, the table of SEQ ID NOs).

[0061] An "amino acid subunit" or "amino acid residue" refers to an alpha amino acid residue (e.g., -CO-CHR-NH-) or a beta- or other amino acid residue (e.g., -CO-(CH2) n CHR—NH—), where R is a side chain (which may contain hydrogen), and n is 1 to 6, preferably 1 to 4.

[0062] "Naturally occurring amino acids" refer to amino acids present in proteins found in nature, such as the 20 (L) amino acids utilized during protein biosynthesis, as well as other amino acids such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. The term "unnatural amino acids" refers to amino acids not present in proteins found in nature, and examples include beta-alanine (β-Ala; or B), 6-aminohexanoic acid (Ahx), and 6-aminopentanoic acid. Further examples of "unnatural amino acids" include, but are not limited to, the (D)-amino acids norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like, which are known to those skilled in the art.

[0063] "Effective amount" or "therapeutically effective amount" refers to the amount of a therapeutic compound, such as an antisense oligomer, administered to a mammalian subject, either as a single dose or as part of a series of doses, that is effective to produce a desired physiological response or therapeutic effect in the subject. One example of a desired physiological response includes an increase in the expression of a relatively functional or biologically active form of dystrophin protein, primarily in muscle tissues or cells that contain defective or non-dystrophin dystrophin protein, compared to the absence of the antisense oligomer or control oligomer. Examples of desired therapeutic effects include, but are not limited to, amelioration of the symptoms or pathology of muscular dystrophy, reduction in the progression of the symptoms or pathology of muscular dystrophy, and delay in the onset of the symptoms or pathology of muscular dystrophy. Examples of such symptoms include fatigue, mental retardation, muscle weakness, difficulty with motor skills (e.g., running, hopping, jumping), frequent falls, and difficulty walking. Muscular dystrophy pathology can be characterized, for example, by muscle fiber damage and membrane leakage. For antisense oligomers, this effect is typically brought about by altering the splicing process of a selected target sequence (e.g., dystrophin), for example, to induce exon skipping.

[0064] "Exon" refers to a defined section of a nucleic acid that encodes a protein, or a nucleic acid sequence that is represented in a mature RNA molecule after any portion of a preprocessed (or precursor) RNA is removed by splicing. A mature RNA molecule can be a messenger RNA (mRNA) or a functional form of non-coding RNA, such as rRNA or tRNA. The human dystrophin gene has approximately 75 exons.

[0065] "Intron" refers to a nucleic acid region (in a gene) that is not translated into protein. Introns are non-coding sections that are transcribed into precursor mRNA (pre-mRNA) and subsequently removed by splicing during formation of the mature RNA.

[0066] "Exon skipping" generally refers to the process by which an entire exon, or a portion thereof, is removed from a given preprocessed RNA, thereby eliminating it from being present in a mature RNA, such as a mature mRNA, that is translated into a protein. Thus, the portion of the protein that would otherwise be encoded by the skipped exon is absent from the expressed form of the protein, typically creating an altered, but still functional, form of the protein. In certain embodiments, the skipped exon is an aberrant exon from the human dystrophin gene, which may contain a mutation or other change in its sequence that would otherwise cause aberrant splicing. In certain embodiments, the skipped exon is any one or more of exons 1-75 of the dystrophin gene, although any one or more of exons 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and / or 55 of the human dystrophin gene are preferred.

[0067] "Dystrophin" is a rod-shaped cytoplasmic protein, an integral part of the protein complex that connects the cytoskeleton of muscle fibers to the surrounding extracellular matrix through the cell membrane. Dystrophin contains multiple functional domains. For example, dystrophin contains an actin-binding domain at approximately amino acids 14–240 and a central rod-shaped domain at approximately amino acids 253–3040. This large central domain is formed by 24 spectrin-like triple-helical elements of approximately 109 amino acids, which share homology with α-actinin and spectrin. The repeats are typically interrupted by four proline-rich non-repetitive segments, also called hinge regions. Repeats 15 and 16 are separated by an 18-amino acid stretch that appears to provide the primary site for proteolytic cleavage of dystrophin. Sequence identity among the various repeats ranges from 10–25%. A single repeat contains three α-helices: 1, 2, and 3. Alpha helices 1 and 3 are each formed by seven helical turns, which likely interact as a coiled coil through a hydrophobic interface. Alpha helix 2 is formed by segments of four or three helical turns separated by glycine or proline residues. Each repeat is encoded by two exons and is typically interrupted by an intron between amino acids 47 and 48 in the first part of alpha helix 2. Other introns are found at different positions within this repeat, usually dispersed throughout helix 3. Dystrophin also contains a cysteine-rich domain at approximately amino acids 3080–3360, which contains a cysteine-rich segment (i.e., 15 cysteines in 280 amino acids) that shows homology to the C-terminal domain of Dictyostelium discoideum alpha actinin. The carboxy-terminal domain is located at approximately amino acids 3361–3685.

[0068] The amino terminus of dystrophin binds to F-actin, and the carboxy terminus binds to the dystrophin-associated protein complex (DAPC) in the muscle cell membrane. DAPC contains dystroglycan, sarcoglycan, integrin, and caveolin, and mutations in any of these components cause autosomal inherited muscular dystrophy. DAPC becomes unstable in the absence of dystrophin, which results in reduced levels of its protein members and, in turn, leads to progressive fiber damage and membrane leakage. In various types of muscular dystrophies, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), muscle cells produce altered and functionally defective forms of dystrophin, or no dystrophin at all, primarily due to mutations in the gene sequence that lead to incorrect splicing. The predominant expression of defective dystrophin proteins or the complete absence of dystrophin or dystrophin-like proteins leads to the rapid progression of muscle degeneration, as described above. In this regard, a "deficient" dystrophin protein may be characterized by the type of dystrophin produced in a particular subject with DMD or BMD, or by the absence of detectable dystrophin, as is known in the art.

[0069] Table A provides examples of various dystrophin domains, the amino acid residues that encompass these domains, and the exons that encode them. [Table A-1]

[0070] [Table A-2]

[0071] As used herein, the terms "function" and "functional" and similar terms refer to biological, enzymatic, or therapeutic function.

[0072] A "functional" dystrophin protein generally refers to a dystrophin protein that has sufficient biological activity to reduce the progressive degradation of muscle tissue otherwise characteristic of muscular dystrophies, compared to altered or "missing" dystrophin proteins typically present in subjects with DMD or BMD. In certain embodiments, a functional dystrophin protein may have approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers therebetween) of the in vitro or in vivo biological activity of wild-type dystrophin, as measured according to routine techniques in the art. As one example, dystrophin-related activity in in vitro muscle cultures can be measured according to myotube size, myofibrillar organization (disorganization), contractility, and spontaneous clustering of acetylcholine receptors (see, e.g., Brown et al., Journal of Cell Science. 112:209-216, 1999). Animal models are also valuable resources for studying disease pathogenesis and provide a means to test dystrophin-associated activities. Two of the most widely used animal models for DMD research are the mdx mouse and the Golden Retriever Muscular Dystrophy (GRMD) dog, both of which are dystrophin-negative (e.g., Collins and Morgan, Int J Exp J Neurosci (2018) 103:131–132). (See Pathol 84:165-172, 2003.) These and other animal models can be used to measure the functional activity of various dystrophin proteins, including truncated forms of dystrophin such as those produced by certain exon-skipping antisense compounds of the invention.

[0073] "Gene" means a unit of heredity occupying a specific locus on a chromosome and consisting of transcriptional and / or translational regulatory sequences, and / or coding regions and / or untranslated sequences (i.e., introns, 5' and 3' untranslated sequences).

[0074] "Isolated" means material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polynucleotide," as used herein, can refer to a polynucleotide that has been purified or removed from the sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from the sequences that normally flank the DNA fragment.

[0075] "Enhance" or "enhancing" or "increase" or "increasing" or "stimulate" or "stimulating" generally refers to the ability of one or more antisense compounds or compositions to produce or cause a greater physiological response (i.e., a downstream effect) in a cell or subject compared to the response caused by the absence of the antisense compound or by either a control compound. A measurable physiological response may include an increase in the expression of a functional form of dystrophin protein or an increase in dystrophin-related biological activity in muscle tissue, among other responses that are apparent from the understanding in the art and the description herein. Increases in muscle function can also be measured, including an increase or improvement in muscle function of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The percentage of muscle fibers expressing functional dystrophin can also be measured, including increasing dystrophin expression in about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of muscle fibers. For example, it has been shown that an improvement in muscle function of about 40% can occur when 25-30% of fibers express dystrophin (see, e.g., DelloRusso et al., Proc Natl Acad Sci USA 99:12979-12984, 2002).An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points in between and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the amount produced in the absence of the antisense compound (absence of agent) or by a control compound.

[0076] The terms "reduce" or "inhibit" generally refer to the ability of one or more antisense compounds of the present invention to "reduce" a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic field. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in the symptoms or pathology of muscular dystrophy, or a reduction in the expression of defective forms of dystrophin, e.g., an altered form of dystrophin expressed in individuals with DMD or BMD. A "reduction" in response may be statistically significant compared to the response produced in the absence of the antisense compound or by a control composition and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction, including all integers in between.

[0077] "Homology" refers to the percentage of amino acids that are identical or that constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of similar or substantially different lengths to those cited herein can be compared by inserting gaps into the alignment, and such gaps are determined, for example, by the comparison algorithm used by GAP.

[0078] As used herein, the term "sequence identity" or, for example, "a sequence that is 50% identical to" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a comparison window. Thus, "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is present in both sequences, resulting in the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to produce the percentage of sequence identity.

[0079] Terms used to describe sequence relatedness between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is at least 8 or 10, but frequently 15-18, and often at least 25, monomeric units in length, inclusive, in both nucleotides and amino acid residues. Two polynucleotides may each contain (1) sequences that are similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) sequences that are mismatched between the two polynucleotides. Sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6, usually about 50 to about 100, more usually about 100 to about 150 contiguous positions, in which one sequence is compared to the reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. For optimal alignment of two sequences, the comparison window may contain about 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions). Optimal alignment of sequences for aligning the comparison window may be performed by computerized algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA), or by examining and selecting the best alignment (i.e., the one that produces the highest percentage of homology across the comparison window) produced by any of the various methods. Reference may also be made to the BLAST family of programs, for example, as disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0080] "Treatment" or "treating" of an individual (e.g., a mammal, such as a human) or cell may include any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be performed either prophylactically or following contact with the initiation or etiology of a pathological event. Treatment may include any desired effect on the symptoms or pathology of a disease or condition associated with dystrophin protein, such as in certain types of muscular dystrophies, and may include, for example, a minimal change or improvement in one or more measurable markers of the disease or condition being treated. "Prophylactic" treatment is also included, which may be directed toward reducing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate a complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0081] Thus, included are methods of treating muscular dystrophies, such as DMD and BMD, by administering one or more antisense oligomers of the present invention (e.g., SEQ ID NOS: 1-569 and 612-635, and variants thereof) to a patient in need thereof, optionally as part of a pharmaceutical formulation or dosage form. Also included are methods of inducing exon skipping in a subject by administering one or more antisense oligomers, wherein the exon is one of exons 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and / or 55 from the dystrophin gene, preferably the human dystrophin gene. "Subject," as used herein, includes any animal exhibiting or at risk of exhibiting symptoms that can be treated with the antisense compounds of the present invention, for example, a subject having or at risk of having DMD or BMD, or any of the symptoms associated with these conditions (e.g., muscle fiber loss). Suitable subjects (patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs), as well as non-human primates and, preferably, human patients.

[0082] Also included are vector delivery systems capable of expressing the oligomeric, dystrophin-targeting sequences of the present invention, such as vectors expressing polynucleotide sequences comprising any one or more of SEQ ID NOS: 1-569 and 612-635, or variants thereof, as described herein. "Vector" or "nucleic acid construct" refers to a polynucleotide molecule, preferably a DNA molecule, derived from, for example, a plasmid, bacteriophage, yeast, or virus, into which a polynucleotide can be inserted or cloned. The vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell, including a target cell or tissue or a progenitor cell or tissue thereof, or may be capable of integrating into the genome of a defined host cell so that the cloned sequence can be reproduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, such as a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may include any means for ensuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

[0083] A vector or nucleic acid construct system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the entire DNA or transposon to be introduced into the genome of a host cell. The choice of vector typically depends on the vector's compatibility with the host cell into which it will be introduced. In the present case, the vector or nucleic acid construct is preferably one that is operably functional in mammalian cells, such as muscle cells. The vector can also contain a selectable marker, such as an antibiotic or drug resistance gene or a reporter gene (i.e., green fluorescent protein, luciferase), which can be used to select or identify suitable transformants or transfectants. Exemplary delivery systems can include viral vector systems (i.e., virus-mediated transduction), including, but not limited to, retroviral (e.g., lentiviral) vectors, adenoviral vectors, adeno-associated viral vectors, and herpes viral vectors, among others known in the art.

[0084] The term "operably linked," as used herein, means placing a sequence encoding an oligomer under the regulatory control of a promoter, which in turn controls transcription of the oligomer.

[0085] A wild-type gene or gene product is that which is most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild-type" form of that gene.

[0086] "Alkyl" or "alkylene" both refer to saturated straight or branched chain hydrocarbon groups containing 1 to 18 carbons. Examples include, but are not limited to, methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl, and n-hexyl. The term "lower alkyl" refers to an alkyl group, as defined herein, containing between 1 and 8 carbons.

[0087] "Alkenyl" refers to an unsaturated, straight or branched chain hydrocarbon group containing 2 to 18 carbons and containing at least one carbon-to-carbon double bond. Examples include, but are not limited to, ethenyl, propenyl, iso-propenyl, butenyl, iso-butenyl, tert-butenyl, n-pentenyl, and n-hexenyl. The term "lower alkenyl" refers to an alkenyl group, as defined herein, containing between 2 and 8 carbons.

[0088] "Alkynyl" refers to an unsaturated, straight or branched chain hydrocarbon group containing 2 to 18 carbons and containing at least one carbon-to-carbon triple bond. Examples include, but are not limited to, ethynyl, propynyl, iso-propynyl, butynyl, iso-butynyl, tert-butynyl, pentynyl, and hexynyl. The term "lower alkynyl" refers to an alkynyl group, as defined herein, containing between 2 and 8 carbons.

[0089] "Cycloalkyl" refers to a mono- or poly-cyclic alkyl group. Examples include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0090] "Aryl" refers to a cyclic aromatic hydrocarbon moiety containing 5 to 18 carbons having one or more closed rings. Examples include, but are not limited to, phenyl, benzyl, naphthyl, anthracenyl, phenanthracenyl, and biphenyl.

[0091] "Aralkyl" refers to a radical of the formula R R b where R R is an alkylene chain as defined above and R b is one or more aryl groups as defined above, e.g., benzyl, diphenylmethyl, and the like.

[0092] "Thioalkoxy" refers to a radical of the formula -SRc, where Rc is an alkyl radical as defined above. The term "lower thioalkoxy" refers to an alkoxy radical containing between 1 and 8 carbons, as defined herein.

[0093] "Alkoxy" refers to a group of formula -ORda, where Rd is an alkyl group, as defined herein. The term "lower alkyl" refers to an alkoxy group, as defined herein, containing between 1 and 8 carbons. Examples of alkoxy groups include, but are not limited to, methoxy and ethoxy.

[0094] "Alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group.

[0095] "Carbonyl" refers to the group -C(=O)-.

[0096] "Guanidinyl" refers to the group H2N(C=NH2)-NH-.

[0097] "Amidinyl" refers to the group H2N(C=NH2)CH-.

[0098] "Amino" refers to the group -NH2.

[0099] "Alkylamino" refers to a group of formula -NHR or -NRR, where each R is independently an alkyl group as defined herein. The term "lower alkylamino" refers to an alkylamino group containing between 1 and 8 carbons, as described herein.

[0100] "Heterocycle" means a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocycle that is either saturated, unsaturated, or aromatic and contains 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the nitrogen and sulfur heteroatoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be bonded via any heteroatom or carbon atom. Heterocycle includes heteroaryl as defined below. Thus, in addition to the heteroaryls listed below, heterocycles also include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiopyranyl, and the like.

[0101] "Heteroaryl" refers to a 5- to 10-membered aromatic heterocycle containing at least one heteroatom selected from nitrogen, oxygen, and sulfur, and at least one carbon atom, including both monocyclic and bicyclic rings. Representative heteroaryls include pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl.

[0102] "Optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkoxy," "optionally substituted thioalkoxy," "optionally substituted alkylamino," "optionally substituted lower alkyl," "optionally substituted lower alkenyl," "optionally substituted lower alkoxy," "optionally substituted lower thioalkoxy," "optionally substituted lower alkylamino," and "optionally substituted heterocyclyl" mean that, when substituted, at least one hydrogen atom is replaced by a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, substituents include deuterium, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted cycloalkyl, oxo, halogen, —CN, —ORx, NRRy, NRC(═O)Ry, NSRySORy, —NRxC(═O)NRxRy, C(═O)Rx, C(═O)ORx, C(═O)NRxRy, —SOmRx, and —SOmNRxRy, where m is 0, 1, or 2, and Rx and Ry are the same or different and independently represent hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted cycloalkyl, oxo, halogen, —CN, —ORx, NRRy, NRC(═O)Ry, NSRySORy, —NRxC(═O)NRxRy, C(═O)Rx, C(═O)ORx, C(═O)NRxRy, —SOmRx, and —SOmNRxRy, where m is 0, 1, or 2, and Rx and Ry are the same or different. and optionally substituted aryl, optionally substituted heterocycle, optionally substituted cycloalkyl, or optionally substituted cycloalkyl, each of which optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted cycloalkyl, and optionally substituted cycloalkyl substituents may be further substituted with one or more of oxo, halogen, —CN, —ORx, NRRy, NRC(=O)Ry, NSRy, —NRxC(=O)NRxRy, C(=O)Rx, C(=O)ORx, C(=O)NRxRy, —SOmRx, and —SOmNRxRy.

[0103] Construction of antisense oligonucleotides Examples of morpholino oligonucleotides with phosphorus-containing backbone linkages are illustrated in Figures 1A-1C. Particularly preferred are phosphorodiamidate-linked morpholino oligonucleotides, as shown in Figure 1C, which, according to one aspect of the present invention, are modified to contain positively charged groups at preferably 10% to 50% of their backbone linkages. Morpholino oligonucleotides with uncharged backbone linkages, including antisense oligonucleotides, and their preparation are described in detail, for example, in Summerton and Weller 1997, and in co-owned U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, and 5,506,337, all of which are expressly incorporated herein by reference.

[0104] Important properties of morpholino-based subunits include: 1) the ability to be linked in oligomeric form by stable, uncharged or positively charged backbone linkages; 2) the ability to support nucleotide bases (e.g., adenine, cytosine, guanine, thymidine, uracil, and inosine) such that the formed polymer can hybridize to complementary base target nucleic acids, including target RNA, at a Tm greater than about 45°C in relatively short oligonucleotides (e.g., 10-15 bases); 3) the ability of the oligonucleotide to be actively or passively transported into mammalian cells; and 4) the ability of the antisense oligonucleotide:RNA heteroduplex to resist RNAse and RNase H degradation, respectively.

[0105] Exemplary backbone structures for the claimed subject antisense oligonucleotides include the morpholino subunit types shown in Figures 1D-1G, in which the subunits are linked by phosphorus-containing linkages, each of which is uncharged or positively charged. Figure 1D shows phosphorus-containing linkages forming a five-atom repeat unit backbone, in which the morpholino rings are linked by one-atom phosphoamide linkages. Figure 1E shows linkages resulting in a six-atom repeat unit backbone. In this structure, the atom Y connecting the 5' morpholino carbon to the phosphorus group may be sulfur, nitrogen, carbon, or, preferably, oxygen. The X moiety pendant from the phosphorus may be fluorine, alkyl or substituted alkyl, alkoxy or substituted alkoxy, thioalkoxy or substituted thioalkoxy, or unsubstituted, monosubstituted, or disubstituted nitrogen, including ring structures such as morpholine or piperidine. Alkyl, alkoxy, and thioalkoxy preferably contain 1 to 6 carbon atoms. The Z moiety is sulfur or oxygen, preferably oxygen.

[0106] The linkages shown in Figures 1F and 1G are referred to as 7-atom unit-length backbones. In Structure 1F, the X moiety is the same as in Structure 1E, and the Y moiety can be methylene, sulfur, or preferably oxygen. In Structure 1G, the X and Y moieties are the same as in Structure 1E. Particularly preferred morpholino oligonucleotides include those composed of morpholino subunit structures of the type shown in Figure 1E, where X = NH2, N(CH3)2, optionally substituted 1-piperazinyl, or other charged group, Y = O, and Z = O.

[0107] As described above, uncharged or substantially uncharged oligonucleotides may be modified in accordance with embodiments of the present invention to contain, for example, up to about one charged linkage for every two to five uncharged linkages, e.g., about four to five linkages for every ten uncharged linkages. Optimal improvement in antisense activity may be seen when about 25% of the backbone linkages are cationic, including about 20% to about 30%. Oligomers in which about 35%, 40%, 45%, 50%, 55%, 60% (including all integers in between) or more of the backbone linkages are cationic are also included. Enhancement may also be seen with smaller numbers, e.g., 5% or 10-20% cationic linkages.

[0108] A substantially uncharged phosphorus-containing backbone in an oligonucleotide analogue is typically one in which the majority of the subunit linkages, e.g., 50% to 100%, typically at least 60% to 100%, or 75% or 80%, are uncharged at physiological pH and contain a single phosphorus atom.

[0109] Further experiments performed in support of the present invention indicate that the enhancement seen with added cationic backbone charge can in some cases be further enhanced by distributing the net charge close to the "center region" backbone linkages of the antisense oligonucleotide, for example, in a 20-mer oligonucleotide having 8 cationic backbone linkages with at least 70% of these charged linkages localized in the 10 most central linkages.

[0110] Antisense compounds can be prepared by stepwise solid-phase synthesis, utilizing the methods detailed in the references cited above and below for the synthesis of oligonucleotides bearing a mixture of uncharged and cationic backbone linkages. In some cases, it may be desirable to add additional chemical moieties to the antisense compound, for example, to enhance pharmacokinetics or facilitate compound capture or detection. Such moieties may typically be covalently attached to the terminus of the oligomer, following standard synthetic methods. For example, the addition of polyethylene glycol moieties or other hydrophilic polymers, e.g., those with 10-100 monomer subunits, may be useful for enhancing solubility. One or more charged groups, e.g., anionic groups such as organic acids, may enhance cellular uptake. Reporter moieties, such as fluorescein or radioactive groups, may be attached for detection purposes. Alternatively, the reporter label attached to the oligomer may be a ligand, such as an antigen or biotin, capable of binding a labeled antibody or streptavidin. In selecting moieties for attachment or modification of antisense compounds, it is of course generally desirable to select groups of chemical compounds that are likely to be biocompatible or tolerated by the subject without undesirable side effects.

[0111] As noted above, antisense compounds can be constructed to contain a selected number of cationic linkages interspersed with uncharged linkages of the types described above. Both uncharged and cationic intersubunit linkages are preferably phosphorus-containing linkages and have the structure (II):

[0112] [ka] Including, where: W is -S- or -O-, preferably -O-; X=-NR 1 R 2 -OR 6 , Y=-O- or -NR 7 , and Each such linkage in the oligomer is selected from: (a) Uncharged linkage (a), where R 1 , R 2 , R 6 , and R 7 each is independently selected from hydrogen and lower alkyl; (b1) Cationic linkage (b1), where X=-NR 1 R 2 and Y=—O—, —NR 1 R 2 represents an optionally substituted piperazinyl moiety, such that R 1 R 2 =-CHRCHRN(R 3 )(R 4 )CHRCHR-, where: each R is independently H or -CH; R 4 is H, -CH3, or an electron pair; and R 3 is H, optionally substituted lower alkyl, -C(=NH)NH2, -ZL-NHC(=NH)NH2, and [-C(=O)CHR'NH] m H, where Z is -C(=O)- or a direct bond, L is any linker up to 18 atoms in length, preferably up to 12 atoms in length, and more preferably up to 8 atoms in length, with a linkage selected from optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylamino, R' is the side chain of a naturally occurring amino acid or its one or two carbon homologue, and m is 1 to 6, preferably 1 to 4; (b2) Cationic linkage (b2), where X=-NR 1 R 2 and Y=-O-, R 1 =H or -CH3, and R 2 =LNR 3 R 4 R 5 where L, R 3 , and R 4is as defined above, and R 5 is H, optionally substituted lower alkyl, or optionally substituted lower (alkoxy) alkyl; and (b3) Cationic linkage (b3), where Y=-NR 7 and X=-OR 6 , and R 7 =-LNR 3 R 4 R 5 where L, R 3 , R 4 , and R 5 is as defined above, and R 6 is H or optionally substituted lower alkyl; and At least one of said linkages is selected from cationic linkages (b1), (b2), and (b3).

[0113] Preferably, the oligomer comprises at least two consecutive linkages of type (a) (i.e., uncharged linkages). In a further embodiment, at least 5% of the linkages in the oligomer are cationic linkages (i.e., type (b1), type (b2), or type (b3)); for example, 10% to 60%, preferably 20% to 50%, of the linkages are cationic linkages.

[0114] In one embodiment, at least one linkage is of type (b1), where preferably each R is H and R 4 is H, -CH3, or an electron pair, and R 3 is H, optionally substituted lower alkyl, —C(═NH)NH, and —C(═O)-L-NHC(═NH)NH. R 3 The latter two embodiments of R provide the guanidino moiety either directly attached to the piperazine ring or pendant to the linker group L, respectively. For ease of synthesis, R 3 The variable Z in is preferably —C(═O)— as shown.

[0115] The linker group L comprises a bond in its backbone selected from optionally substituted alkyl, optionally substituted alkoxy, and optionally substituted alkylamino, as described above, where the terminal atom in L (e.g., the one adjacent to the carbonyl or nitrogen) is a carbon atom. Although branched linkages are possible, the linker is preferably unbranched. In one embodiment, the linker is a linear alkyl linker. Such linkers have the structure -(CH2) n -, where n is 1 to 12, preferably 2 to 8, and more preferably 2 to 6.

[0116] The morpholino subunit has the following structure (III):

[0117] [ka] where Pi is a base-pairing moiety and the linkage shown above connects the nitrogen atom of (III) to the 5' carbon of the adjacent subunit. The base-pairing moieties Pi can be the same or different and are generally designed to provide a sequence that binds to the target nucleic acid.

[0118] The use of embodiments of linkage types (b1), (b2), and (b3) above to link morpholino subunits (III) may be illustrated diagrammatically as follows:

[0119] [ka] Preferably, all cationic linkages in the oligomer are of the same type; ie, all of type (b1), all of type (b2), or all of type (b3).

[0120] In further embodiments, the cationic linkage is selected from linkages (b1') and (b1'') shown below, where (b1') is referred to herein as a "Pip" linkage and (b1'') is referred to herein as a "GuX" linkage.

[0121] [ka] In the above structure, W is S or O, and preferably O; R 1 and R 2 is independently selected from hydrogen and optionally substituted lower alkyl, and is preferably methyl; and A represents hydrogen or a non-interfering substituent (i.e., a substituent that does not adversely affect the ability of the oligomer to bind to its intended target) on one or more carbon atoms in (b1') and (b1"). Preferably, the ring carbons in the piperazine ring are unsubstituted; however, the ring carbons of the piperazine ring may contain non-interfering substituents such as methyl or fluorine. Preferably, a maximum of one or two carbon atoms are so substituted.

[0122] In a further embodiment, at least 10% of the linkages are of type (b1') or (b1''); for example, 10% to 60%, preferably 20% to 50%, of the linkages may be of type (b1') or (b1'').

[0123] In other embodiments, the oligomer does not contain linkages of type (b1') above. Alternatively, the oligomer does not contain linkages of type (b1), where each R is H and R 3 is H or -CH3, and R 4 is H, -CH3, or an electron pair.

[0124] The morpholino subunits may also be linked by non-phosphorus-based intersubunit linkages, as further described below, where at least one linkage is modified with a pendant cationic group as described above.

[0125] Other oligonucleotide analog linkages can be used that are uncharged in the unmodified state but can also have pendant amine substituents. For example, the 5' nitrogen atom on the morpholino ring can be utilized in a sulfamide or urea linkage (where phosphorus is replaced by carbon or sulfur, respectively), modified in a manner similar to the 5' nitrogen atom in structure (b3) above.

[0126] Oligomers having any number of cationic linkages are provided, including fully cationic linked oligomers. Preferably, however, the oligomers are partially charged, e.g., 10% to 80%. In preferred embodiments, about 10% to 60%, and preferably 20% to 50%, of the linkages are cationic.

[0127] In one embodiment, the cationic linkages are dispersed along the backbone. The partially charged oligomer preferably contains at least two consecutive uncharged linkages; i.e., the oligomer does not have a strictly alternating pattern along its entire length.

[0128] Oligomers having blocks of cationic linkages and blocks of uncharged linkages are also contemplated; for example, a central block of uncharged linkages may be flanked by blocks of cationic linkages, or vice versa. In one embodiment, the oligomer has 5', 3', and central regions of approximately equal length, and the percentage of cationic linkages in the central region is greater than about 50%, preferably greater than about 70%.

[0129] Oligomers for use in antisense applications generally range in length from about 10 to about 40 subunits, more preferably from about 10 to 30 subunits, typically 15 to 25 bases, including those having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases. In certain embodiments, oligomers of the invention having 19 to 20 subunits, a useful length for antisense compounds, will ideally have 2 to 10, e.g., 4 to 8, cationic linkages and remaining uncharged linkages. Oligomers having 14 to 15 subunits will ideally have 2 to 7, e.g., 3 to 5, cationic linkages and remaining uncharged linkages.

[0130] Each morpholino ring structure supports a base-pairing moiety to form a sequence of base-pairing moieties that are typically designed to hybridize to a selected antisense target in a treated cell or subject. The base-pairing moieties may be purines or pyrimidines (e.g., A, G, C, T, or U) found in native DNA or RNA, or analogs such as hypoxanthine (the base component of the nucleoside inosine) or 5-methylcytosine.

[0131] Peptide transporters The antisense compounds of the present invention may comprise an oligonucleotide moiety conjugated to an arginine-rich peptide transport moiety effective to enhance transport of the compound into cells. The transport moiety is preferably attached to the terminus of the oligomer, as shown, for example, in Figures 1B and 1C. The peptide transport moiety preferably comprises 6 to 16 subunits selected from X', Y', and Z' subunits, wherein: (a) each X′ subunit independently represents a lysine, an arginine, or an arginine analog, said analog having the structure R 1 N=C(NH2)R2 where R is a cationic α-amino acid containing a side chain of 1 is H or R; R 2 is R, -NH, -NHR, or -NR, where R is optionally substituted lower alkyl or optionally substituted lower alkenyl; R 1 and R 2 may be joined together to form a ring; and the side chain is R 1 or R 2 linked to the amino acid via (b) each Y' subunit independently comprises the neutral amino acid -C(=O)-(CHR) n represents —NH—, where n is 2 to 7, and each R is independently H or methyl; and (c) each Z' subunit independently represents an α-amino acid having a neutral aralkyl side chain; where the peptide is (X'Y'X') p , (X'Y') m , and / or (X'Z'Z') p where p is 2 to 5 and m is 2 to 8. Particular embodiments include sequences represented by at least one of (X'Y'X') p , (X'Y') m , and / or (X'Z'Z') p and (X'Y'X')(X'Z'Z')(X'Y'X')(X'Z'Z') (SEQ ID NO: 637).

[0132] In selected embodiments, for each X', the side chain moiety is guanidyl, as in the amino acid subunit arginine (Arg). In certain embodiments, each Y' is independently -C(=O)-(CH2). n-CHR-NH-, where n is 2-7 and R is H. For example, when n is 5 and R is H, Y' is a 6-aminohexanoic acid subunit, abbreviated herein as Ahx; when n is 2 and R is H, Y' is a β-alanine subunit, abbreviated herein as B. Certain embodiments relate to carrier peptides with combinations of different neutral amino acids, including, for example, a peptide comprising the sequence -RahxRRBRRAhxRRBRAhxB- (SEQ ID NO: 578), which contains both β-alanine and 6-aminohexanoic acid.

[0133] Preferred peptides of this type include those containing arginine dimers alternating with a single Y' subunit, where Y' is preferably Ahx or B or both. Examples include peptides of the formula (RY'R): p and / or formula (RRY') p where p is 1-2-5, and where Y' is preferably Ahx. In one embodiment, Y' is a 6-aminohexanoic acid subunit, R is arginine, and p is 4. Particular embodiments include peptides having at least two (RY'R) p and (RRY') p and m is 3 or 4, including, for example, exemplary peptides having the sequence (RY'R)(RRY')(RY'R)(RRY') (SEQ ID NO: 638) or (RRY')(RY'R)(RRY') (SEQ ID NO: 639). Other combinations are contemplated. In a further exemplary embodiment, each Z' is phenylalanine and m is 3 or 4.

[0134] The conjugated peptide is preferably linked to the terminus of the oligomer via the linker Ahx-B, where Ahx is a 6-aminohexanoic acid subunit and B is a β-alanine subunit, as shown, for example, in Figures 1B and 1C.

[0135] In selected embodiments, for each X', the side chain moiety is independently selected from the group consisting of guanidyl (HN=C(NH2)NH-), amidinyl (HN=C(NH2)CH-), 2-aminodihydropyrimidyl, 2-aminotetrahydropyrimidyl, 2-aminopyridinyl, and 2-aminopyrimidonyl, preferably guanidyl and amidinyl. In one embodiment, the side chain moiety is guanidyl, as in the amino acid subunit arginine (Arg).

[0136] In certain embodiments, the Y' subunits may be contiguous in that no X' subunits intervene between the Y' subunits or are solely dispersed between the X' subunits. In certain embodiments, linking subunits may be located between the Y' subunits. In one embodiment, the Y' subunits are at the termini of the transporter; in other embodiments, they are flanked by X' subunits. In a further preferred embodiment, each Y' is -C(=O)-(CH2) n The formula is -CHR-NH-, where n is 2-7 and R is H. For example, when n is 5 and R is H, Y' is a 6-aminohexanoic acid subunit, abbreviated herein as Ahx. In selected embodiments of this group, each X' contains a guanidyl side chain moiety, as in an arginine subunit. Preferred peptides of this type include those containing arginine dimers alternating with single Y' subunits, where Y' is preferably Ahx. Examples include peptides having the formula (RY'R)4 or (RRY')4, where Y' is preferably Ahx. In the latter case, the nucleic acid analog is preferably linked to the terminal Y' subunit, preferably at the C-terminus, as shown, for example, in Figures 1B and 1C. A preferred linker is of the structure AhxB, where Ahx is a 6-aminohexanoic acid subunit and B is a β-alanine subunit.

[0137] Such transport moieties have been shown to greatly enhance cellular entry of the conjugated oligomer compared to uptake of the oligomer in the absence of the conjugated transport oligomer and compared to uptake by a conjugated transport moiety lacking the hydrophobic subunit Y'. Such enhanced uptake is evidenced by preferably at least a two-fold increase, and preferably a four-fold increase, in uptake of the compound into mammalian cells compared to uptake of the drug by a conjugated transport moiety lacking the hydrophobic subunit Y'. Uptake is preferably enhanced by at least 20-fold, and more preferably 40-fold, compared to the unconjugated compound.

[0138] A further advantage of the transport moiety is its predicted ability to stabilize the duplex between the antisense compound and its target nucleic acid sequence, presumably through electrostatic interactions between the positively charged transport moiety and the negatively charged nucleic acid. The number of charged subunits in the transporter is less than 14, as noted above, and preferably between 8 and 11, because too many charged subunits can lead to reduced sequence specificity.

[0139] The use of arginine-rich peptide transporters (i.e., cell-penetrating peptides) is particularly useful in the practice of the present invention. Certain peptide transporters have been shown to be highly effective in delivering antisense compounds to primary cells, including muscle cells (Marshall, Oda et al., 2007; Jearawiriyapaisarn, Moulton et al., 2008; Wu, Moulton et al., 2008). Furthermore, compared to other peptide transporters, such as penetratin and Tat peptides, the peptide transporters described herein demonstrate enhanced ability to alter the splicing of some gene transcripts when conjugated to antisense PMOs (Marshall, Oda et al., 2007). Particularly preferred are the P007, CP06062, and CP04057 transport peptides listed below in Table 3 (SEQ ID NOs: 573, 578, and 577, respectively).

[0140] Exemplary peptide transporters including a linker (B or AhxB) are provided below in Table B. Preferred sequences are those designated CP06062 (SEQ ID NO: 578), P007 (SEQ ID NO: 573), and CP04057 (SEQ ID NO: 577). [Table B]

[0141] formulation In certain embodiments, the present invention provides formulations or compositions suitable for therapeutic delivery of antisense oligomers as described herein. Thus, in certain embodiments, the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the oligomers described herein, formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. While the oligomers of the present invention cannot be administered alone, it is preferable to administer the compounds as pharmaceutical formulations (compositions).

[0142] Methods for delivery of nucleic acid molecules are described, for example, in Akhtar et al., 1992, Trends Cell Bio., 2:139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, edited by Akhtar; Sullivan et al., PCT WO 94 / 02595. These and other protocols can be utilized for delivery of virtually any nucleic acid molecule, including the isolated oligomers of the present invention.

[0143] As detailed below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained release formulation; (3) topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin; (4) intravaginally or rectally, e.g., as a pessary, cream, or foam; (5) sublingually; (6) ophthalmic; (7) transdermal; or (8) nasal.

[0144] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment and are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, and in accordance with a reasonable benefit-to-risk ratio.

[0145] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium, or zinc stearate, or steric acid), or solvent encapsulating agent, involved in carrying or transporting a compound of interest from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0146] Some examples of substances that can serve as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) propolis; (11) glycols such as propylene glycol; (12) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (13) esters such as ethyl oleate and ethyl laurate; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic compatible materials utilized in pharmaceutical formulations.

[0147] Further non-limiting examples of drugs suitable for formulation with the antisense oligomers of the invention include: PEG-conjugated nucleic acids, phospholipid-conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance drug entry into various tissues; biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, DF et al., 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge; and loaded nanoparticles, such as those made from polybutylcyanoacrylate, which can deliver drugs across the blood-brain barrier and alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).

[0148] The present invention also features the use of compositions containing surface-modified liposomes (PEG-modified, branched or unbranched, or a combination thereof, or long-circulating or stealth liposomes) containing poly(ethylene glycol) lipids. The oligomers of the present invention can also contain covalently attached PEG molecules of various molecular weights. These formulations provide a method for increasing drug accumulation in target tissues. This class of drug carriers is resistant to opsonization and elimination by the mononuclear phagocyte system (MPS or RES), thereby allowing for longer blood circulation times and enhanced tissue exposure of encapsulated drugs (Lasic et al., Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to selectively accumulate in tumors, likely through extravasation and entrapment in neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared with conventional cationic liposomes, which are known to accumulate in MPS tissues (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., PCT International Publication No. WO 96 / 10391; Ansell et al., PCT International Publication No. WO 96 / 10390; Holland et al., PCT International Publication No. WO 96 / 10392). Long-circulating liposomes also likely protect drugs from nuclease degradation to a greater extent than cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

[0149] In a further embodiment, the present invention comprises oligomer compositions prepared for delivery, as described in U.S. Patent Nos. 6,692,911, 7,163,695, and 7,070,807. In this regard, in one embodiment, the present invention provides oligomers of the present invention in compositions comprising lysine and histidine (HK) copolymers, as described in U.S. Patent Nos. 7,163,695, 7,070,807, and 6,692,911, either alone or in combination with PEG (e.g., branched or unbranched PEG, or a mixture of both), or in combination with any of the foregoing in combination with PEG and a targeting moiety or a crosslinking agent. In certain embodiments, the present invention provides antisense oligomers in compositions comprising gluconate-modified polyhistidine or gluconyl-polyhistidine / transferrin-polylysine. Those skilled in the art will also recognize that amino acids with properties similar to His and Lys may be substituted in the compositions.

[0150] Certain embodiments of the oligomers described in this invention may contain a basic functional group, such as amino or alkylamino, and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts," in this regard, refers to relatively non-toxic, inorganic and organic acid addition salts of the compounds of the invention. These salts can be prepared in situ during the administration vehicle or dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with an appropriate organic or inorganic acid and isolating the salt thus formed during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and lauryl sulfonate salts, and the like (see, e.g., Berge et al., (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66:1-19).

[0151] Pharmaceutically acceptable salts of the subject oligomers include the conventional non-toxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids such as propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothioic acid, and the like.

[0152] In certain embodiments, the oligomers of the present invention may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these examples, the term "pharmaceutically acceptable salts" refers to relatively non-toxic, inorganic and organic base addition salts of the compounds of the present invention. These salts can likewise be prepared in situ during the administration vehicle or dosage form manufacturing process, or can be prepared by separately reacting a purified compound of the present invention in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, e.g., Berge et al., supra).

[0153] Wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0154] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) fat-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0155] The formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. These formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of compound that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0156] In certain embodiments, formulations of the invention comprise an excipient selected from cyclodextrins, celluloses, liposomes, micelle-forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides; and an oligomer of the invention. In certain embodiments, the formulations described above render the oligomer of the invention orally bioavailable.

[0157] Methods of preparing these formulations and compositions include the step of bringing into association an oligomer of the present invention with a carrier and, optionally, one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0158] Formulations of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of a compound of the present invention as an active ingredient. The oligomers of the present invention may also be administered as a bolus, electuary, or paste.

[0159] In the solid dosage forms of the present invention for oral administration (capsules, tablets, pills, dragees, powders, granules, lozenges, and the like), the active ingredient may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, Certain silicic acids and sodium carboxylates; (5) solution retardants, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as poloxamers and sodium lauryl sulfate; (7) wetting agents, such as cetyl alcohol, glycerol monostearate, and nonionic surfactants; (8) adsorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) coloring agents; and (11) controlled-release agents, such as crospovidone or ethylcellulose. In the case of capsules, tablets, and pills, pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0160] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents, or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0161] Tablets and other dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills, and granules, can be optionally obtained and prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical arts. They can also be formulated to provide delayed or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They can also be formulated for rapid release, for example, lyophilized. They can be sterilized, for example, by a bacteria-retaining filter or by incorporating the sterile agent in the form of a sterile solid composition that can be dissolved in sterile water or some other injectable medium immediately before use. These compositions can also optionally contain opacifying agents and can be compositions that release the active ingredient only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0162] The liquid dosage form for oral administration of the compound of the present invention includes pharmaceutically acceptable emulsion, microemulsion, solution, suspension, syrup and elixir.In addition to active ingredient, liquid dosage form may contain commonly used in the art inert diluent such as water or other solvent, solubilizer and emulsifier such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed oil, peanut oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, and mixtures thereof.

[0163] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0164] Suspensions may contain, in addition to the active ingredient, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0165] Formulations for rectal or vaginal administration may be presented as suppositories, which may be prepared by mixing one or more compounds of the present invention with a suitable non-inflammatory carrier or excipient including, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, which is solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity to release the active compound.

[0166] The preparations or dosage forms for topical or transdermal administration of the oligomers provided herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.The active oligomers may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.Ointments, pastes, and gels may contain, in addition to the active compounds of the present invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0167] Powders and sprays can contain, in addition to the oligomer of the invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0168] Transdermal patches have the additional advantage of providing controlled delivery of the oligomers of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the oligomers of the present invention in a suitable medium. Absorption enhancers can also be used to increase the flux of the drug across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the drug in a polymer matrix or gel, among other methods known in the art.

[0169] Pharmaceutical compositions suitable for parenteral administration may contain one or more oligomers of the present invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes that make the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0170] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action on the target oligomer may be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0171] In some cases, it may be desirable to delay the absorption of a drug from subcutaneous or intramuscular injection in order to prolong the effect of the drug.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility, among other methods known in the art.The rate of absorption of the drug then depends on its dissolution rate, which in turn may depend on the crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0172] Injectable depot formulations may be made by forming microencapsulated matrices of the target oligomer in biodegradable polymers such as polylactide-polyglycolide. The rate of oligomer release can be controlled depending on the ratio of oligomer to polymer and the properties of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations may also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0173] When the oligomers of the present invention are administered as pharmaceuticals to humans and animals, they can be given on their own or in combination with a pharmaceutically acceptable carrier, for example, as a pharmaceutical composition containing 0.1 to 99% (more preferably, 10 to 30%) of the active ingredient.

[0174] As mentioned above, the formulations or preparations of the present invention may be administered orally, parenterally, topically, or rectally.They are typically administered in a form suitable for each administration route.For example, they are administered in tablet or capsule form, injection, inhalation, eyewash, ointment, suppository, etc., by injection, infusion, or inhalation; topically by lotion or ointment; and rectally by suppository.

[0175] The phrases "parenteral administration" and "parenterally administered," as used herein, mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0176] The phrases "systemic administration," "administered systemically," "peripheral administration," and "peripherally administered," as used herein, refer to the administration of a compound, drug, or other substance other than directly into the central nervous system so that it enters the patient's system and is therefore subject to metabolism and other similar processes, such as subcutaneous administration.

[0177] Regardless of the route selected, the oligomers of the invention, which may be used in a suitable hydrated form, and / or pharmaceutical compositions of the invention may be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without unacceptable toxicity to the patient.

[0178] The selected dosage level will depend upon a variety of factors, including the activity of the particular oligomer of the invention, or ester, salt, or amide thereof, employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular oligomer employed, the rate or extent of absorption, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular oligomer employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0179] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician or veterinarian can start the dosage of the compound of the present invention utilized in the pharmaceutical composition at a level lower than that required to achieve the desired effect, and gradually increase the dosage until the desired effect is achieved. Generally, a suitable daily dose of a compound of the present invention is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Generally, oral, intracerebroventricular, and subcutaneous doses of the compound of the present invention for a patient, when used for the indicated effect, will range from about 0.0001 to about 100 mg / kilogram of body weight / day.

[0180] If desired, the effective daily dose of active compound can be administered as 2, 3, 4, 5, 6 or more sub-doses that are administered separately at appropriate intervals throughout the day, optionally in unit dosage form.In certain circumstances, administration is 1 time per day.In certain circumstances, administration is 1 time per day or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, as needed, to maintain the desired expression of functional dystrophin protein.

[0181] Nucleic acid molecules can be administered to cells by a variety of methods known to those skilled in the art, including, but not limited to, encapsulation in liposomes, iontophoresis, or incorporation into other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, as described herein and known in the art. In certain embodiments, microemulsification techniques may be utilized to improve the bioavailability of lipophilic (water-insoluble) pharmaceutical agents. Examples include trimetrine (Dordunoo, SK et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991) and REV 5901 (Sheen, PC et al., J. Pharm Sci 80(7), 712-714, 1991). Among other advantages, microemulsification provides enhanced bioavailability by preferentially directing absorption into the lymphatic system instead of the circulatory system, thereby bypassing the liver and preventing breakdown of the compound in the hepatobiliary circulation.

[0182] In one aspect of the invention, a formulation comprises micelles formed from an oligomer as provided herein and at least one amphiphilic carrier, wherein the micelles have an average diameter of less than about 100 nm. More preferred embodiments provide micelles having an average diameter of less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter of less than about 30 nm, or even less than about 20 nm.

[0183] While any suitable amphiphilic carrier is contemplated, presently preferred carriers are generally those that are Generally Recognized as Safe (GRAS) status and are capable of both solubilizing and microemulsifying the compounds of the present invention at later stages where the solution comes into contact with complex aqueous phases (e.g., those found in the human gastrointestinal tract). Typically, amphiphilic components that meet these requirements have an HLB (hydrophilic to lipophilic balance) value of 2 to 20, and their structures contain linear aliphatic groups ranging from C-6 to C-20. Examples are polyethylene-glycolized fatty glycerides and polyethylene glycols.

[0184] Examples of amphiphilic carriers include saturated and monounsaturated polyethylene glycolated fatty acid glycerides, such as those obtained from various fully or partially hydrogenated vegetable oils. Such oils may advantageously comprise tri-, di-, and mono-fatty acid glycerides and di- and mono-polyethylene glycol esters of the corresponding fatty acids; a particularly preferred fatty acid composition includes 4-10% capric acid, 3-9% capric acid, 40-50% lauric acid, 14-24% myristic acid, 4-14% palmitic acid, and 5-15% stearic acid. Another useful class of amphiphilic carriers includes partially esterified sorbitan and / or sorbitol with saturated or monounsaturated fatty acids (SPAN series) or their corresponding ethoxylated analogs (TWEEN series).

[0185] Commercially available amphiphilic carriers may be particularly useful, including the Gelucire series, Labrafil, Labrasol, or Lauroglycol (all manufactured and sold by Gattefosse Corporation, Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono- and di-laurate, lecithin, polysorbate 80, and the like (manufactured and sold by numerous companies in the United States and around the world).

[0186] In certain embodiments, delivery may be achieved through the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., for the introduction of the compositions of the present invention into suitable host cells. In particular, the compositions of the present invention may be formulated for delivery encapsulated in any of lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc. The preparation and use of such delivery vehicles can be carried out using known and conventional techniques.

[0187] Hydrophilic polymers suitable for use in the present invention are those that are readily soluble in water, can be covalently attached to vesicle-forming lipids, and are tolerated in vivo without toxic effects (i.e., are biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic acid (also called polylactide), polyglycolic acid (also called polyglycolide), polylactic acid-polyglycolic acid copolymers, and polyvinyl alcohol. In certain embodiments, the polymer has a molecular weight of about 100 or 120 daltons to about 5,000 or 10,000 daltons, or about 300 daltons to about 5,000 daltons. In other embodiments, the polymer is polyethylene glycol having a molecular weight of about 100 to about 5,000 daltons, or about 300 to about 5,000 daltons. In certain embodiments, the polymer is 750 dalton polyethylene glycol (PEG(750)). Polymers may also be defined by the number of monomers therein; preferred embodiments of the present invention utilize polymers of at least about three monomers, such PEG polymers consisting of three monomers (approximately 150 daltons).

[0188] Other hydrophilic polymers that may be suitable for use in the present invention include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0189] In certain embodiments, the formulations of the present invention comprise a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

[0190] Cyclodextrins are cyclic oligosaccharides consisting of six, seven, or eight glucose units, designated by the Greek letters α, β, or γ, respectively. These glucose units are linked by α-1,4-glucosidic bonds. As a result of the chair conformation of the sugar units, all secondary hydroxyl groups (at C-2 and C-3) are located on one side of the ring, while all primary hydroxyl groups at C-6 are located on the other side. As a result, the exterior surfaces are hydrophilic, making cyclodextrins water-soluble. In contrast, the cavities of cyclodextrins are hydrophobic because they are lined by hydrogen atoms at C-3 and C-5 and by ether-like oxygens. These matrices allow complexation with a variety of relatively hydrophobic compounds, including steroid compounds such as 17α-estradiol (see, e.g., van Uden et al., Plant Cell Tiss. Org. Cult. 38:1-3-113 (1994)). This complexation occurs through Van der Waals interactions and through hydrogen bond formation. For a general review of cyclodextrin chemistry, see Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822 (1994).

[0191] The physicochemical properties of cyclodextrin derivatives depend strongly on the type and degree of substitution. For example, their water solubility ranges from insoluble (triacetyl-β-cyclodextrin) to 147% soluble (w / v) (G-2-β-cyclodextrin). In addition, they are soluble in many organic solvents. The properties of cyclodextrins allow for control over the solubility of various formulation components by increasing or decreasing their solubility.

[0192] Numerous cyclodextrins and methods for their preparation have been described. For example, Parmeter (I) et al. (U.S. Pat. No. 3,453,259) and Gramera et al. (U.S. Pat. No. 3,459,731) described electrically neutral cyclodextrins. Other derivatives include cyclodextrins with cationic character [Parmeter (II) U.S. Pat. No. 3,453,257], insoluble crosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), and cyclodextrins with anionic character [Parmeter (III), U.S. Pat. No. 3,426,011]. Among cyclodextrins with anionic character, carboxylic acids, phosphorous acids, phosphinic acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfinic acids, and sulfonic acids are added to the parent cyclodextrin [Parmeter (III), supra]. Additionally, sulfoalkyl ether cyclodextrin derivatives have been described by Stella et al. (US Pat. No. 5,134,127).

[0193] Liposomes consist of at least one lipid bilayer surrounding an aqueous interior compartment. Liposomes can be characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and typically range in diameter between 0.02 and 0.05 μm; large unilamellar vesicles (LUVs) are typically larger than 0.05 μm. Large oligolamellar vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 μm. Liposomes with several non-concentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are called multivesicular vesicles.

[0194] One aspect of the present invention relates to a formulation comprising liposomes containing an oligomer of the present invention, wherein the liposome membrane is formulated to provide liposomes with increased transport capacity. Alternatively, or in addition, the compound of the present invention may be contained in or adsorbed to the liposome bilayer of the liposome. The oligomer of the present invention may be aggregated with a lipid surfactant and transported into the interior space of the liposome; in these cases, the liposome membrane is formulated to resist the disruptive effects of the active agent-surfactant aggregates.

[0195] According to one embodiment of the invention, the lipid bilayer of the liposome comprises lipids derivatized with polyethylene glycol (PEG), such that the polyethylene glycol (PEG) chains extend from the interior surface of the lipid bilayer into the interior space encapsulated by the liposome and from the exterior of the lipid bilayer into the surrounding environment.

[0196] The active agent contained in the liposomes of the present invention is in a solubilized form. Aggregates of surfactant and active agent (e.g., emulsions or micelles containing the active agent of interest) may be entrapped within the interior space of liposomes of the present invention. The surfactant acts to disperse and solubilize the active agent and may be selected from any suitable aliphatic, alicyclic, or aromatic surfactant, including, but not limited to, biocompatible lysophosphatidylcholines (LPCs) of various chain lengths (e.g., about C14 to about C20). Polymer-derivatized lipids, such as PEG-lipids, may also be used for micelle formation because they inhibit micelle / membrane fusion, and the addition of polymers to the surfactant molecule reduces the CMC of the surfactant, aiding micelle formation. Surfactants with CMCs in the micromolar range are preferred; surfactants with higher CMCs may be used to prepare micelles entrapped in the liposomes of the present invention.

[0197] Liposomes according to the present invention may be prepared by any of a variety of techniques known in the art. See, for example, U.S. Patent No. 4,235,871; PCT Publication WO 96 / 14057; New RRC, Liposomes: A Practical Approach, IRL Press, Oxford (1990), pp. 33-104; and Lasic DD, Liposomes from Physics to Applications, Elsevier Science Publishers BV, Amsterdam, 1993. For example, liposomes according to the present invention may be prepared by dispersing a lipid derivatized with a hydrophilic polymer into preformed liposomes, e.g., by exposing preformed liposomes to micelles composed of lipid-grafted polymers at a lipid concentration corresponding to the final molar percentage of derivatized lipid desired in the liposomes. Liposomes containing hydrophilic polymers can also be formed by homogenization, lipid field hydration, or extrusion techniques, as are known in the art.

[0198] In another exemplary formulation procedure, the active agent is first dispersed by ultrasonication in lysophosphatidylcholine or other low CMC surfactants (including polymer-grafted lipids), which easily dissolve hydrophobic molecules.The resulting micellar suspension of the active agent is then used to rehydrate a dried lipid sample containing an appropriate mole percentage of polymer-grafted lipids or cholesterol.The lipid and active agent suspension is then formed into liposomes using extrusion techniques, as known in the art, and the resulting liposomes are separated from the non-encapsulated solution by standard column separation.

[0199] In one embodiment of the present invention, liposomes are prepared to have a substantially uniform size within a selected size range. One effective sizing method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes with a selected uniform pore size; the pore size of the membrane roughly corresponds to the maximum size of the liposomes produced by extrusion through the membrane. See, for example, U.S. Patent No. 4,737,323 (April 12, 1988). In certain embodiments, reagents such as DharmaFECT® and Lipofectamine® may be used to introduce polynucleotides or proteins into cells.

[0200] The release characteristics of the formulations of the present invention depend on the encapsulation material, the concentration of encapsulated drug, and the presence of release modifiers. For example, release can be engineered to be pH-dependent using a pH-sensitive coating that releases only at low pH, such as in the stomach, or only at higher pH, such as in the intestine. Enteric coatings can be used to prevent release from occurring until after passage through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain early release in the stomach, followed by later release in the intestine. Release can also be engineered by including salts or pore-forming agents, which can increase water uptake or drug release upon dispersion from the capsule. Excipients that modify drug solubility can also be used to control the release rate. Agents that enhance matrix degradation or release from the matrix can also be incorporated. These can be added to the drug, added as a separate phase (i.e., as microparticles), or co-dissolved in the polymer phase, depending on the compound. In many cases, the amount should be between 0.1 and 30 percent (w / w polymer). Types of degradation accelerators include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine, and surfactants such as Tween® and Pluronic®. Pore-forming agents (i.e., water-soluble compounds such as inorganic salts and sugars) that add microstructure to the matrix are added as microparticles, typically ranging from 1 to 30% (w / w polymer).

[0201] Uptake can also be manipulated by altering the residence time of the particles in the intestine. This can be achieved, for example, by coating the particles with a mucoadhesive polymer or by selecting a mucoadhesive polymer as the encapsulating material. Examples include many polymers with free carboxyl groups, such as chitosan, cellulose, and polyacrylates, among others (as used herein, polyacrylate refers to polymers containing acrylate groups and modified acrylate groups, e.g., cyanoacrylate and methacrylate).

[0202] The oligomer may be formulated to be contained in or adapted to be released by a surgical or medical device or implant. In certain embodiments, an implant may be coated with or otherwise treated with the oligomer. For example, hydrogels or other polymers, such as biocompatible and / or biodegradable polymers, may be used to coat an implant with the compositions of the present invention (i.e., the composition may be adapted for use with a medical device by using hydrogels or other polymers). Polymers and copolymers for coating medical devices with drugs are well known in the art. Examples of implants include, but are not limited to, stents, drug-eluting stents, sutures, prostheses, vascular catheters, dialysis catheters, vascular grafts, artificial heart valves, cardiac pacemakers, implantable cardioverter-defibrillators, intravenous needles, devices for bone setting and formation, such as pins, screws, plates, and other devices, and artificial tissue matrices for wound healing.

[0203] In addition to the methods provided herein, oligomers for use in accordance with the invention may be formulated for administration in any convenient manner for use in human or veterinary medicine, by analogy with other pharmaceuticals. Antisense oligomers and their corresponding formulations may be administered alone or in combination with other therapeutic strategies in the treatment of muscular dystrophy, such as myoblast transplantation, stem cell therapy, administration of aminoglycoside antibiotics, proteosome inhibitors, and upregulation therapy (e.g., upregulation of utrophin, an autosomal paralog of dystrophin).

[0204] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0205] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made to the present invention without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only, and not for limiting purposes. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially similar results.

[0206]

number

[0207]

number

[0208]

number

[0209] material and method Cell and tissue culture treatment conditions Low-passage human rhabdomyosarcoma cells (ATCC, CCL-136; RD cells) stored in 5% DMSO solution (Sigma) were thawed in a 37°C water bath until the ice silver color was no longer visible. Cells were cultured at 1.5 × 10 cells / well in 24 mL of warmed DMEM supplemented with L-glutamine (HyClone), 10% fetal bovine serum, and 1% penicillin-streptomycin antibiotic solution (CelGro). 6 Cells / flask were seeded into tissue-culture-treated T75 flasks (Nunc); after 24 hours, the medium was aspirated, the cells were washed once with warm PBS, and fresh medium was added. Cells were grown in a 37°C incubator with 5.0% CO2 until 80% confluent.

[0210] The medium was aspirated from the T75 flasks; the cells were washed once with warmed PBS and aspirated. 3 mL of trypsin / EDTA, prewarmed in a 37°C water bath, was added to each T75 flask. The cells were incubated at 37°C for 2–5 minutes with gentle agitation five times until they were released from the flask. The cell suspension was transferred to a 15.0 mL conical tube; the flask was rinsed with 1.0 mL of trypsin / EDTA solution to collect any remaining cells. Cells were counted using a Vi-Cell XR cell counter (Beckman Coulter). Cells were plated at 2.0 × 10 cells per well in 1.0 mL of medium. 5 Viable cells were seeded into tissue culture-treated 12-well plates (Falcon) and incubated overnight in a 37°C incubator with 5.0% CO2.

[0211] Twelve-well seeded plates were tested for uniform cell distribution and plate adhesion. Lyophilized peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) were resuspended at 2.0 mM in nuclease-free water (Ambion) and kept on ice during cell treatment; to confirm molar concentration, PPMOs were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Immediately prior to PPMO treatment, the medium was aspirated and cells were rinsed with warmed PBS. PPMOs were diluted with warmed medium to the desired molar concentration; cells were treated in a total volume of 1.0 mL PPMO per well. PPMOs were tested in triplicate. For untreated controls, fresh, warmed medium was added in a total volume of 1.0 mL. Cells were incubated for 48 hours in a 37°C incubator with 5.0% CO2.

[0212] RNA extraction The medium was aspirated, and the cells were rinsed with warmed PBS. RNA was extracted using the QuickGene-Mini80 system, the QuickGene RNA Cultured Cell HC Kit S, and the MagNAlyser, using the manufacturer's recommended protocol with ceramic bead homogenization. Briefly, cells were cultured in 350 μL LRP (100 μL The homogenates were lysed in a treatment plate with lysis buffer (plus 10 μL β-mercaptoethanol per LRP); the homogenates were gently triturated to ensure complete lysis and transferred to MagNAlyser tubes. The tubes were centrifuged in the MagNAlyser at 2800 rpm for 30 seconds to ensure complete homogenization and briefly chilled on ice. 50 μL SRP lysis buffer was added, and the homogenates were vortexed for 15 seconds. 170 μL >99% ethanol was added to each tube, and the homogenates were vortexed for 60 seconds. The homogenates were briefly centrifuged and transferred to a Mini80 RNA cartridge; the sample was pressurized and the flow-through was discarded. The cartridge was washed in 750 μL WRP wash buffer and pressurized. 40 μL of DNase solution (1.25 μL Qiagen DNase I, 35 μL RDD buffer, 3.75 μL nuclease-free water) was added directly to the cartridge membrane; the cartridge was incubated at room temperature for 4 minutes. The cartridge was washed twice with 750 μL WRP and pressurized after each wash. The cartridge was placed on a nuclease-free tube. 50 μL CRP elution buffer was added to each membrane; the membrane was incubated at room temperature for 5 minutes. The cartridge was pressurized and the eluate was collected. RNA was stored at -80°C pending quantification. RNA was quantified using a NanoDrop™ 2000 spectrophotometer.

[0213] Nested RT-PCR Primer-specific, exon-specific, optimized nested RT-PCR amplification was performed using primer pair sets for each dystrophin exon as shown below in Table 1.

[0214] [Table 1] The primer pairs shown are either forward or reverse (F / R) and either external or internal (I / O) primer pairs corresponding to the primary or secondary amplification, respectively. The primer target locations are indicated in the exon column, and the purpose is to detect exon skipping events. For example, primers PS170 and PS176 amplify the region of exons 48-53 in the primary amplification. Then, primers PS172 and PS174 amplify the region of exons 49-52 in the secondary amplification. This nested PCR reaction detects exon skipping of both exon 50 and / or exon 51. Specific nested RT-PCR reaction conditions are provided below.

[0215] RNA extracted from treated cells (described above) was diluted to 20 ng / μl for all samples.

[0216] [Table 2]

[0217] [Table 3]

[0218] [Table 4]

[0219] [Table 5] Gel electrophoresis analysis Ten microliters of 5x Ficoll loading dye was added to each 50 microliter nested RT-PCR reaction. Fifteen microliters of the PCR / dye mixture was run on a 10% TBE gel at 300 volts for 30 minutes. After electrophoresis, the gel was washed in diH2O for at least 1 hour, with water changes every 30 minutes. The gel was then scanned on a Typhoon Trio Variable Mode Imager (GE Healthcare). For exon 44 skipping, the nested RT-PCR product from the full-length dystrophin transcript was 571 bp, compared with 423 bp from the exon 44-skipped mRNA (exon 44 is 148 bp). For exon 45, the nested RT-PCR product from the full-length dystrophin transcript was 571 bp, compared with 395 bp from the exon 45-skipped mRNA (exon 45 is 176 bp). For exon 53, the PCR product from the full-length dystrophin transcript is 365 bp and from the exon 53 skipped mRNA is 153 bp (exon 53 is 212 bp).

[0220] Gel images were subjected to quantitative analysis by measuring the band intensity of the full-length PCR product compared to the exon-skipping product. In some cases, the percent skipping at a fixed PPMO concentration (e.g., 3 micromolar) determined the relative activity of a series of PPMOs used to induce exon skipping of a given exon. In other situations, a range of PPMO doses (e.g., 0.1, 0.3, 1.0, 3.0, and 10 micromolar) were used to treat cells, and the EC 50 was calculated based on the percentage skipping induced at each concentration.

[0221] Example 1 Exon 51 scanning A series of overlapping antisense PPMOs targeting human dystrophin exon 51 were designed, synthesized, and used to treat either human rhabdomyosarcoma cells (RD cells) or primary human skeletal muscle cells. This strategy, called "exon scanning," was similarly used for several other dystrophin exons, as described below. All PPMOs were synthesized as peptide-conjugated PMOs (PPMOs) using the CP06062 peptide (SEQ ID NO: 578) and a 3'-terminal PMO linkage. For exon 51, a series of 26 PPMOs, each 26 bases long, was generated (SEQ ID NOs: 309-311, 314, 316, 317, 319, 321, 323, 324, 326, 327, 329-331, 333, 335, 336, 338-345), as shown in Figure 2A. These PPMOs were evaluated for exon skipping efficacy by treating RD cells at various concentrations, as described above in Materials and Methods. Three PPMOs (SEQ ID NOs: 324, 326, and 327) were identified as effective in inducing exon skipping and selected for further evaluation. Dose-ranging experiments in RD cells and primary human skeletal muscle cells were used to confirm the relative potency of these three PPMO sequences. SEQ ID NO: 327 was shown to be the most effective in inducing exon 51 skipping, as shown in Figures 2B and 2C.

[0222] A comparison of the relative efficacy of SEQ ID NO:327 to other exon 51-targeting antisense sequences was performed in RD cells and primary human skeletal muscle cells, as described above. All evaluated sequences were generated as peptide-conjugated PMOs using the CP06062 peptide (SEQ ID NO:578). This allowed for a direct comparison of the relative efficacy of antisense sequences, regardless of antisense chemistry or cellular delivery. The relative positions of specific exon 51-targeting oligos compared to SEQ ID NO:327 are shown in Figure 2D. As shown in Figure 2C, there is a hierarchical hierarchy of exon skipping efficacy, with SEQ ID NO:327 being the most effective, by a factor of at least several-fold compared to the other sequences.

[0223] Example 2 Exon 50 scan We designed and synthesized a series of overlapping antisense PPMOs targeting human dystrophin exon 50. For exon 50, we generated a series of 17 PPMOs, each 25 bases long, as shown in Figure 3A (SEQ ID NOS: 267, 269, 271, 273, 275, 277, 279, 280, 282, and 284-291). These PPMOs were evaluated for exon skipping efficacy by treating RD cells at various concentrations, as described above in Materials and Methods. Four PPMOs (SEQ ID NOS: 277, 287, 290, and 291) were identified as effective in inducing exon skipping and were selected for further evaluation. Dose-ranging experiments in RD cells were used to confirm the relative potencies of these four PPMO sequences. SEQ ID NOs: 584 (AVI-5656) and 287 (AVI-5038) were shown to be the most effective in inducing exon 50 skipping, as shown in Figure 3B. 50 Values ​​are derived from dose-ranging experiments and represent the calculated concentration at which 50% of the PCR product is produced from mRNA lacking exon 50 compared to PCR product produced from mRNA containing exon 50. Compared to other sequences (see, e.g., SEQ ID NOs: 584 and 585 correspond to SEQ ID NOs: 173 and 175, respectively, in WO2006 / 000057), AVI-5038 (SEQ ID NO: 287) is equivalent or better at inducing exon skipping activity in the RD cell assay, as shown in Figure 3B.

[0224] Example 3 Exon 53 scanning We designed and synthesized a series of overlapping antisense PPMOs targeting human dystrophin exon 53. For exon 53, we generated a series of 24 PPMOs, each 25 bases long, as shown in Figure 4A (SEQ ID NOS: 416, 418, 420, 422, 424, 426, 428, 429, 431, 433, 434, 436, 438-440, and 443-451). These PPMOs were evaluated for exon skipping efficacy by treating RD cells and primary human skeletal muscle cells at various concentrations, as described above in Materials and Methods. Three PPMOs (SEQ ID NOS: 428, 429, and 431) were identified as effective in inducing exon skipping and were selected for further evaluation. Dose-ranging experiments in RD cells were used to confirm the relative potencies of these three PPMO sequences. SEQ ID NO:429 was shown to be the most effective in inducing exon 53 skipping, as shown in Figures 4B-F. However, when compared with other exon 53 antisense sequences, SEQ ID NO:429 was found to be identical to H53A(+23+47), listed as SEQ ID NO:195 in WO2006 / 000057, and SEQ ID NO:609 in the present application. Other sequences compared to SEQ ID NO:429 included H53A(+39+69) and H53A(-12+10) (listed as SEQ ID NO:193 and 199, respectively, in WO2006 / 000057) and h53AON1 (listed as SEQ ID NO:39 in U.S. Application Serial No. 11 / 233,507), listed as SEQ ID NO:608, 611, and 610, respectively, in the present application. All evaluated sequences were generated as peptide-conjugated PMOs using the CP06062 peptide (SEQ ID NO:578). This allowed for a direct comparison of the relative efficacy of antisense sequences, regardless of antisense chemistry or cellular delivery. As shown in Figures 4I and 4G-H, SEQ ID NO: 429 was shown to be superior to each of these four sequences.

[0225] Example 4 Exon 44 scan A series of overlapping antisense PPMOs targeting human dystrophin exon 44 were designed and synthesized. For exon 44, a series of PPMOs, each 25 bases long, was generated (SEQ ID NOS: 1-20), as shown in Figure 5A. These PPMOs were evaluated for exon skipping efficacy by treating RD cells at various concentrations, as described above in Materials and Methods. Five PPMOs (SEQ ID NOS: 4, 8, 11, 12, and 13) were identified as effective in inducing exon skipping and selected for further evaluation. Dose-ranging experiments in RD cells were used to confirm the relative potency of these five PPMO sequences, as shown in Figure 5C-H. SEQ ID NOS: 8, 11, and 12 were shown to be the most effective in inducing exon 44 skipping, as shown in Figure 5H, with SEQ ID NOS: 12 providing the highest potency.

[0226] Comparison of SEQ ID NO: 12 to other exon 44 antisense sequences was performed in both RD cells and primary human skeletal muscle cells. All evaluated sequences were produced as peptide-conjugated PMOs using the CP06062 peptide (SEQ ID NO: 578). This allowed for a direct comparison of the relative efficacy of antisense sequences, regardless of antisense chemistry or cellular delivery.

[0227] Alignment of the sequences (SEQ ID NOS: 600, 601, 602, and 603) with SEQ ID NOS: 4, 8, 11, and 12 is shown in Figure 5B. SEQ ID NOS: 601 and 603 are listed as SEQ ID NOS: 165 and 167 in WO2006 / 000057. SEQ ID NOS: 602 is listed in WO2004 / 083446 and as SEQ ID NOS: 21 in U.S. Patent Application 11 / 233,507. SEQ ID NOS: 600 was published in 2007 (Wilton, Fall et al., 2007). Comparison in RD cells showed that SEQ ID NOS: 602 and 603 were superior to SEQ ID NOS: 12 (Figure 5I). However, as shown in Figure 5J, in human primary skeletal muscle cells, SEQ ID NOS: 12 (8.86% exon skipping) was superior to SEQ ID NOS: 602 (6.42%). Similar experiments were also performed with SEQ ID NOS: 603.

[0228] Example 5 Exon 45 scan We designed and synthesized a series of overlapping antisense PPMOs targeting human dystrophin exon 45. For exon 45, we generated a series of 22 PPMOs, each 25 bases long, as shown in Figure 6A (SEQ ID NOS: 21, 23, 25, 27, 29, 31, 32, 34, 35, 37, 39, 41, 43, and 45-53). These PPMOs were evaluated for exon skipping efficacy by treating RD cells and human primary skeletal muscle cells at various concentrations, as described above in Materials and Methods. Five PPMOs (SEQ ID NOS: 27, 29, 34, and 39) were identified as effective in inducing exon skipping and selected for further evaluation. Dose-ranging experiments in RD cells were used to confirm the relative potency of these four PPMO sequences, as shown in Figures 6C-G and summarized in Figure 6H. SEQ ID NOS: 49 was used as a negative control in these experiments. SEQ ID NOs: 29 and 34 were shown to be the most effective in inducing exon 45 skipping, as shown in Figure 6H.

[0229] Comparison of SEQ ID NO:34 to other exon 45-targeting antisense sequences was performed in both RD cells and primary human skeletal muscle cells. All evaluated sequences were generated as peptide-conjugated PMOs using the CP06062 peptide (SEQ ID NO:578). This allowed for a direct comparison of the relative efficacy of antisense sequences, regardless of antisense chemistry or cellular delivery. Alignment of sequences (SEQ ID NOs:604, 605, 606, and 607) with SEQ ID NOs:27, 29, 34, and 39 is shown in Figure 6B. SEQ ID NOs:604 and 607 are listed as SEQ ID NOs:211 and 207, respectively, in WO 2006 / 000057. SEQ ID NOs:605 and 606 are listed as SEQ ID NOs:23 and 1, respectively, in U.S. Application Serial No. 11 / 233,507. Comparison in RD cells showed that SEQ ID NO:34 outperformed all four evaluated sequences, as shown in Figure 6I. Testing of these compounds in different populations of human primary skeletal muscle cells has been performed as described above.

[0230] Sequence Listing Sequences are shown using the common nucleotide base symbols for DNA: A, G, C, and T. Other antisense chemistries, such as 2'-O-methyl, use U instead of T. Any base may be substituted with inosine (I), especially in stretches of three or more G residues.

[0231] [Table 6-1]

[0232] [Table 6-2]

[0233] [Table 6-3]

[0234] [Table 6-4]

[0235] Table 6-5

[0236] Table 6-6

[0237] Table 6-7

[0238] Table 6-8

[0239] Table 6-9

[0240] Table 6-10

[0241] Table 6-11

[0242] Table 6-12

[0243] Table 6-13

[0244] Table 6-14

[0245] Table 6-15

[0246] Table 6-16

[0247] Table 6-17

[0248] Table 6-18

[0249] Table 6-19

[0250] Table 6-20

[0251] Table 6-21

[0252] Table 6-22

[0253] Table 6-23

[0254] Table 6-24

[0255] Table 6-25

Claims

[Claim 1] A kit as described in the specification.

Images