Multi-exon skipping compositions for DMD

Abstract

Provided are antisense molecules capable of binding to a selected target site in the human muscular dystrophin gene in order to induce exon skipping, and methods of treating muscular dystrophy using the same.

Description

[0001] Related Applications

[0002] This application is a divisional application of Chinese Patent Application No. 201610137572.3, which is a divisional application of Chinese Patent Application No. 200980142321.2. This application claims priority to U.S. Provisional Patent Application No. 61 / 108,416 filed on October 24, 2008, which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The application provides a text format of the relevant sequence table for replacing paper copy, and these sequence tables are incorporated into this specification by reference. The name of the text file containing these sequence tables is 120178_410PC_SEQUENCE_LISTING.txt. This text file is 156KB, created on October 23, 2009, and submitted electronically through EFS-Web. Field of the Invention

[0005] The present application relates to novel antisense compounds and compositions suitable for promoting exon skipping of the human dystrophin gene. Methods of inducing exon skipping using antisense compositions suitable for use in the methods of the present invention are also provided. Background of the Invention

[0007] Antisense technology has been developed using a variety of chemicals to affect gene expression at different levels (transcription, splicing, stability, translation). Many of these studies focus on using antisense compounds to correct or compensate for abnormal or disease-related genes within a wide range of indications. Antisense molecules can specifically inhibit gene expression, and therefore, many studies, which are dedicated to oligonucleotides as gene expression regulators, have focused on inhibiting the expression of targeted genes or the function of cis-acting elements. In the case of some viral RNA targets, antisense oligonucleotides are usually directed to RNA, or positive strands (such as mRNA) or negative strands. In order to achieve the desired effect of downregulation of specific genes, oligonucleotides usually promote the degradation of targeted mRNA, block the translation of mRNA or block the function of cis-acting RNA elements, thereby effectively preventing the de novo synthesis of target proteins or the replication of viral RNA.

[0008] However, these techniques do not work if 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 transcripts will not undergo targeted degradation or steric inhibition, so antisense oligonucleotide chemistries will not promote degradation of the target mRNA or block translation.

[0009] In many genetic diseases, the impact of mutation on the final expression of genes can be regulated by the process of targeted exon skipping in the splicing process. The splicing process is guided by a complex multi-component mechanism, which makes the adjacent exons-introns in the pre-mRNA (pre-mRNA) connect closely and cut the phosphodiester bond at the end of the intron, accompanied by their subsequent transformation between the exons to be spliced ​​together. This complex and highly accurate process is mediated by the sequence motifs in the pre-mRNA, which are relatively semi-conservative RNA fragments, and many nuclear splicing factors participating in the splicing reaction afterwards are combined with it. By changing the splicing mechanism to read or identify the mode of the motifs involved in the processing of the pre-mRNA, differentiated splicing mRNA molecules can be produced. It has now been recognized that most human genes are spliced ​​alternatively during normal gene expression, although the relevant mechanism has not yet been identified.

[0010] In cases where normal functional proteins are terminated prematurely due to mutations, the use of antisense technology to restore the production of some functional proteins has shown that it is possible to interfere with the splicing process, and if the exons associated with pathogenic mutations can be specifically deleted from some genes, shortened protein products can sometimes be produced that have biological properties similar to those of the native protein or have biological activities sufficient to improve 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. (U.S. Pat. Nos. 5,627,274, 5,916,808, 5,976,879 and 5,665,593) disclose methods of using modified antisense oligonucleotide analogs that do not promote degradation of targeted mRNA precursors to combat aberrant splicing. Bennett et al. (U.S. Pat. No. 6,210,892) describe antisense regulation of wild-type cell mRNA processing also using antisense oligonucleotide analogs that do not induce RNAse H-mediated cleavage of target RNA.

[0011] The process of targeted exon skipping may be particularly useful for long genes in which there are many exons and introns, where the genetic components or proteins with redundant exons can function without one or more specific exons. Efforts to redirect gene processing for the treatment of genetic diseases associated with truncations caused by mutations in various genes have focused on the use of antisense oligonucleotides that (1) fully or partially overlap with an element involved in the splicing process or (2) bind to the mRNA precursor in sufficient proximity to the element to prevent the binding and function of splicing factors that normally mediate a specific splicing reaction occurring at that element.

[0012] Duchenne muscular dystrophy (DMD) is caused by a defect in the expression of the dystrophin protein. The gene encoding the protein contains 79 exons spread across more than 2 million nucleotides of DNA. Any exon mutation, which changes the exon reading frame, introduces a stop codon, or is characterized by the removal of a complete out-of-frame exon or multiple exons or copies of one or more exons, can potentially interfere with the production of functional dystrophin, leading to DMD.

[0013] A less severe form of muscular dystrophy is Becker muscular dystrophy (BMD), which is found to occur because a mutation, usually a deletion of one or more exons, results in a correct reading frame along the entire dystrophin transcript, so that translation of the mRNA into protein does not terminate prematurely. If, in the processing of the mutant dystrophin pre-mRNA, the joining of upstream and downstream exons maintains the correct reading frame of the gene, the result is an mRNA encoding a protein with a short internal deletion that retains some activity leading to the Becker phenotype.

[0014] The deletion of one or more exons does not change the reading frame of dystrophin, but will cause the BMD phenotype, while the deletion of exons that causes frameshift will cause DMD (Monaco, Bertelson et al. 1988). In general, dystrophin mutations include point mutations and exon deletions that change the reading frame, thereby blocking normal protein translation, thus leading to DMD. It should also be noted that some BMD and DMD patients have exon deletions covering multiple exons.

[0015] Although antisense molecules may provide a tool for treating Duchenne muscular dystrophy (DMD), attempts to induce exon skipping using antisense molecules have had mixed results. Dystrophin exon 19 was successfully skipped from the dystrophin pre-mRNA using a variety of antisense molecules directed to flanking splice sites or motifs within an exon that is included in the exon definition as described by Errington et al. (Errington, Mann et al. 2003).

[0016] Wilton et al. (Wilton, Lloyd et al. 1999) first reported an example of specific, reproducible exon skipping in the mdx mouse model. By directing antisense molecules to the donor splice site, exon 23 skipping was induced in the dystrophin mRNA within 6 hours of treating the cultured cells. Wilton et al. also described targeting the receptor region of the mouse dystrophin mRNA precursor with longer antisense oligonucleotides. Although the first antisense oligonucleotide directed to the intron 23 donor splice site induced exon skipping in primary cultured myoblasts, it was found that the compound was much less effective in immortalized cell cultures expressing higher levels of dystrophin.

[0017] Despite these efforts, there remains a need for improved antisense oligomers targeting multiple dystrophin exons and improved myo-delivery compositions and methods for DMD therapeutic applications. SUMMARY OF THE INVENTION

[0019] 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.

[0020] In certain embodiments, exon skipping of single or multiple exons can be improved by covalently linking two or more antisense oligonucleotide molecules together (see, eg, Aartsma-Rus, Janson et al. 2004).

[0021] In certain embodiments, the antisense compounds of the invention induce exon skipping in the human dystrophin gene, thereby allowing muscle cells to produce functional dystrophin.

[0022] The antisense oligonucleotide compounds of the present invention (also referred to herein as oligomers) typically: (i) comprise morpholino subunits and phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit, (ii) contain 10-40 bases, preferably 20-35 bases, and (iii) comprise a base sequence that can effectively hybridize with at least 12 consecutive bases of a target sequence in a dystrophin mRNA precursor and induce exon skipping.

[0023] In certain embodiments, antisense compounds of the invention may comprise a phosphorus-containing intersubunit linkage linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit according to the following structure (I):

[0024]

[0025] in:

[0026] Y1 is -O-, -S-, -NH- or -CH2-;

[0027] Z is O or S;

[0028] Pj is a purine or pyrimidine base pairing moiety that efficiently binds to a polynucleotide base by base-specific hydrogen bonding; and

[0029] X is fluoro, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, amino, optionally substituted alkylamino, or optionally substituted heterocyclyl.

[0030] In certain embodiments, the uncharged intersubunit linkages described above may be interspersed with positively charged linkages at physiological pH, wherein 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 linkages may have the above structure, wherein X is an optionally substituted 1-piperazinyl. In other embodiments, the positively charged linkages may have the above structure, wherein X is a substituted 1-piperazinyl, wherein the 1-piperazinyl is substituted at the 4-position with an optionally substituted alkylguanidinyl moiety.

[0031] If the antisense compound administered can effectively target the splice site of preprocessed human dystrophin, it can have a base sequence complementary to a target region containing 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.

[0032] In certain embodiments, the antisense sequence of the invention is comprised of:

[0033] (a) any sequence 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 producing exon 44 skipping in the processing of human dystrophin pre-processed mRNA;

[0034] (b) any sequence 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 producing exon 45 skipping in the processing of human dystrophin pre-processed mRNA;

[0035] (c) any sequence 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, 99 and 101, for use in producing exon 46 skipping in the processing of human dystrophin pre-processed mRNA;

[0036] (d) any sequence 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 producing exon 47 skipping in the processing of human dystrophin pre-processed mRNA;

[0037] (e) any sequence 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 producing exon 48 skipping in the processing of human dystrophin pre-processed mRNA;

[0038] (f) any sequence 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 producing exon 49 skipping in the processing of human dystrophin pre-processed mRNA;

[0039] (g) any sequence 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 producing exon 50 skipping in the processing of human dystrophin pre-processed mRNA;

[0040] (h) any sequence 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 producing exon 51 skipping in the processing of human dystrophin pre-processed mRNA;

[0041] (i) any sequence identified by SEQ ID NOS:372-415, preferably SEQ ID NOS:372-397, and more preferably SEQ ID NOS:379-382, 384, 390 and 392-395, for use in producing exon 52 skipping in the processing of human dystrophin pre-processed mRNA;

[0042] (j) any sequence 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 producing exon 53 skipping in the processing of human dystrophin pre-processed mRNA;

[0043] (k) any sequence 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 producing exon 54 skipping in the processing of human dystrophin pre-processed mRNA;

[0044] (1) any sequence 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 producing exon 55 skipping in the processing of human dystrophin pre-processed mRNA;

[0045] In certain embodiments, the compound may be conjugated to an arginine-rich polypeptide effective to promote cellular uptake of the compound. Exemplary peptides, in addition to other peptides described herein, include peptides identified by SEQ ID NOS: 570-578.

[0046] In an exemplary embodiment, the arginine-rich polypeptide is covalently coupled at its N-terminal or C-terminal residue to the 3' or 5' end of the antisense compound. In another exemplary embodiment, the antisense compound consists of morpholino subunits and a phosphorus-containing intersubunit linkage linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit.

[0047] Typically, the peptide-oligomer conjugate may also include a homing peptide that is selective for a selected mammalian tissue, i.e., the same tissue targeted by the cell-penetrating peptide. The conjugate may be in the form of a cell-penetrating peptide-homing peptide-antisense oligomer, or more preferably in the form of a homing peptide-cell-penetrating peptide-antisense oligomer. For example, the peptide-conjugated compound for treating Duchenne muscular dystrophy as described above may also include a homing peptide that is selective for muscle tissue, such as a peptide having a sequence determined by SEQ ID NO: 579, which is conjugated to a cell-penetrating peptide. Exemplary conjugates of this type include those represented herein as CP06062-MSP-PMO (cell-penetrating peptide-homing peptide-antisense oligomer) and represented as MSP-CP06062-PMO (homing peptide-cell-penetrating peptide-antisense oligomer) (see SEQ ID NOs: 580-583).

[0048] In some embodiments, the peptide is coupled to the oligomer via a linker moiety. In certain embodiments, the linker moiety may comprise an optionally substituted piperazine moiety. In other embodiments, the linker moiety may also comprise a β-alanine and / or a 6-aminocaproic acid subunit. Still in other embodiments, the peptide is directly coupled to the oligomer without a linker moiety.

[0049] The coupling of the peptide to the oligomer can be located at any position 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 coupling of the peptide can be located at the 3' end of the oligomer. In other embodiments, the coupling of the peptide to the oligomer can be located at the 5' end of the oligomer. Still in other embodiments, the peptide can be coupled to the oligomer through any intersubunit connection.

[0050] In some embodiments, the peptide is coupled to the oligomer at the 5' end of the oligomer. In embodiments comprising phosphorus-containing intersubunit linkages, the peptide can be coupled to the oligomer via a covalent bond to the phosphorus of the terminal linking group. This manner of coupling may or may not require the linker moiety described above.

[0051] In still other embodiments, the peptide is coupled to the oligomer at the 3' end of the oligomer. In some other embodiments, the peptide is coupled to the nitrogen atom of the morpholino group at the 3' end of the oligomer. In this regard, the peptide can be coupled to the oligomer directly or through the above-mentioned linker part.

[0052] In some embodiments, the oligomer can be coupled to a moiety that enhances the solubility of the oligomer in an aqueous medium. In some embodiments, the moiety that enhances the solubility of the oligomer in an aqueous medium is polyethylene glycol. Still in other embodiments, the moiety that enhances the solubility of the oligomer in an aqueous medium is triethylene glycol. For example, in some embodiments, the moiety that enhances the solubility in an aqueous medium can be coupled to the oligomer at the 5' end of the oligomer. The coupling of the moiety that enhances the solubility of the oligomer in an aqueous medium to the oligomer can be direct or through the above-mentioned linker moiety.

[0053] Certain embodiments of the present invention provide antisense molecules selected and / or adapted to assist in the prophylactic or therapeutic treatment of a genetic disease, comprising at least one antisense molecule in a form suitable for delivery to a patient.

[0054] Certain embodiments of the present invention provide methods for treating patients with genetic diseases, wherein there is a mutation in a gene encoding a specific protein and the effects of the mutation can be eliminated by exon skipping, comprising the steps of: (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 purified and isolated antisense oligonucleotides of the present invention in the preparation of a medicament for treating genetic diseases.

[0055] Certain embodiments provide methods of treating muscular dystrophy, such as a disease state characterized by Duchenne muscular dystrophy, comprising administering to a patient in need of treatment an effective amount of an appropriately designed antisense oligonucleotide as described herein, which is associated with a specific genetic lesion in the patient. Furthermore, certain embodiments provide methods of prophylactically treating a patient in order to prevent or at least minimize muscular dystrophy, including Duchenne muscular dystrophy, comprising the steps of administering to a patient an effective amount of an antisense oligonucleotide or a pharmaceutical composition comprising one or more of these biomolecules.

[0056] Certain embodiments are directed to a method of treating muscular dystrophy in an individual comprising administering to the patient an effective amount of a substantially uncharged antisense compound comprising 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, the antisense compound comprising a sequence selected from the group consisting of SEQ ID NOS: 1-569 and 612-635 and capable of forming a heteroduplex with a complementary mRNA sequence in an exon of a dystrophin gene between the compound and the mRNA having a Tm of at least 45°C, wherein the exon is selected from exons 44-55.

[0057] In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD). In certain embodiments, the muscular dystrophy is Becker muscular dystrophy (BMD).

[0058] In certain embodiments, the sequence is selected from SEQ ID NOS: 1-20, and the exon is exon 44. In certain embodiments, the sequence is selected from SEQ ID NOS: 21-76 and 612-624, and the exon is exon 45.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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 NOS:287.

[0063] Certain embodiments provide a kit for treating a genetic disease, the kit comprising at least one antisense oligonucleotide of the invention packaged in a suitable container, and instructions for use thereof.

[0064] These and other objects and features will be more fully understood from the following detailed description of the present invention read in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1A Exemplary morpholino oligomer structures with phosphodiamidate linkages are shown.

[0067] Figure 1B Conjugates of arginine-rich peptides and antisense oligomers according to embodiments of the present invention are shown.

[0068] Figure 1C Shows Figure 1B A conjugate wherein the main chain contains one or more positively charged groups.

[0069] Figure 1D -G shows a repeating subunit segment of an exemplary morpholino oligonucleotide, which is designated DG.

[0070] Figure 2A The relative positions and results of exon 51 scanning of antisense oligomers designed to induce exon 51 skipping of human dystrophin are shown.

[0071] Figure 2B -C shows the relative activity of three optimal oligomers selected from exon 51 scanning (SEQ ID NOs: 324, 326 and 327) relative to sequences that can effectively induce exon 51 skipping (AVI-5658; SEQ ID NO: 588 and h51AON1; SEQ ID NO: 594) in cultured human rhabdomyosarcoma (RD) cells and human primary skeletal muscle cells. Figure 2D The relative positions of three selected oligomers within exon 51 compared to certain sequences are shown.

[0072] Figure 3A The relative positions and results of exon 50 scanning of antisense oligomers designed to induce exon 50 skipping of human dystrophin compared to other sequences that induce exon 50 skipping are shown.

[0073] Figure 3B The relative position and activity 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) are shown.

[0074] Figure 4A The relative positions and results of exon 53 scanning by antisense oligomers designed to induce exon 53 skipping of human dystrophin are shown. Figure 4B The relative positions of certain sequences used to compare the exon skipping activity of those oligomers selected as most active in exon 53 scanning are shown.

[0075] Figure 4C -F shows the results of a dose-ranging study using oligomers selected as most effective in scanning exon 53 (SEQ ID NOS: 422, 428, 429 and 431), which are summarized in Figure 4G middle.

[0076] Figure 4H and 4I Shown are the relative activities of certain sequences (SEQ ID NOS:608-611) compared to the activity of the most active exon 53 skipping oligomer (SEQ ID NO:429) in RD cells and human primary skeletal muscle cells.

[0077] Figure 5A Shown are the relative positions and results of exon 44 scanning with antisense oligomers designed to induce exon 44 skipping of human dystrophin. Figure 5B The relative positions within exon 44 of certain sequences used to compare the exon skipping activity of those oligomers selected as most active in scanning exon 44 are shown.

[0078] Figure 5C -G shows the results of a dose-ranging study using oligomers selected as most effective in exon 44 scanning (SEQ ID NOS: 4, 8, 11, 12 and 13), which are summarized in Figure 5H middle.

[0079] Fig.5I and 5J Shown are the relative activities of certain sequences (SEQ ID NOS: 600-603) compared to the activity of the most active exon 53 skipping oligomer (SEQ ID NO: 12) in RD cells and human primary skeletal muscle cells.

[0080] Fig. 6A The relative positions and results of exon 45 scanning by antisense oligomers designed to induce exon 45 skipping of human dystrophin are shown. Figure 6B The relative positions within exon 45 of certain sequences used to compare the exon skipping activity of those oligomers selected as most active in exon 45 scanning are shown.

[0081] Figure 6C -F shows the results of a dose-ranging study using oligomers selected as most effective in exon 45 scanning (SEQ ID NOS: 27, 29, 34 and 39), which are summarized in Figure 6H middle. Figure 6G A relatively inactive oligomer (SEQ ID NO: 49) was used as a negative control.

[0082] Fig.6I and 6JShown are the relative activities of certain sequences (SEQ ID NOS: 604-607) compared to the activity of the most active exon 53 skipping oligomer (SEQ ID NO: 34) in RD cells and human primary skeletal muscle cells.

[0083] Detailed Description of the Invention

[0084] Embodiments of the present invention generally relate to improved antisense compounds specifically designed to induce exon skipping in the dystrophin gene, and methods of use thereof. The dystrophin protein plays a vital role in muscle function, and various muscle-related diseases are characterized by mutant forms of this gene. Therefore, in certain embodiments, the improved antisense compounds described herein induce exon skipping in mutant forms of the human dystrophin gene. For example, the mutant dystrophin gene found in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

[0085] Due to abnormal mRNA splicing events caused by the mutation, these mutant human dystrophin genes either express defective dystrophin or express almost unmeasurable dystrophin, which is a disease condition that leads to various forms of muscular dystrophy. To treat such disease conditions, the antisense compounds of the present invention generally hybridize to selected regions of preprocessed RNA of the mutant human dystrophin gene, induce exon skipping and differential splicing in the otherwise abnormally spliced ​​dystrophin mRNA, thereby causing muscle cells to produce mRNA transcripts encoding functional dystrophin. In certain embodiments, the resulting dystrophin is not necessarily a "wild-type" form of dystrophin, but rather a truncated, yet functional or semi-functional form of dystrophin.

[0086] By increasing the level of functional dystrophin in muscle cells, these and related embodiments can be used to prevent and treat muscular dystrophy, in particular, those forms of muscular dystrophy, such as DMD and BMD, which are characterized by the expression of defective dystrophin due to aberrant mRNA splicing. The specific oligomers described herein also provide improved dystrophin exon-specific targeting over other oligomers in use, thereby providing a clear practical advantage over alternative approaches to treating related forms of muscular dystrophy.

[0087] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar to or equivalent to the methods and materials described herein can be used to practice or test the present invention, preferred methods and materials are still described. For purposes of the present invention, the following terms are defined below.

[0088] definition

[0089] As used herein, the articles "a" and "an" refer to one or to more than one (ie, 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.

[0090] "About" means a quantity, level, value, amount, 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% of the referenced quantity, level, value, amount, frequency, percentage, dimension, size, amount, weight or length.

[0091] "Coding sequence" refers to any nucleotide sequence that contributes to the encoding of a polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleotide sequence that does not contribute to the encoding of a polypeptide product of a gene.

[0092] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" should be understood to imply the inclusion of stated steps or elements or groups of steps or elements but not the exclusion of any other steps or elements or groups of steps or elements.

[0093] "Consisting of" means including and limited to whatever is within the phrase "consisting of." Thus, the phrase "consisting of" means that the listed elements are required or mandatory, and that no other elements are present. "Consisting essentially of" means including any elements listed within the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" means that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they materially affect the activity or action of the listed elements.

[0094] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., sequences of nucleotides) that are associated by base pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA". Complementarity can be "partial", in which only some nucleic acid bases match according to the base pairing rules. Or, there can be "complete" or "complete" complementarity between nucleic acids. The degree of complementarity between nucleic acid chains has a significant effect on the hybridization efficiency and intensity between nucleic acid chains. Although perfect complementarity is generally desired, some embodiments may include one or more but preferably 6, 5, 4, 3, 2 or 1 mismatches with the target RNA. Included in variations at any position within the oligomer. In certain embodiments, variations on sequences close to the ends of the oligomer are generally preferred over internal variations, and if present, are generally within about 6, 5, 4, 3, 2 or 1 nucleotides of the 5' and / or 3' ends.

[0095] The term "cell penetrating peptide" or "CPP" is used interchangeably and refers to a cationic cell penetrating peptide, also known as a transport peptide, a carrier peptide, or a peptide transduction domain. The peptides shown herein can induce cell penetration in 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of a given cell culture population, including all integers in between, and allow macromolecules to migrate within multiple tissues in the body after systemic administration.

[0096] The term "antisense oligomer" or "antisense compound" is used interchangeably and refers to a sequence of cyclic subunits connected by inter-subunit linkages, each subunit carrying a base pairing moiety that hybridizes with a target sequence in a nucleic acid (usually RNA) by Watson-Crick base pairing to form a nucleic acid: oligomer heteroduplex within the target sequence. The cyclic subunits are based on ribose or other pentose sugars, or in a preferred embodiment, on morpholino groups (see description of morpholino oligomers below).

[0097] Such antisense oligomers can be designed to block or inhibit the translation of mRNA or inhibit the splicing processing of natural mRNA precursors, and can be considered as "directed" or "targeted" target sequences hybridized therewith. In certain embodiments, the target sequence includes the region of the 3' or 5' splicing site or branch point of the AUG start codon of the mRNA, the pre-processed mRNA. The target sequence can be located in an exon or intron. The target sequence of the splicing site can include an mRNA sequence of 1 to about 25 base pairs at its 5' end with a normal splicing site receptor junction downstream of the pre-processed mRNA. The preferred target sequence for splicing is any region of the pre-processed mRNA including the splicing site or being integrally contained within the exon coding sequence or across a splicing acceptor or donor site. When the oligomer targets the nucleic acid of the target in the above manner, the oligomer is more commonly referred to as a "targeted" biologically relevant target, such as a protein, virus or bacteria. Included are antisense oligomers comprising one or more of SEQ ID NOS: 1-569 and 612-635, or consisting essentially of one or more of SEQ ID NOS: 1-569 and 612-635, or consisting of one or more of SEQ ID NOS: 1-569 and 612-635. Also included are variants of these antisense oligomers, including variants having 80%, 85%, 90%, 95%, 97%, 98% or 99% (including all integers in between) sequence identity or sequence homology to any 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 variants 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, as described herein, comprising an appropriate number of charged linkages, e.g., up to about 1 for every 2-5 uncharged linkages, e.g., about 4-5 for every 10 uncharged linkages, and / or as described herein, comprising an Arg-rich peptide attached thereto.

[0098] The term "morpholino oligomer" or "PMO" (phosphoramidate or phosphodiamidate morpholino oligomer) refers to an oligonucleotide analog composed of morpholino subunit structures, wherein (i) the structures are linked by phosphorus-containing linkages, which are 1-3 atoms in length, preferably 2 atoms, preferably uncharged or cationic, linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit, and (ii) each morpholino ring carries a purine or pyrimidine base pairing moiety that effectively binds to a base on a polynucleotide by base-specific hydrogen bonding. See, e.g. Figure 1AThe structure of , which shows a preferred phosphorodiamidate connection type. This connection can be changed as long as it does not interfere with binding or activity. For example, the oxygen attached to the phosphorus can be replaced by sulfur (thiophosphorodiamidate). The 5' oxygen can be replaced by an amino or lower alkyl substituted amino group. The pendant nitrogen attached to the phosphorus can be non-substituted or mono- or di-substituted (optionally substituted) by a lower alkyl group. See the discussion of cationic connection below. The synthesis, structure and binding characteristics of morpholino oligomers are described in detail in U.S. Patents Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063 and 5,506,337 and PCT Application No. PCT / US07 / 11435 (cationic linker), all of which are incorporated herein by reference.

[0099] The purine or pyrimidine base pairing moiety is usually adenine, cytosine, guanine, uracil, thymine or hypoxanthine. Also included are bases such as 4-pyridone, 2-pyridone, phenyl, pseudouracil, 2,4,6-trimethoxybenzene (trimeth115 thoxy benzene), 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (such as 5-methylcytidine), 5-alkyluridine (such as ribothymidine), 5-halouridine (such as 5-bromouridine) or 6-azapyrimidine or 6-alkylpyrimidine (such as 6-methyluridine), propyne, Q nucleoside (quesosine), 2-thiouridine, 4-thiouridine, wybutoxosine, wybutoxosine, 4-acetylcytidine (acetyltidine), 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, β-D-galactoside Q nucleoside (queosine), 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonyhnethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, β-D-mannosyl Q nucleoside (queosine), uridine-5-oxoacetic acid, 2-thiocytosine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). In this regard, "modified bases" refer to nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), as described above; these bases can be used in any position of antisense molecules. It will be appreciated by those skilled in the art that Ts and Us are interchangeable, depending on the oligomer used. For example, other antisense chemicals that are more like RNA, such as 2'-O-methyl antisense oligonucleotides, T bases can be represented as U (see, for example, Sequence ID listings (Sequence ID listings, identifier lists)).

[0100] "Amino acid subunit" or "amino acid residue" refers to an α-amino acid residue (such as -CO-CHR-NH-) or a β- or other amino acid residue (such as -CO-(CH2) n CHR-NH-), wherein R is a side chain (which may include hydrogen) and n is 1-6, preferably 1-4.

[0101] The term "naturally occurring amino acid" refers to amino acids found in proteins in nature, such as the 20 (L)-amino acids used during protein biosynthesis and precursor amino acids such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine. The term "unnatural amino acid" refers to amino acids not found in proteins in nature, examples of which include β-alanine (β-Ala; or B), 6-aminohexanoic acid (Ahx) and 6-aminovaleric acid. Other examples of "unnatural amino acids" include, but are not limited to, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, etc., which are known to those skilled in the art.

[0102] "Effective amount" or "effective therapeutic amount" refers to the amount of a therapeutic compound, such as an antisense oligomer, administered to a mammalian subject as a single dose or as part of a series of doses, which is effective to produce a desired physiological response or therapeutic effect in the subject. An example of a desired physiological response includes enhanced expression of a relatively functional or biologically active form of dystrophin compared to an antisense oligomer or a control oligomer, primarily in muscle tissue or cells containing a defective dystrophin or no dystrophin. Examples of desired therapeutic effects include, but are not limited to, improvement of muscular dystrophy symptoms or conditions, reduction in the development of muscular dystrophy symptoms or conditions, and slowing of the onset of muscular dystrophy symptoms or conditions, among others. Examples of such symptoms include fatigue, mental retardation, muscle weakness, difficulty with motor skills (such as running, bouncing, jumping), frequent falls, and difficulty walking. Muscular dystrophy pathology is characterized by, for example, muscle fiber damage and membrane leakage. For antisense oligomers, this effect is generally brought about by altering the splicing processing of a selected target sequence (such as dystrophin), such as by inducing exon skipping.

[0103] "Exon" refers to a defined portion of a nucleic acid encoding a protein, or a nucleic acid sequence represented by a mature form of an RNA molecule after any portion of a preprocessed RNA (precursor) has been removed by splicing. The mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as rRNA or tRNA. The human dystrophin gene has about 75 exons.

[0104] "Intron" refers to a region of nucleic acid (within a gene) that is not translated into protein. Introns are non-coding portions that are removed by splicing during formation of mature RNA after transcription into pre-mRNA (pre-mRNA).

[0105] "Exon skipping" is generally a process by which an entire exon or a portion thereof is removed from a given pre-processed RNA and thereby excluded from a mature RNA, such as a mature mRNA that is translated into a protein. Therefore, the portion of the protein encoded by the skipped exon is not present in the expressed form of the protein, typically forming an altered but still functional form of the protein. In certain embodiments, the skipped exon is an abnormal exon from the human dystrophin gene, which may contain mutations or other changes in its sequence that otherwise lead to abnormal 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.

[0106] "Dystrophin" is a rod-shaped cytoplasmic protein and is an important part of a protein complex that connects the cellular backbone of the muscle fiber to the surrounding extracellular matrix through the cell membrane. Dystrophin contains multiple functional domains. For example, dystrophin contains an actin binding domain at about amino acids 14-240 and a central rod-shaped domain at amino acids 253-3040. This large central domain is formed by 24 membrane contractile protein-like triple helical elements of about 109 amino acids, which have homology with α-actin and membrane contractile proteins. These repeats are usually interrupted by four proline-rich non-repeating segments, which are also called hinge regions. Repeats 15 and 16 are separated by a sequence of 18 amino acids, which appears to provide the main site for proteolytic cleavage of dystrophin. The sequence identity between most repeats ranges from 10-25%. One repeat contains three α helices: 1, 2 and 3. Alpha helices 1 and 3 are each formed by 7 helical turns, which may interact as coiled coils through hydrophobic interfaces. Alpha helix 2 has a more complex structure and is formed by segments of four and three helical turns separated by glycine or proline residues. Two exons encode one repeat, which are usually interrupted by an intron between amino acids 47 and 48 in the first part of alpha helix 2. Other introns are found at other positions in the repeat, usually scattered in helix 3. Dystrophin also contains a cysteine-rich domain at about amino acids 3080-3360, which includes a cysteine-rich segment (i.e., 15 cysteines in 280 amino acids) showing homology to the C-terminal domain of the slime mold (Dictyostelium discoideum) alpha actin. The carboxyl-terminal domain is located at about amino acids 3361-3685.

[0107] The amino terminus of dystrophin binds to F-actin and the carboxyl terminus binds to the dystrophin-associated protein complex (DAPC) on the sarcolemma. DAPC includes dystrophin, sarcomeric protein, integrins, and caveolin, and mutations in any of these components lead to autosomal inherited muscular dystrophy. DAPC is unstable in the absence of dystrophin, which causes reduced membrane protein levels and then progressive fiber damage and membrane leakage. In many forms of muscular dystrophy, 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, mainly due to mutations in the gene sequence that lead to incorrect splicing. The dominant expression of defective dystrophin, or the complete lack of dystrophin or dystrophin-like proteins, leads to the rapid development of muscle degeneration, as described above. In this regard, a "defective" dystrophin protein is characterized by a form of dystrophin produced in certain DMD or BMD individuals known in the art or by the absence of detectable dystrophin protein.

[0108] Table A provides a description of various dystrophin domains, the amino acid residues comprising these domains, and the exons encoding them.

[0109] Table A

[0110]

[0111]

[0112] As used herein, the terms "function" and "functional" and the like refer to a biological, enzymatic or therapeutic function.

[0113] "Functional" dystrophin generally refers to a dystrophin having biological activity sufficient to reduce the progressive degeneration of muscle tissue, which is characteristic of muscular dystrophy, generally compared to an altered or "defective" form of dystrophin present in certain individuals with DMD or BMD. In certain embodiments, a functional dystrophin may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (including all integers in between) of the in vivo or in vitro biological activity of wild-type dystrophin, which can be measured according to routine techniques in the art. As an example, dystrophin-related activity in in vitro muscle cultures can be measured according to myotube size, myofibril organization (or decomposition), contractile activity, and spontaneous clustering of acetylcholine receptors (see, e.g., Brown et al., Journal of Cell Science. 112: 209-216, 1999). Animal models are also an important resource for studying disease pathology, providing a means for detecting dystrophin-related activity. The two 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 (see, e.g., Collins & Morgan, Int J Exp Pathol 84: 165-172, 2003). These and other animal models can be used to measure the functional activity of various dystrophins, including truncated forms of dystrophin, such as those produced by certain exon skipping antisense compounds of the present invention.

[0114] "Gene" means a genetic unit occupying a specific location on a chromosome and consisting of transcriptional and / or translational regulatory sequences and / or coding regions and / or untranslated sequences (ie, introns, 5' and 3' untranslated sequences).

[0115] "Isolated" means a material that is substantially or essentially free of components that normally accompany it in its natural state. For example, "isolated polynucleotide" as used herein refers to a polynucleotide that has been purified or removed from sequences that flank it in its naturally occurring state, such as a DNA fragment that has been removed from sequences that are normally adjacent to it.

[0116] "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 individual compared to the response caused by no antisense compound or a control compound. Measurable physiological responses may include increased expression of a functional form of dystrophin, or an increase in dystrophin-related biological activity in muscle tissue, in addition to other responses that are apparent from the understanding of the art and the description herein. Increased 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 an increase in 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 the muscle fibers. For example, it has been shown that if 25-30% of the fibers express dystrophin, about 40% improvement in muscle function will occur (see, e.g., DelloRusso et al, Proc Natl Acad Sci USA 99: 12979–12984, 2002). An "increase" or "enhancement" amount is generally a "statistically significant" amount and may include an increase of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g., 500, 1000 times) (including all integers and decimal points greater than 1 in between), such as 1.5, 1.6, 1.7, 1.8, etc.) the amount produced by a non-antisense compound (lacking an agent) or a control compound.

[0117] The terms "reduce" or "inhibit" generally refer to the ability of one or more antisense compounds of the invention to "reduce" a relevant physiological or cellular response, such as a disease symptom or condition described herein, which can be measured according to conventional techniques in the diagnostic arts. 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 a muscular dystrophy symptom or condition, or a reduction in the expression of a defective form of dystrophin, such as an altered form of dystrophin expressed in an individual with DMD or BMD. A "reduction" in a response compared to a no antisense compound or a control compound is statistically significant and can 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.

[0118] "Homology" refers to the percentage of amino acids that are identical or constitute conservative substitutions. Homology can be determined using a sequence comparison program 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 in the alignment, such gaps determined by, for example, the alignment algorithm used by GAP.

[0119] As used herein, "sequence identity" or, for example, "a sequence that is 50% identical to ..." refers to a sequence that is identical on a nucleotide-to-nucleotide basis or an amino acid-to-amino acid basis in a comparison window. Therefore, the "percentage of sequence identity" can be calculated as follows: compare two optimally aligned sequences in a comparison window, determine the number of positions at which 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 to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiply the result by 100 to produce the percentage of sequence identity.

[0120] Terms used to describe sequence relationships 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 often 15-18 and usually at least 25 monomer units in length, including nucleotides and amino acid residues. Because two polynucleotides each contain (1) a sequence that is similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence), and (2) a sequence that is different between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the two polynucleotide sequences in a "comparison window" to determine and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 consecutive positions, usually about 50-100, more usually about 100-150, in which, after optimal alignment, the sequence is compared to a reference sequence having the same number of consecutive positions. For optimal alignment of the 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). The optimal alignment for comparing sequences over a comparison window can be performed by computer-implemented algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment generated by any of a variety of selected methods (i.e., obtaining the highest percentage homology in the comparison window). Reference can also be made to, for example, the BLAST family of programs disclosed in Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0121] "Treatment" or "treating" of an individual (e.g., a mammal, e.g., a human) or a cell may include any type of intervention intended to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be prophylactic, or performed after the onset of disease or exposure to a pathogen. Treatment includes any desired effect on the symptoms or pathology of a disease or condition, such as dystrophin-related disease in certain forms of muscular dystrophy, and may include, for example, minimal changes or improvements in one or more measurable markers of the disease or condition being treated. "Preventative" treatment is also included, which may involve reducing the rate of development of the disease or condition being treated, or delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily mean complete eradication, cure, or prevention of a disease or condition, or its associated symptoms.

[0122] Thus, methods of treating muscular dystrophy, such as DMD and BMD, are included by administering to an individual in need thereof one or more antisense oligomers of the invention (e.g., SEQ ID NOS: 1-569 and 612-635, and variants thereof), optionally as part of a pharmaceutical formulation or dosage form. Also included are methods of inducing exon skipping in an individual 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 of a muscular dystrophy gene, preferably a human muscular dystrophy gene. As used herein, "individual" includes any animal that exhibits symptoms, or is at risk of exhibiting symptoms and can be treated with the antisense compounds of the invention, such as an individual suffering from DMD or BMD or any symptoms associated with these disease conditions (e.g., muscle fiber loss) or at risk of suffering from DMD or BMD and any symptoms associated with these disease conditions (e.g., muscle fiber loss). Suitable subjects (patients) include experimental animals (eg, mice, rats, rabbits or guinea pigs), farm animals and domestic animals or pets (eg, cats or dogs), including non-human primates and preferably human patients.

[0123] Also included are vector delivery systems capable of expressing the oligomeric dystrophin targeting sequence of the invention, such as a vector that expresses a polynucleotide sequence comprising any one 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, from, for example, a plasmid, phage, yeast or virus into which a polynucleotide can be inserted or cloned. The vector preferably contains one or more unique restriction sites and is capable of autonomous replication in a defined host cell, including a target cell or tissue or its progenitor cell or tissue, or is capable of integrating with the genome of a defined host cell so that the cloned sequence can be propagated. Thus, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, whose replication 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 contain any means to ensure self-replication. Alternatively, the vector can be a vector that can be integrated into the genome and replicated together with the chromosome into which it is integrated when introduced into a host cell.

[0124] The vector or nucleic acid construct system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA or transposon to be introduced into the host cell genome. The selection of the vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced. In this case, the vector or nucleic acid construct preferably has a vector that can be operably functioned in mammalian cells such as muscle cells. The vector may also include a selective 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 may include viral vector systems (i.e., viral-mediated transduction) including but not limited to retroviral (e.g., lentiviral) vectors, adenoviral vectors, adeno-associated viral vectors, and herpesvirus vectors, except other vectors known in the art.

[0125] As used herein, the term "operably linked" means that the oligomer coding sequence is placed under the regulatable control of a promoter, which in turn controls the transcription of the oligomer.

[0126] A wild-type gene or gene product is the gene that is most commonly found in a population and is thus arbitrarily designated the "normal" or "wild-type" form.

[0127] "Alkyl" or "alkylene" both refer to saturated straight or branched chain hydrocarbon groups containing 1-18 carbons. Examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, n-pentyl, and n-hexyl. The term "lower alkyl" refers to an alkyl group containing 1-8 carbons as defined herein.

[0128] "Alkenyl" refers to an unsaturated straight or branched hydrocarbon group containing 2-18 carbons and at least one carbon-carbon double bond. Examples include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl, n-pentenyl, and n-hexenyl. The term "lower alkenyl" refers to an alkenyl group containing 2-8 carbons as defined herein.

[0129] "Alkynyl" refers to an unsaturated straight or branched chain hydrocarbon group containing 2-18 carbons and comprising at least one carbon-carbon triple bond. Examples include, but are not limited to, ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, tert-butynyl, pentynyl, and hexynyl. The term "lower alkynyl" refers to an alkynyl group containing 2-8 carbons as defined herein.

[0130] "Cycloalkyl" refers to a mono- or polycyclic alkyl group. Examples include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0131] "Aryl" refers to a cyclic aromatic hydrocarbon moiety containing 5-18 carbon atoms forming one or more closed rings. Examples include, but are not limited to, phenyl, benzyl, naphthyl, anthracenyl, phenanthracenyl, and biphenyl.

[0132] "Aralkyl" refers to a radical of the formula RaRb where Ra is an alkylene chain as defined above and Rb is one or more aryl radicals as defined above, for example, benzyl, benzhydryl, and the like.

[0133] "Thioalkoxy" refers to a radical of the formula -SRc, wherein Rc is an alkyl radical as defined herein. The term "lower thioalkoxy" refers to an alkoxy radical as defined herein containing 1 to 8 carbons.

[0134] "Alkoxy" refers to a radical of the formula -ORda, wherein Rd is an alkyl radical as defined herein. The term "lower alkoxy" refers to an alkoxy radical as defined herein containing 1-8 carbons. Examples of alkoxy radicals include, but are not limited to, methoxy and ethoxy.

[0135] "Alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group.

[0136] "Carbonyl" refers to a -C(=O)- group.

[0137] "Guanidynyl" refers to a H2N(C=NH2)-NH- group.

[0138] "Amidinyl" refers to a H2N(C=NH2)CH- group.

[0139] "Amino" refers to the -NH2 group.

[0140] "Alkylamino" refers to a radical of the formula -NHRd or -NRdRd, wherein each Rd is independently an alkyl radical as defined herein. The term "lower alkylamino" refers to an alkylamino radical as defined herein containing 1 to 8 carbons.

[0141] "Heterocycle" means a 5-7 yuan monocyclic or 7-10 yuan bicyclic, heterocycle, which is saturated, unsaturated or aromatic, and contains 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, wherein nitrogen and sulfur heteroatoms are optionally oxidized, and nitrogen heteroatoms are optionally quaternized, including any of the above-mentioned heterocycles fused to the bicyclic ring of a benzene ring. Heterocycle can be connected by any heteroatom or carbon atom. Heterocycle includes heteroaryl as defined below. Thus, in addition to the heteroaryl listed below, heterocycle also includes morpholinyl, pyrrolidonyl, pyrrolidinyl, piperidinyl, piperazinyl (piperizynyl), hydantoinyl, valerolactamyl (valerolactamyl), oxirane, oxetanyl (oxetanyl), tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiopyranyl etc.

[0142] "Heteroaryl" means a 5-10 membered aromatic heterocyclic ring having at least one heteroatom selected from nitrogen, oxygen and sulfur and containing at least 1 carbon atom, including monocyclic and bicyclic ring systems. Representative heteroaryl groups are pyridyl, furanyl, benzofuranyl, phenylthio, benzophenylthio, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolinyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl and quinazolinyl.

[0143] The terms "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 oxygen-containing substituent (=O), both hydrogen atoms are substituted. In this regard, substituents include: deuterium, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted cycloalkyl, oxygen (oxo), halogen, -CN, -ORx, NRxRy, NRxC(=O)Ry, NRxSO2Ry, -NRxC(=O)NRxRy, C(=O)Rx, C(=O)ORx, C(=O)NRxRy, -SOmRx and -SOmNRxRy, wherein m is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, optionally substituted alkyl, optionally substituted wherein the substituents are optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle or optionally substituted cycloalkyl, and each of the optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle and optionally substituted cycloalkyl substituents may be further substituted by one or more of oxygen, halogen, -CN, -ORx, NRxRy, NRxC(=O)Ry, NRxSO2Ry, -NRxC(=O)NRxRy, C(=O)Rx, C(=O)ORx, C(=O)NRxRy, -SOmRx and -SOmNRxRy.

[0144] Construction of antisense oligonucleotides

[0145] Examples of morpholino oligonucleotides having phosphorus-containing backbone linkages include Figures 1A-1C Particularly preferred are phosphodiamidate-linked morpholino oligonucleotides such as Figure 1C As shown, according to one aspect of the invention, it is modified to preferably contain positively charged groups on 10-50% of its main chain linkages. Morpholino oligonucleotides including antisense oligonucleotides with uncharged main chain linkages and their preparation are described in detail, for example, (Summerton and Weller 1997) and co-owned U.S. Patents 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, which are expressly incorporated herein by reference.

[0146] 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, thymine, uracil, and hypoxanthine) so that the formed polymer can hybridize with base-complementary target nucleotides, including target RNA, with Tm values ​​above about 45°C in relatively short oligonucleotides (e.g., 10-15 bases); 3) the ability of oligonucleotides to be actively or passively transported into mammalian cells; and 4) the ability of antisense oligonucleotide:RNA heteroduplexes to resist degradation by RNAse and RNaseH, respectively.

[0147] Exemplary backbone structures of the claimed antisense oligonucleotides include Figure 1D -G shows the type of morpholino subunits, each linked by either an uncharged or positively charged phosphorus-containing subunit linker. Figure 1D The phosphorus-containing linkages forming the five atom repeat unit backbone are shown, with the morpholino rings connected by 1-atom phosphoramidite linkages. Figure 1E Shown is the connection that produces 6 atomic repeating unit main chain.In this structure, the atom Y that connects 5' morpholino carbon and phosphorus group can be sulfur, nitrogen, carbon, or preferably oxygen.The X part hanging on phosphorus can be fluorine, alkyl or substituted alkyl, alkoxy or substituted alkoxy, thioalkoxy or substituted thioalkoxy, or non-substituted, monosubstituted or disubstituted nitrogen, including cyclic structure such as morpholine or piperidine.Alkyl, alkoxy and thioalkoxy preferably include 1-6 carbon atom.Z part is sulfur or oxygen, preferably oxygen.

[0148] Figure 1F and 1G The linkage shown is designed for a 7-atom unit length backbone. In Structure 1F, the X moiety is as shown in Structure 1E, and the Y moiety can be methylene, sulfur, or preferably oxygen. In Structure 1G, the X and Y moieties are as shown in Structure 1E. Particularly preferred morpholino oligonucleotides include those consisting of Figure 1E Those consisting of the morpholino subunit structure shown, wherein X=NH2, N(CH3)2, optionally substituted 1-piperazinyl, or other charged groups, Y=O, and Z=O.

[0149] As described above, according to one aspect of the present invention, uncharged or substantially uncharged oligonucleotides are modified to include charged connections, such as up to about 1 charged connection in every 2-5 uncharged connections, such as about 4-5 charged connections in every 10 uncharged linkers. When about 25% of the main chain connections are cationic, including about 20%-30%, the best improvement in antisense activity can be observed. Also included are about 35%, 40%, 45%, 50%, 55%, 60% (including all integers in between) or more main chain connections in the oligomer are cationic. A small number, such as 5% or 10%-20% of the cationic connections, can also be observed to be enhanced.

[0150] The substantially uncharged phosphorus-containing backbone in an oligonucleotide analog is typically where a majority of the subunit linkages at physiological pH, such as where 50%-100% of the linkages, typically at least 60%-100% or 75% or 80% of the linkages are uncharged and contain a single phosphorus atom.

[0151] Additional experiments performed in support of the present invention indicate that the enhancement observed with the addition of cationic backbone charges can in some cases be distributed by having the majority of the charges close to the "central region" backbone linkers of the antisense oligonucleotide, such as in a 20mer oligonucleotide having 8 cationic backbone linkers, which has at least 70% of these charged linkers located in the 10 most central linkers.

[0152] Antisense compounds can be prepared by stepwise solid phase synthesis, using the methods detailed in the references cited above and the following about the synthesis of oligonucleotides with mixed uncharged and cationic main chain connections. In some cases, it is desirable to add other chemical parts to the antisense compounds, such as to enhance pharmacokinetics or promote the capture or detection of compounds. According to standard synthesis methods, these parts can be covalently linked to, usually to the ends of the oligomer. For example, adding polyethylene glycol parts or other hydrophilic polymers such as hydrophilic polymers with 10-100 monomer subunits can be used to enhance solubility. One or more charged groups such as charged groups, such as anionic charged groups such as organic acids, can enhance cellular uptake. For detection purposes, a reporter part such as fluorescein or a radiolabeled group can be connected. Alternatively, the reporter label connected to the oligomer can be a ligand capable of binding labeled antibodies or streptavidin, such as an antigen or antibiotic. In the part used to connect or modify the antisense compound, it is of course generally desirable to select a biocompatible compound collection that may be tolerated by an individual and has no undesirable side effects.

[0153] As indicated above, antisense compounds can be constructed to contain a selected number of cationic linkages interspersed with the uncharged linkage types described above. Intersubunit linkages, including uncharged and cationic linkages, are preferably phosphorus-containing linkages having structure (II):

[0154]

[0155] in:

[0156] W is -S- or -O-, and preferably -O-,

[0157] X=-NR 1 R 2 OR 6 ,

[0158] Y=-O- or -NR 7 ,and

[0159] Each of the linkages in the oligomer is selected from:

[0160] (a) No electrical connection (a), where R 1 , R 2 , R 6 and R 7 Each of which is independently selected from hydrogen and lower alkyl;

[0161] (b1) Cationic linker (b1), wherein X = -NR 1 R 2 and Y=-O-, and -NR 1 R 2 represents an optionally substituted piperazine moiety, such that R 1 R 2 =-CHRCHRN(R 3 )(R 4 )CHRCHR-, where:

[0162] Each R is independently H or -CH3,

[0163] R 4 is H, -CH3 or an electron pair, and

[0164] R 3 is selected from H, optionally substituted lower alkyl, -C(=NH)NH2, -ZL-NHC(=NH)NH2 and [-C(=O)CHR'NH] m H, wherein: Z is -C(=O)- or a direct bond, L is an optional linker of up to 18 atoms in length, preferably up to 12 atoms in length, more preferably up to 8 atoms in length, having a bond selected from optionally substituted alkyl, optionally substituted alkoxy and optionally substituted alkylamino, R' is a side chain of a naturally occurring amino acid or a one- or two-carbon analog thereof, and m is 1-6, preferably 1-4.

[0165] (b2) Cationic linkage (b2), wherein X = -NR 1 R 2 and Y=-O-,R1 =H or -CH3, and R 2 =LNR 3 R 4 R 5 , where L, R 3 and R 4 As defined above, and R 5 is H, optionally substituted lower alkyl or optionally substituted lower (alkoxy)alkyl; and

[0166] (b3) Cationic bonding (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 As defined above, and R 6 is H or optionally substituted lower alkyl; and

[0167] At least one of the linkages is selected from cationic linkages (b1), (b2) and (b3).

[0168] Preferably, the oligomer comprises at least two consecutive linkages of type (a) (i.e., uncharged linkages). In other embodiments, at least 5% of the linkages of the oligomer are cationic linkages (i.e., type (b1), (b2), or (b3)); for example, 10%-60%, preferably 20-50%, of the linkages may be cationic linkages.

[0169] In one embodiment, at least one linkage is of type (b1), wherein preferably each R is H, R 4 is H, -CH3 or an electron pair, and R 3 R is selected from H, optionally substituted lower alkyl, -C(=NH)NH2 and -C(=O)-L-NHC(=NH)NH2. 3 The latter two embodiments provide the guanidine moiety either directly attached to the piperazine ring or pendant from a linker group L, respectively. For ease of synthesis, R 3 The variable Z in is preferably -C(=O)- as shown.

[0170] As described above, the linker group L contains a bond selected from optionally substituted alkyl, optionally substituted alkoxy and optionally substituted alkylamino in its backbone, wherein the terminal atoms of L (such as those adjacent to the carbonyl or nitrogen) are carbon atoms. Although branched connections are possible, the linker is preferably unbranched. In one embodiment, the linker is a linear alkyl linker. Such a linker may have the structure -(CH2)n -, wherein n is 1-12, preferably 2-8, more preferably 2-6.

[0171] The morpholino subunit has the following structure (III):

[0172]

[0173] Where Pi is a base pairing moiety, the above described linkage 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.

[0174] The morpholino subunit (III) is linked using the above embodiments of the (b1), (b2) and (b3) linking types, as illustrated below:

[0175]

[0176] Preferably, all cationic linkages in an oligomer are of the same type; ie, all of type (b1), all of type (b2), or all of type (b3).

[0177] In other embodiments, the cationic linkage is selected from linkages (b1′) and (b1″) shown below, wherein (b1′) is referred to herein as a “Pip” linkage and (b1″) is referred to herein as a “GuX” linkage:

[0178]

[0179]

[0180] In the above structure, W is S or O, preferably O; R 1 and R 2 Each of (b1′) and (b1″) 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 negatively 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 carbon atoms in the piperazine ring are unsubstituted; however, the ring carbon atoms of the piperazine ring may include non-interfering substituents, such as methyl or fluorine. Preferably, no more than one or two carbon atoms are so substituted.

[0181] In other embodiments, at least 10% of the linkages are of type (b1′) or (b1″); for example, 10%-60%, preferably 20%-50%, of the linkages may be of type (b1′) or (b1″).

[0182] In other embodiments, the oligomer does not contain a linkage of the type described in (b1′) above. Alternatively, the oligomer does not contain a linkage of the type described in (b1) above, wherein each R is first H, R 3 is H or -CH3, and R 4 It is H, -CH3, or an electron pair.

[0183] As described below, the morpholino subunits can also be linked via non-phosphorus based intersubunit linkages, wherein at least one linkage is modified with a pendant cationic group as described above.

[0184] Other oligonucleotide analogue linkages can be used which are uncharged in their non-modified state but which may also carry pendant amino substituents. For example, the 5' nitrogen atom on the morpholino ring can be used in a sulfonamide linkage or a urea linkage (wherein the phosphorus is replaced by carbon or sulfur, respectively) and modified in a manner similar to the 5' nitrogen atom in structure (b3) above.

[0185] Oligomers with any number of cationic linkages are provided, including oligomers that are completely cationic linkages. However, preferably, the oligomer is partially charged, with, for example, 10%-80%. In a preferred embodiment, about 10%-60%, preferably 20%-50%, of the linkages are cationic.

[0186] In one embodiment, the cationic linkages are dispersed along the backbone. A partially charged oligomer preferably contains at least two consecutive uncharged linkages; in other words, the oligomer preferably does not have a strictly alternating pattern along its entire length.

[0187] Oligomers with cationic linker blocks and uncharged linker blocks are also contemplated; for example, uncharged linker center wings can be cationic linker blocks, or vice versa. In one embodiment, the oligomer has approximately equal lengths of 5', 3', and center regions, and the percentage of cationic linker in the center domain is greater than about 50%, preferably greater than about 70%.

[0188] The length of oligomers for antisense applications is generally about 10 to about 40 subunits, more preferably about 10 to about 30 subunits, and generally 15 to 25 bases, including those with 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, the invention has oligomers of 19 to 20 subunits, which can be used in antisense compounds, and may ideally have 2 to 10, e.g., 4 to 8 cationic linkages, with the remainder being uncharged linkages. Oligomers with 14 to 15 subunits ideally have 2 to 7, e.g., 3 to 5 cationic linkages, with the remainder being uncharged linkages.

[0189] Each morpholino ring structure supports a base pairing moiety so as to form a sequence of base pairing moieties that is typically designed to hybridize to a selected antisense target in a cell or individual to be treated. The base pairing moiety can be a purine or pyrimidine found in natural DNA or RNA (such as A, G, C, T or U) or an analog such as hypoxanthine (a component of the nucleoside inosine) or 5-methylcytosine.

[0190] Peptide transporters

[0191] The antisense compounds of the present invention may include an oligonucleotide portion coupled to an arginine-rich peptide transport moiety that is effective in enhancing transport of the compound into a cell. The transport moiety is preferably attached to the end of the oligomer, such as Figure 1B and 1C The peptide transport portion preferably comprises 6-16 subunits selected from the group consisting of X' subunits, Y' subunits and Z' subunits,

[0192] in:

[0193] (a) Each X' subunit independently represents lysine, arginine or an arginine analog, wherein the analog is a amino acid comprising the structure R 1 N=C(NH2)R 2 The side chain of a cationic α-amino acid, where R 1 is H or R; R 2 is R, -NH2, -NHR or -NR2, wherein R is optionally substituted lower alkyl or optionally substituted lower alkenyl; R 1 and R 2 can be connected together to form a ring; and the side chain is through R 1 or R 2 connected to the amino acid;

[0194] (b) Each Y' subunit independently represents a neutral amino acid -C(=O)-(CHR) n -NH-, wherein n is 2-7, and each R is independently H or methyl; and

[0195] (c) each Z' subunit independently represents an α-amino acid having a neutral aralkyl side chain;

[0196] The peptide comprises (X'Y'X') p 、(X'Y') m and / or (X'Z'Z') p wherein p is 2-5 and m is 2-8. Specific embodiments include independently selected from (X'Y'X') p 、(X'Y') m and / or (X'Z'Z')p Various combinations of, including, for example, a peptide having the sequence (X'Y'X')(X'Z'Z')(X'Y'X')(X'Z'Z')(SEQ ID NO:637).

[0197] In selected embodiments, for each X', the side chain moiety is a guanidine group, such as in the amino acid subunit arginine (Arg). In certain embodiments, each Y' is independently -C(=O)-(CH2) n- CHR-NH-, wherein 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 having different combinations of 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.

[0198] Preferred peptides of this type include those comprising arginine dimers alternating with a single Y' subunit, wherein Y' is preferably Ahx or B or both. Examples include peptides having the formula (RY'R) p And / or formula (RRY') p A peptide wherein p is 1-2-5 and wherein Y' is preferably Ahx. In one embodiment, Y' is a 6-aminohexanoic acid subunit, R is arginine and p is 4. Certain embodiments include at least two (RY'R) p and (RRY') p Various linear combinations of, including, for example, illustrative 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 another illustrative embodiment, each Z' is phenylalanine, and m is 3 or 4.

[0199] The coupled peptide is preferably linked to the end of the oligomer via a linker Ahx-B, wherein Ahx is a 6-aminohexanoic acid subunit and B is a β-alanine subunit, such as Figure 1B and 1C shown.

[0200] In selected embodiments, for each X', the side chain moiety is independently selected from: guanidino (HN=C(NH2)NH-), amidino (HN=C(NH2)CH-), 2-aminodihydropyrimidine, 2-aminotetrahydropyrimidine, 2-aminopyrimidine and 2-aminopyrimidine, which is preferably selected from guanidino and amidino. In one embodiment, the side chain moiety is guanidino as in the amino acid subunit arginine (Arg).

[0201] In some embodiments, the Y' subunits can be continuous, in that there are no intervening X' subunits between the Y' subunits, or interspersed individually between the X' subunits. In some embodiments, the connecting subunits can be between the Y' subunits. In one embodiment, the Y' subunits are at the ends of the transporter; in other embodiments, they are flanked by X' subunits. In another preferred embodiment, each Y' is -C(=O)-(CH2) n- CHR-NH-, wherein 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 these groups, each X' comprises a guanidinyl side chain portion such as an arginine subunit. Preferred peptides of this type include those comprising arginine dimers alternating with a single Y' subunit, wherein Y' is preferably Ahx. Examples include peptides having the formula (RY'R)4 or the formula (RRY')4, wherein Y' is preferably Ahx. In the latter case, the nucleic acid analog is preferably attached to the terminal Y' subunit, preferably at the C-terminus, such as Figure 1B and 1C A preferred linker has the structure AhxB, wherein Ahx is a 6-aminohexanoic acid subunit and B is a β-alanine subunit.

[0202] The above-described transport moieties are shown to greatly enhance the entry of the attached oligomer into cells relative to the uptake of the oligomer in the absence of the attached transport moiety, and relative to the uptake of the attached transport moiety lacking the hydrophobic subunit Y'. This enhanced uptake is demonstrated to be preferably at least a 2-fold increase, preferably a 4-fold increase, in the uptake of the compound by mammalian cells relative to the uptake of the agent by the attached transport moiety lacking the subunit Y'. The uptake is preferably enhanced by at least 20-fold, more preferably 40-fold relative to the unconjugated compound.

[0203] Another advantage of the transport moiety is its expected ability to stabilize the duplex between the antisense compound and its target nucleic acid sequence, presumably by means of electrostatic interactions between the positively charged transport moiety and the negatively charged nucleic acid. As mentioned above, the number of charged subunits in the transport protein is less than 14, preferably between 8 and 11, because too high a number of charged subunits can lead to reduced sequence specificity.

[0204] The use of arginine-rich peptide transporters (i.e., cell penetrating peptides) is particularly useful in practicing the present invention. Certain peptide transporters have been shown to be very effective in delivering antisense compounds to primary cells, including muscle cells (Marshall, Oda et al. 2007; Jearawiriyapaisarn, Moulton et al. 2008; Wu, Moultonet al. 2008). Moreover, compared to other peptide transporters such as Penetratin and Tat peptides, the peptide transporters described herein, when coupled to antisense PMOs, exhibit an enhanced ability to alter the splicing of several gene transcripts (Marshall, Oda et al. 2007). Particularly preferred are the P007, CP06062 and CP04057 transport peptides listed in Table 3 below (SEQ ID NOS: 573, 578 and 577, respectively).

[0205] Exemplary peptide transporters, including the linker (B or AhxB), are given 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).

[0206] Table B. Exemplary peptide transporters for intracellular delivery of PMOs

[0207]

[0208]

[0209] preparation

[0210] In certain embodiments, the present invention provides preparations and compositions suitable for therapeutic delivery of antisense oligomers described herein. Therefore, in certain embodiments, the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more oligomers described herein, the oligomers being formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. Although the oligomers of the present invention can be administered alone, it is preferred to administer the compound in a pharmaceutical formulation (composition).

[0211] Methods for delivering nucleic acid molecules are described, for example, in Akhtar et al., 1992, Trends Cell Bio., 2:139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar; Sullivan et al., PCT WO 94 / 02595. These or other schemes can be used to deliver virtually any nucleic acid molecule, including the isolated oligomers of the invention.

[0212] As described in detail below, the pharmaceutical compositions of the present invention can be particularly formulated for administration in solid or liquid form, including those suitable for the following: (1) oral administration, such as a drench (aqueous or non-aqueous solution or suspension), tablets, such as those targeted for buccal, sublingual and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration by subcutaneous, intramuscular, intravenous or intrathecal injection, such as sterile solutions or suspensions, or sustained-release formulations; (3) topical application, such as as a cream, ointment, or controlled-release patch or spray applied to the skin; (4) vaginal or rectal, such as a vaginal suppository, cream or foam; (5) sublingual; (6) ocular; (7) transdermal; or (8) nasal.

[0213] The term "pharmaceutically acceptable" is used herein to refer to those compounds, substances, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, inflammation, allergic response or other problems or complications, commensurate with a reasonable advantage / risk ratio.

[0214] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing auxiliary (such as a lubricant, mica, magnesium, calcium or zinc stearate, or stearic acid), or a solvent encapsulating substance, which participates in carrying or transporting the subject compound from one organ, or part of the body to another organ, or other part 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.

[0215] 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) mica; (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 ) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (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 substances used in pharmaceutical formulations.

[0216] Other non-limiting examples of agents 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 (e.g., Pluronic P85) that can enhance drug entry into various tissues; biodegradable polymers, such as poly (DL-lactic acid-cobalt glycolide) microspheres for sustained release delivery after implantation (Emerich, DF et al., 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as nanoparticles made of polybutylcyanoacrylate, which can deliver drugs across the vascular-brain barrier and alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).

[0217] The present invention is also characterized by the use of surface-modified liposomes (PEG-modified, branched and unbranched or a combination thereof, or long-circulating liposomes or stealth liposomes) containing poly(ethylene glycol) lipids. The oligomers of the present invention may also contain covalently linked PEG molecules of various molecular weights. These formulations provide methods for enhancing drug accumulation in target tissues. Such drug carriers resist conditioning and clearance by the mononuclear phagocytic system (MPS or RES), thereby achieving longer blood circulation time 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). It has been shown that these liposomes selectively accumulate in tumors, presumably by means of 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). In particular, long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA compared to conventional cationic liposomes known to accumulate in MPS tissues (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96 / 10391; Ansell et al., International PCT Publication No. WO 96 / 10390; Holland et al., International PCT Publication No. WO 96 / 10392). Based on their ability to avoid accumulation in metabolically active MPS tissues such as the liver and spleen, long-circulating liposomes may also protect drugs from nuclease degradation to a greater extent than cationic liposomes.

[0218] In another embodiment, the present invention includes oligomer compositions prepared for delivery, as described in U.S. Pat. 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 containing lysine and histidine (HK) copolymers as described in U.S. Pat. Nos. 7,163,695, 7,070,807, and 6,692,911, which are alone or in combination with PEG (such as branched or unbranched PEG or a mixture of the two), in combination with PEG and a targeting moiety, or any of the foregoing in combination with a cross-linking agent. In certain embodiments, the present invention provides antisense oligomers in compositions comprising gluconic acid-modified polyhistidine or glucuronic acid-polyhistidine / transferrin-polyhistidine. It will also be appreciated by those skilled in the art that amino acids having properties similar to His and Lys within the composition can be substituted.

[0219] Certain embodiments of the oligomers described herein may contain basic functional groups, such as amino or alkylamino groups, and thus may be capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. In this regard, the term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic acid addition salts of the compounds of the invention. These salts may be prepared in situ during the preparation of the administration vehicle or dosage form, or by reacting the purified compounds of the invention in their free alkaline form with suitable organic or inorganic acids individually and isolating the acid formed during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, disulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, napthylate, methanesulfonate, gluconate, lactobionate, and dodecylsulfonate, among others (see, e.g., Berge et al. (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66: 1-19).

[0220] The pharmaceutically acceptable salts of the subject oligomers include conventional non-toxic salts or quaternary ammonium salts of the compounds, such as from non-toxic organic or inorganic acids. For example, these conventional non-toxic salts include salts derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfanilic acid, phosphoric acid, nitric acid, etc.; and salts prepared from organic acids, such as acetic acid, 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, alicyclic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isosulfuric acid, etc.

[0221] In certain embodiments, the oligomer of the present invention may contain one or more acidic functional groups, so that it can form a pharmaceutically acceptable salt with a pharmaceutically acceptable base. In these cases, the term "acceptable salt" refers to relatively nontoxic inorganic and organic base addition salts of the compounds of the present invention. These salts can also be prepared in situ during the preparation of a dosing medium or a dosage form, or by reacting the purified compound in its free acid form with a suitable base such as a pharmaceutically acceptable metal cation hydroxide, carbonate or bicarbonate, with ammonia, or with a pharmaceutically acceptable organic primary amine, secondary amine or tertiary amine individually to prepare. Representative alkaline or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts, etc. Representative organic amines that can be used to form base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc. (see, e.g., Berge et al., supra).

[0222] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coatings, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0223] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0224] The preparations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The preparations can be conveniently present in unit dosage forms and prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary according to the host to be treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the compound that produces the therapeutic effect. Usually, in percentage, the range of this amount is generally about 0.1%-99% of the active ingredient, preferably about 5%-70%, and most preferably about 10%-30%.

[0225] In certain embodiments, the formulation of the present invention comprises an excipient selected from cyclodextrin, cellulose, liposome, a micelle forming agent such as bile acid, and a polymeric carrier such as polyester and polyanhydride; and an oligomer of the present invention. In certain embodiments, the aforementioned formulation makes the oligomer of the present invention orally bioavailable.

[0226] The method for preparing these preparations or compositions comprises the step of combining the oligomer of the present invention with a carrier and optionally one or more auxiliary ingredients. In general, the preparation is prepared by uniformly and intimately combining the compound of the present invention with a liquid carrier or a fine solid carrier or both, and then shaping the product if necessary.

[0227] The formulations of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using flavor bases, usually sucrose and gum arabic or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as lozenges (using inert bases, such as gelatin and glycerin, or sucrose and gum arabic) and / or as mouthwashes, etc., each containing a predetermined amount of the compound of the present invention as an active ingredient. The oligomers of the present invention may also be administered as boluses, licking agents or patches.

[0228] In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules, trouches, etc.), 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, polyvinyl pyrrolidone, sucrose and / or acacia; (3) wetting agents such as glycerol; (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, Certain silicates and sodium carbonate; (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, glyceryl monostearate and nonionic surfactants; (8) absorbents such as kaolin and bentonite; (9) lubricants such as mica, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid and mixtures thereof; (10) colorants; and (11) controlled release agents such as crospovidone or ethylcellulose. In the case of capsules / tablets and pills, the pharmaceutical composition may also contain a buffer. Similar types of solid compositions may also be used as fillers for soft and hard shell capsules using excipients such as lactose or milk sugars and high molecular weight polyethylene glycols.

[0229] Tablets can be prepared by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (such as gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surface active or dispersing agent. Molded tablets can be prepared by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0230] Tablets and other solid dosage forms of the pharmaceutical composition of the present invention, such as sugar-coated tablets, capsules and granules can be optionally impressed or prepared with coatings and shells such as enteric coatings and other coatings known in the field of pharmaceutical preparations. They can also be formulated using, for example, hydroxypropylmethylcellulose, other polymeric matrices, liposomes and / or microspheres in different proportions that provide the desired release characteristics, so as to provide a sustained release or controlled release of the active ingredient therein. They can be formulated to be released quickly, such as freeze-dried. They can be sterilized by filtering through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain an opacifying agent, which can be a composition that optionally releases the active ingredient only in a delayed manner, or preferably in certain parts of the gastrointestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can be in microcapsule form, if appropriate, in microcapsule form with one or more of the above-mentioned excipients.

[0231] Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as water, or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0232] 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.

[0233] Suspensions, in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0234] Formulations for rectal or vaginal administration may be presented as suppositories which may be prepared by mixing one or more of the compounds of the invention with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylic acid, which are solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active compound.

[0235] Preparations or dosage forms for topical or transdermal administration of the oligomers provided herein include powders, sprays, ointments, pastes, emulsions, lotions, gels, solutions, patches and inhalants. The active oligomers can be mixed with pharmaceutically acceptable carriers under aseptic conditions, and with any necessary preservatives, buffers, propellants. In addition to the active compounds of the present invention, ointments, pastes, emulsions and gels can contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth gum, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, mica and zinc oxide, or mixtures thereof.

[0236] In addition to the active oligomers of the invention, powders and sprays may contain excipients such as lactose, mica, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants, such as chlorofluorocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.

[0237] Transdermal patches have the additional advantage of controlling the delivery of the oligomers of the present invention to the body. Such dosage forms can be prepared by dissolving or dispersing the oligomers in a suitable medium. Absorption enhancers can also be used to increase the flow of the reagent through the skin. In addition to other methods known in the art, the speed of this flow can also be controlled by providing a speed control membrane or dispersing the reagent in a polymer matrix or gel.

[0238] Pharmaceutical compositions suitable for parenteral administration may comprise one or more oligomers of the invention and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders reconstituted into sterile injectable solutions or dispersions immediately before use, the injectable solutions or dispersions may contain sugars, alcohols, antioxidants, buffers, antibacterial agents, solutes or suspending or thickening agents that make the preparation isotonic with the blood of the intended recipient. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the 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. Appropriate fluidity can be maintained by using coating materials, such as lecithin, by maintaining the necessary particle size in the case of dispersions, and by using surfactants.

[0239] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Preventing the effects of microorganisms on the subject oligomers can be ensured by including a variety of antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc. It is also desirable to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, extended absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0240] In some cases, in order to prolong the drug effect, it is desirable to slow down the absorption of the drug of subcutaneous or intramuscular injection. Except for additives known in the art, this can be achieved by using a liquid suspension of a crystal or amorphous substance with weak water solubility. The absorption rate of the drug depends on its dissolution rate subsequently, and the dissolution rate depends on crystal size and crystalline form in turn. Alternatively, the delayed absorption of the drug form given parenterally can be achieved by dissolving the drug or being suspended in an oil medium.

[0241] Injectable depot forms can be prepared by forming a microcapsule matrix of the subject oligomer in a biodegradable polymer such as polylactic acid-polyethylene glycol. Depending on the ratio of oligomer to polymer, and the characteristics of the specific polymer used, the oligomer release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0242] When the oligomers of the present invention are administered to humans and animals as drugs, they can be administered per se or as a pharmaceutical composition containing, for example, 0.1-99% (preferably 10-30%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0243] As mentioned above, the preparations or articles of the present invention can be administered orally, parenterally, topically or rectally. They are administered in a form suitable for each route of administration. For example, they are administered in tablet or capsule form by injection, inhalation, eye lotion, ointment, suppository, etc., by injection, infusion or inhalation; topical administration by lotion or ointment; and rectal administration by suppository.

[0244] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0245] As used herein, the phrases "systemic administration," "systemically administering," "peripheral administration," and "peripherally administering" mean the administration of a compound, drug, or other substance, other than direct administration to the central nervous system, e.g., subcutaneously, so that it enters the patient's entire body and thereby undergoes metabolism and other similar processes.

[0246] Regardless of the selected route of administration, the oligomer of the present invention used in a suitable hydrated form, and / or the pharmaceutical composition of the present invention can be formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied so as to obtain an amount of the active ingredient that is effective in achieving a desired therapeutic response for a specific patient, composition and mode of administration and is not unacceptably toxic to the patient.

[0247] The selected dosage level will depend upon a variety of factors, including the activity of the particular oligomer of the present invention employed, or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion or metabolism of the particular oligomer employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and / or substances used in combination with the particular oligomer employed, the age, sex, weight, disease condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0248] A doctor or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a doctor or veterinarian can start the dosage of the compound of the present invention used in the pharmaceutical composition at a level lower than the required level, so as to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Usually the daily dosage suitable for the compound of the present invention is the minimum dosage at which the compound effectively produces a therapeutic effect. This effective dose usually depends on the above factors. Usually, when used for a specified effect, for a patient, the oral, intravenous, intracerebroventricular and subcutaneous dosage of the compound of the present invention is about 0.0001 mg to about 100 mg per kilogram of body weight per day.

[0249] If desired, the effective daily dose of active compound may be administered throughout the day, optionally in unit dosage form, as two, three, four, five, six or more sub-doses administered separately at appropriate intervals. In some cases, dosing is performed once a day. In certain embodiments, one or more doses are administered every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months as needed to maintain the desired expression of functional dystrophin.

[0250] Nucleic acid molecules can be administered to cells by a variety of methods known to those familiar with the art, including but not limited to iontophoresis as known in the art and described herein, or by incorporation into other media, such as hydrosols, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres encapsulated in liposomes. In certain embodiments, microemulsion technology can be used to improve the bioavailability of lipophilic (water-insoluble) agents. Examples include Trimetrine (Dordunoo, SK, et al., Drug Development and Industrial Pharmacy, 17 (12), 1685-1713, 1991 and REV5901 (Sheen, PC, et al., J Pharm Sci 80 (7), 712-714, 1991). Among other advantages, microemulsion provides enhanced bioavailability by preferentially directing absorption to the lymphatic system rather than the circulatory system, which thereby bypasses the liver and prevents the compound from being destroyed in the hepatobiliary circulation.

[0251] In one aspect of the invention, the preparation contains the micelle formed by the oligomer provided herein and at least one amphoteric carrier, wherein the average diameter of the micelle is less than about 100 nm. A more preferred embodiment provides micelles with an average diameter less than about 50 nm, and an even more preferred embodiment provides micelles with an average diameter less than about 30 nm, or even less than about 20 nm.

[0252] Although all suitable amphiphilic carriers are contemplated, currently preferred carriers are generally those having Generally Recognized As Safe (GRAS) status and capable of dissolving the compounds of the invention and microemulsifying them at a later stage when the solution comes into contact with a complex water phase such as that found in the human gastrointestinal tract. Typically, amphiphilic ingredients that meet these requirements have an HLB (hydrophilic to lipophilic balance) of 2-20 and their structures contain straight chain aliphatic groups from C-6 to C-20. Examples are polyethylene-glycolized fatty acid glycerides and polyethylene glycol.

[0253] Examples of amphoteric carriers include saturated and monounsaturated polyethylene glycol fatty acid glycerides, such as those obtained from fully or partially hydrogenated various vegetable oils. Such oils are preferably composed of tri-, di- and mono-fatty acid glycerides and di- and mono-polyethylene glycol esters of the corresponding fatty acids, and particularly preferred fatty acid compositions include capric acid 4-10, capric acid 3-9, lauric acid 40-50, myristic acid 14-24, palmitic acid 4-14 and stearic acid 5-15%. Alternative-available amphoteric carriers include partially esterified sorbitan and / or sorbitol, with saturated or monounsaturated fatty acids (SPAN series) or corresponding ethoxylated analogs (TWEEN series (TWEEN-series)).

[0254] Particularly useful are commercially available amphoteric carriers, 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-laurate and di-laurate, lecithin, polysorbate 80, etc. (manufactured and sold by many companies in the USA and around the world).

[0255] In certain embodiments, delivery can be performed by using liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., for introducing the compositions of the present invention into suitable host cells. In particular, the compositions of the present invention can be formulated for delivery by encapsulation in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc. The formulation and use of such delivery vehicles can be carried out using known and conventional techniques.

[0256] Suitable hydrophilic polymers for use in the present invention are hydrophilic polymers that are readily soluble in water, can be covalently attached to the lipids that form the vesicles, and can be tolerated and non-toxic (i.e., biocompatible) in vivo. Suitable polymers include polyethylene glycol (PEG), polylactic acid (also known as polylactide), polyglycolic acid (also known as polyglycolide), polylactic-polyglycolic acid copolymers, and polyvinyl alcohol. In certain embodiments, the molecular weight of the polymer is about 100 or 120 daltons, up to about 5000 or 10000 daltons, or about 300 daltons to about 5000 daltons. In other embodiments, the polymer is a polyethylene glycol having a molecular weight of about 100 to about 5000 daltons, or about 300 to about 5000 daltons. In certain embodiments, the polymer is a polyethylene glycol having a molecular weight of 750 daltons (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 3 monomers, such PEG polymers being composed of three monomers (about 150 daltons).

[0257] Other hydrophilic polymers suitable for use in the present invention include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0258] 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 methacrylates, polyethylene polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactic acid-cobalt-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and blends, mixtures or copolymers thereof.

[0259] Cyclodextrin is a cyclic oligosaccharide composed of 6, 7 or 8 glucose units, respectively referred to as the Greek letters α, β or γ. The glucose units are connected by 1,4 glycosidic bonds. As a result of the chair-type conformation of the sugar unit, all secondary hydroxyls (at C-2, C-3) are located on one side of the ring, and all primary hydroxyls at C-6 are located on the other side. As a result, the external face is hydrophilic, making cyclodextrin water-soluble. On the contrary, the cavity of cyclodextrin is hydrophobic because they are connected by hydrogen of C-3 and C-5 atoms and by ester-like oxygen. These matrices achieve complexes with a variety of relatively hydrophobic compounds, including, for example, steroid compounds, such as 17α-estradiol (see, e.g., van Uden et al. Plant Cell Tiss. Org. Cult. 38: 1-3-113 (1994)). Complexation is achieved by van der Waals interactions and the formation of hydrogen bonds. For an overview of cyclodextrin chemistry, see Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822 (1994).

[0260] The physicochemical properties of cyclodextrin derivatives strongly depend on the type and degree of substitution. For example, their solubility in water ranges from insoluble (such as 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 make it possible to control the solubility of various formulation ingredients by increasing or decreasing their solubility.

[0261] Many cyclodextrins and methods for their preparation have been described. For example, Parmeter (I), et al. (US Pat. No. 3,453,259) and Gramera, et al. (US Pat. No. 3,459,731) describe electrically neutral cyclodextrins. Other derivatives include cyclodextrins with cationic properties [Parmeter (II), US Pat. No. 3,453,257], insoluble cross-linked cyclodextrins (Solms, US Pat. No. 3,420,788) and cyclodextrins with anionic properties [Parmeter (III), US Pat. No. 3,426,011]. Among the cyclodextrin derivatives with anionic properties, carboxylic acids, phosphorous acids, trivalent phosphonic acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfonic acids and sulfonic acids can be attached to the parent cyclodextrin [see Parmeter (III), supra]. Furthermore, Stella, et al. described sulfanyl ether cyclodextrin derivatives (U.S. Pat. No. 5,134,127).

[0262] Liposomes consist of at least one lipid bilayer membrane that seals an aqueous interior space. Liposomes are characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane, and their diameter is usually between 0.02-0.05 μm; large unilamellar vesicles (LUVs) are usually larger than 0.05 μm. Several-layer large vesicles and multi-layer large vesicles have multiple layers of usually concentric membrane layers and are usually larger than 0.1 μm. Liposomes with several non-concentric membranes, that is, several smaller vesicles contained in a larger vesicle, are called multivesicular vesicles.

[0263] One aspect of the invention relates to formulations comprising liposomes containing oligomers of the invention, wherein the liposome membrane is formulated to provide liposomes with enhanced carrying properties. Alternatively or additionally, the compounds of the invention may be contained within or adsorbed on the liposome bilayer of the liposome. The oligomers of the invention may be aggregated with lipid surfactants and carried within the liposome interior space; in these cases, the liposome membrane is formulated to resist the damaging effects of active agent-surfactant aggregates.

[0264] According to one embodiment of the present invention, the lipid bilayer of the liposome contains lipids derivatized with polyethylene glycol (PEG) such that PEG chains extend from the inner surface of the lipid bilayer to the interior space enclosed by the liposome and from the outside of the lipid bilayer to the surrounding environment.

[0265] The active agent contained within the liposomes of the present invention is in dissolved form. Aggregates of surfactants and active agents (e.g., emulsions or micelles containing the active agent of interest) can be entrapped in the interior space of the liposomes of the present invention. The role of the surfactant is to disperse and dissolve the active agent, and can be selected from any suitable aliphatic, alicyclic or aromatic surfactants, including but not limited to biocompatible lysophosphatidylcholines (LPCs) of different chain lengths (e.g., from about C14 to about C20). Polymer-derived lipids, such as PEG-lipids, can also be used for micelle formation because they act to inhibit micelle / membrane fusion because the addition of polymers to the surfactant molecule reduces the CMC of the surfactant and assists in the formation of micelles. Preferred are surfactants with CMCs in the micromolar range; higher CMC surfactants can be used to prepare micelles entrapped within the liposomes of the present invention.

[0266] The liposomes of the present invention can be prepared by any of a variety of techniques known in the art. See, e.g., U.S. Pat. No. 4,235,871, published PCT application WO 96 / 14057, New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104; Lasic DD, Liposomes from physics to applications, Elsevier Science Publishers BV, Amsterdam, 1993. For example, the liposomes of the present invention can be prepared by diffusing the lipids derived from the hydrophilic polymer into preformed liposomes, for example, at a lipid concentration corresponding to the final molar percentage of the derived lipids required for the liposomes, by exposing the preformed liposomes to micelles composed of lipid-grafted polymers. Liposomes containing hydrophilic polymers can also be formed by homogenization, lipid-field hydration or extrusion techniques known in the art.

[0267] In another exemplary formulation procedure, first by dispersing the active agent by ultrasonic treatment in the lysophosphatidylcholine or other low CMC surfactants (including polymer grafted lipids) that are easy to dissolve hydrophobic molecules. Then the micellar suspension of the active agent obtained is used for rehydrating the dried lipid sample, and the dried lipid sample contains the suitable molar percentage of polymer grafted lipid or cholesterol. Then using extrusion technology known in the art, lipid and active agent suspension are formed in liposomes, and the resulting liposomes are separated from the unencapsulated solution by standard column separation.

[0268] In one aspect of the invention, liposomes are prepared to have a substantially uniform size within a selected size range. One effective method for controlling size involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes having a selected uniform pore size; the pore size of the membranes will roughly correspond to the maximum size of the liposomes produced by extrusion through the membranes. See, e.g., U.S. Pat. No. 4,737,323 (April 12, 1988). In certain embodiments, such as and The reagents can be used to introduce polynucleotides or proteins into cells.

[0269] The release characteristics of the formulation of the present invention depend on the concentration of the encapsulated material, the encapsulated drug, and the presence of a release modifier. For example, the release can be manipulated to be pH-dependent, for example, using a pH-selective coating that releases only at low pH, such as in the stomach, or at higher pH, such as in the intestine. Enteric coatings can be used to prevent the release from occurring until after passing through the stomach. Multiple coatings or cyanamide mixtures encapsulated in different materials can be used to obtain initial release in the stomach, followed by later release in the intestine. Release can also be manipulated by including salts or pore formers, which can enhance the water uptake or release of the drug by diffusion from the capsule. Excipients that modify the solubility of the drug can also be used to control the release rate. Agents that enhance matrix degradation or release from the matrix can also be incorporated. Depending on the compound, they can also be added to the drug, added as a separate phase (i.e., microparticles), or co-dissolved in the polymer phase. In most cases, the amount should be between 0.1-30% (w / w polymer). Types of degradation enhancers 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 and Pore ​​formers (ie water soluble compounds such as inorganic salts and sugars) are added as particles which add microstructure to the matrix. The range is typically 1% - 30% (w / w polymer).

[0270] Absorption can also be manipulated by changing the residence time of the particles in the digestive tract. This can be achieved, for example, by coating the particles with or selecting as an encapsulating material, a mucoadhesive polymer. Examples include most polymers with free carboxyl groups, such as chitosan, cellulose, and particularly polyacrylates (as used herein, polyacrylates refer to polymers comprising acrylate groups and modified acrylate groups, such as cyanoacrylates and methacrylates).

[0271] By, oligomer can be configured to be contained in surgical operation or medical device or implant, or be suitable for being released by surgical operation or medical device or implant.In some aspects, implant can be coated or treated with oligomer.For example, hydrogel or other polymers, such as biocompatible and / or biodegradable polymers, can be used to coat implants with the composition of the present invention (i.e., by using hydrogel or other polymers, the composition can be suitable for use with medical device).Polymers and copolymers for coating medical devices with reagents are well known in the art.The example of implant includes but is not limited to stent, drug eluting stent, suture, prosthesis, vascular catheter, dialysis catheter, vascular graft, artificial heart valve, cardiac pacemaker, implantable cardioverter defibrillator, IV needle, for skeletal framework and the device formed such as pin, screw, plate and other devices, and artificial tissue matrix for wound healing.

[0272] In addition to the methods provided herein, by analogy with other drugs, the oligomers used in the present invention can be formulated to be administered in any conventional manner for use in human and animal medicine. In the treatment of muscular dystrophy, antisense oligomers and their corresponding preparations can be administered alone or in combination with other therapeutic strategies, such as myoblast transplantation, stem cell therapy, aminoglycoside antibiotic administration, proteosome inhibitors, and up-regulation therapy (such as up-regulation of dystrophin-related proteins, i.e., autosomal paralogues of dystrophin).

[0273] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0274] Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clear understanding, it will be apparent to those of ordinary skill in the art based on the teachings of the present invention that certain changes and modifications may be made to the present invention without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not limitation. Those skilled in the art will readily recognize that many non-critical parameters may be varied or modified to produce substantially similar results.

[0275] References

[0276] Aartsma-Rus, A., AA Janson, et al. (2004). "Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense." Am J Hum Genet 74(1): 83-92.

[0277] Dunckley, MG, ICEperon, et al. (1997). "Modulation of splicing in the DMD gene by antisense oligoribonucleotides." Nucleosides & Nucleotides 16(7-9):1665-1668.

[0278] Dunckley, MG, M. Manoharan, et al. (1998). "Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides." HumMolGenet 7(7):1083-90.

[0279] Errington, SJ, CJMann, et al. (2003). "Target selection for antisense oligonucleotide induced exon skipping in the dystrophin gene." JGene Med 5(6):518-27.

[0280] Jearawiriyapaisarn, N., HM Moulton, et al. (2008). "Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice." Mol Ther .

[0281] Lu, QL, CJ Mann, et al. (2003). "Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse." Nat Med 9(8):1009-14.

[0282] Mann, CJ, K. Honeyman, et al. (2002). "Improved antisense oligonucleotideinduced exon skipping in the mdx mouse model of muscular dystrophy." J Gene Med 4(6):644-54.

[0283] Marshall, NB, SKOda, et al. (2007). "Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing." Journal of Immunological Methods 325(1-2):114-126.

[0284] Matsuo, M., T. Masumura, et al. (1991). "Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe." J Clin Invest 87(6):2127-31.

[0285] Monaco, AP, CJ Bertelson, et al. (1988). "An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus." Genomics 2(1):90-5.

[0286] Pramono, ZA, Y.Takeshima, et al. (1996). "Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence." Biochem Biophys Res Commun 226(2):445-9.

[0287] Sazani, P., R. Kole, et al. (2007). Splice switching oligomers for the TNF superfamily receptors and their use in treatment of disease. PCT WO2007058894,University of North Carolina

[0288] Sierakowska, H., M. S. Ambassadore, et al. (1996). "Repair of thalassemic human beta-globin mRNA in mammalian cells by antisense oligonucleotides." Proc Natl Acad Sci U SA 93(23):12840-4.

[0289] Summerton, J. and D. Weller (1997). "Morpholino antisense oligomers: design, preparation, and properties." Antisense Nucleic Acid Drug Dev 7(3):187-95.

[0290] Takeshima, Y., H. Nishio, et al. (1995). "Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe." J Clin Invest 95(2):515-20.

[0291] van Deutekom, JC, M. Bremmer-Bout, et al. (2001). "Antisense-induced exonskipping restores dystrophin expression in DMD patient derived muscle cells." Hum Mol Genet 10(15):1547-54.

[0292] van Deutekom, JC, AA Janson, et al. (2007). "Local dystrophin restoration with antisense oligonucleotide PRO051." NEnglJ Med 357(26):2677-86.

[0293] Wilton, SD, A. M. Fall, et al. (2007). "Antisense oligonucleotide-induced exon skipping across the human dystrophin gene transcript." Mol Ther 15(7):1288-96.

[0294] Wilton, SD, F. Lloyd, et al. (1999). "Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides." Neuromuscul Disord 9(5):330-8.

[0295] Wu, B., HM Moulton, et al. (2008). "Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer." ProcNatlAcadSciU SA 105(39):14814-9.

[0296] Yin, H., HM Moulton, et al. (2008). "Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function." Hum Mol Genet 17(24):3909-18. Example

[0297] Materials and Methods

[0298] Cell and tissue culture conditions

[0299] Human rhabdomyosarcoma cells (ATCC, CCL-136; RD cells) stored at low passage number in 5% DMSO solution (Sigma) were thawed in a 37°C water bath until the ice sliver was no longer visible. The cells were plated at 1.5 x 10 6 Cells / flask were seeded into tissue culture treated T75 flasks (Nunc) containing 24 mL of heated DMEM with L-glutamine (HyClone), 10% fetal bovine serum and 1% penicillin-streptomycin antibiotic solution (CelGro); after 24 hours, the medium was aspirated and the cells were washed once with heated PBS and fresh medium was added. The cells were grown to 80% confluence in a 37°C incubator at 5.0% CO2.

[0300] Aspirate the medium from the T75 flask; wash the cells once with heated PBS and aspirate. Add 3 mL of trypsin / EDTA heated in a 37°C water bath to each T75. Incubate the cells at 37°C for 52-5 minutes with gentle shaking until they release from the flask. Transfer the cell suspension to a 15.0 mL conical tube; wash the flask with 1.0 mL of trypsin / EDTA solution to collect the remaining cells. Count the cells using a Vi-Cell XR cell counter (Beckman Coulter). Count 2.0 x 10 cells per well. 5 For live cells, cells were seeded in a 12-well tissue culture treated plate (Falcon) with 1.0 mL of culture medium per well and incubated overnight in a 37°C incubator under 5.0% CO2.

[0301] Check the flat cell distribution and plate adhesion of the twelve-well inoculated plates. Lyophilized peptide-coupled phosphodiamidate morpholino oligomers (PPMOs) were resuspended in nuclease-free water (Ambion) at 2.0 mM and kept on ice during cell treatment; to verify the molar concentration, the PPMOs were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Before PPMO treatment, the culture medium was directly aspirated and the cells were washed with heated PBS. PPMOs were diluted to the desired molar concentration with heated culture medium; cells were treated with a total of 1.0 mL PPMO per well. PPMOs were detected in triplicate. For untreated controls, a total volume of 1.0 mL of fresh heated culture medium was added. The cells were incubated for 48 hours in a 37°C incubator at 5.0% CO2.

[0302] RNA extraction

[0303] Aspirate the culture medium and wash the cells with heated PBS. RNA was extracted using the QuickGene-Mini80 system, QuickGene RNA cultured cell HC kit S, and MagNAlyser with ceramic bead homogenization according to the supplier's recommended protocol. In short, cells were lysed in a treatment plate with lysis buffer of 350uL LRP (10uL β-mercaptoethanol was added to every 100uL LRP); the homogenate was gently ground to ensure complete lysis and transferred to a MagNAlyser tube. The tube was rotated at 2800rpm for 30 seconds in the MagNAlyser to ensure complete homogenization and briefly frozen. 50uL SRP solubilization buffer was added and the homogenate was vortexed for 60 seconds. 170uL>99% ethanol was added to each tube and the homogenate was vortexed for 60 seconds. The homogenate was quickly spun and transferred to a Mini80RNA box, the sample was pressurized and the flow-through was discarded. The box was washed and pressurized with 750uL WRP wash buffer. Add 40uL DNase (deoxyribonuclease) solution (1.25uL Qiagen DNasel, 35uL RDD buffer, 3.75uL nuclease-free water) directly to the box membrane; incubate the box at room temperature for 4 minutes. Wash the box twice with 750uL WRP, pressurizing after each wash. Place the box on a nuclease-free tube. Add 50uL CRP's elution buffer to each membrane; incubate the membrane at room temperature for 5 minutes. Pressurize the box and collect the eluate. Store the RNA to be quantified at -80°C. Use NanoDrop TM RNA was quantified using a 2000 spectrophotometer.

[0304] Nested RT-PCR

[0305] Primer-specific, exon-specific optimal nested RT-PCR amplification was performed using the primer pair set for each dystrophin exon shown in Table 1 below.

[0306] Table 1. Primer pairs used for PCR amplification of human dystrophin mRNA to detect exon skipping

[0307]

[0308] The primer pairs shown are represented as forward or reverse (F / R) and external or internal primer pairs (I / O) corresponding to the primary or secondary amplification, respectively. The positions of the primer targets are represented in the Exon column and the purpose represents the exon skipping events that can be detected. For example, the PS170 and PS176 primers amplify the exon 48-53 region in the primary amplification. Then the primers PS172 and PS174 amplify the exon 49-52 region in the secondary amplification. This nested PCR reaction will detect exon skipping of both exon 50 and / or exon 51. Specific nested RT-PCR reaction conditions are provided below.

[0309] For all samples, RNA extracted from treated cells (as described above) was diluted to 20 ng / ul.

[0310] Table 2: RT-PCR and primary amplification reaction setup (50 ul reaction)

[0311] 2x reaction mixture 25μl PS XXX forward primer (30 μM) (see Table 1) 0.5μl PS XXX reverse primer (30 μM) (see Table 1) 0.5μl Superscript III Platinum Taq Mix 2μl Template RNA (20 ng / μl) 10μl Nuclease-free water (50 μl total volume) 12μl

[0312] Table 3: RT-PCR and primary amplification procedures

[0313]

[0314] Table 4: Nested secondary amplification reaction settings (50 ul reaction)

[0315]

[0316]

[0317] Table 5: Nested secondary amplification procedure

[0318]

[0319] Gel electrophoresis analysis

[0320] 10 microliters of 5x Ficoll loading dye was added to each 50 microliter nested RT-PCR reaction. 15 microliters of PCR / dye mixture was run in a 10% TBE gel at 300 volts for 30 minutes. After electrophoresis, the gel was washed with diH2O for at least one hour, changing the water every 30 minutes. The gel was then scanned in a Typhoon TrioVariable Mode Imager (GE Healthcare). For exon 44 skipping, the nested RT-PCR product from the full-length dystrophin transcript was 571bp, and 423bp came from the mRNA of exon 44 skipping (exon 44 is 148bp). For exon 45, the nested RT-PCR product from the full-length dystrophin transcript was 571bp, and 395bp came from the mRNA of exon 45 skipping (exon 45 is 176bp). For exon 53, the nested RT-PCR product from the full-length dystrophin transcript was 365 bp, and 153 bp from the exon 53 skipped mRNA (exon 53 is 212 bp).

[0321] Gel images were quantitatively analyzed by measuring the band intensity of the full-length PCR product compared to the exon skipping product. In some cases, the percentage of skipping at a fixed PPMO concentration (e.g., 3 micromolar) was used to determine the relative activity of a range of PPMOs in inducing exon skipping of a given exon. In other cases, a range of PPMO doses were used to treat cells (e.g., 0.1, 0.3, 1.0, 3.0, and 10 micromolar), and the EC was calculated based on the percentage of skipping induced at each concentration. 50 .

[0322] Example 1

[0323] Exon 51 scanning

[0324] A series of overlapping antisense PPMOs targeting exon 51 of human dystrophin were designed and synthesized and used to treat human rhabdomyosarcoma cells (RD cells) or primary human skeletal muscle cells. This strategy is called "exon scanning" and was also used for several other dystrophin exons as described below. All PPMOs were synthesized as peptide-coupled PMOs (PPMOs) using the CP06062 peptide (SEQ ID NO:578) and a 3' terminal PMO ligation. For exon 51, a series of 26 PPMOs (SEQ ID NOS:309-311, 314, 316, 317, 319, 321, 323, 324, 326, 327, 329-331, 333, 335, 336, 338-345) were prepared, each with a length of 26 bases, as shown in FIG. Figure 2A The exon skipping potency of PPMOs was evaluated by treating RD cells at various concentrations as described in the Materials and Methods above. Three PPMOs (SEQ ID NOS:324, 326, and 327) that were effective in inducing exon skipping were identified and selected for additional 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 NOS:327 was shown to be the most effective in inducing exon 51 skipping, as shown in Figure 2. Figure 2B and 2C shown.

[0325] As described above, a comparison of the relative potency of SEQ ID NOS:327 with other antisense sequences targeting exon 51 was performed in RD cells and primary human skeletal muscle cells. All evaluated sequences were prepared as peptide-coupled PMOs using the CP06062 peptide (SEQ ID NO:578). This allowed for a direct comparison of the relative potency of the antisense sequences without regard to antisense chemistry or cellular delivery. The relative positions of certain oligomers targeting exon 51 compared to SEQ ID NO:327 are shown in Table 1. Figure 2D As shown. Figure 2C As shown, there is a hierarchy of exon skipping effectiveness, with SEQ ID NO:327 being the most effective, at least several fold compared to the other sequences.

[0326] Example 2

[0327] Exon 50 scanning

[0328] A series of overlapping antisense PPMOs targeting human dystrophin exon 50 were designed and synthesized. For exon 50, a series of 17 PPMOs (SEQ ID NOS: 267, 269, 271, 273, 275, 277, 279, 280, 282, and 284-291) were prepared, each with a length of 25 bases, such as Figure 3A As shown. The exon skipping potency of PPMOs was evaluated by treating RD cells at different concentrations as described in the Materials and Methods above. Four PPMOs (SEQ ID NOS: 277, 287, 290, and 291) that were effective in inducing exon skipping were identified and selected for additional evaluation. Dose ranging experiments in RD cells were used to confirm the relative potency of these four PPMO sequences. SEQ ID NOs: 584 (AVI-5656) and 287 (AVI-5038) were shown to be most effective in inducing exon 50 skipping, as shown. Figure 3B EC derived from dose-ranging experiments 50The value represents the calculated concentration when 50% of the PCR products are from mRNA lacking exon 50 relative to the PCR products produced by mRNA containing exon 50. Other sequences (see, e.g., SEQ ID NOs: 584 and 585 corresponding to SEQ ID NOs: 173 and 175 in WO2006 / 000057, respectively), AVI-5038 (SEQ ID NO: 287) are equivalent or better in inducing exon skipping activity in the RD cell assay, as shown in FIG. Figure 3B shown.

[0329] Example 3

[0330] Exon 53 scanning

[0331] A series of overlapping antisense PPMOs targeting human dystrophin exon 53 were designed and synthesized. For exon 53, a series of 24 PPMOs (SEQ ID NOS: 416, 418, 420, 422, 424, 426, 428, 429, 431, 433, 434, 436, 438-440, and 443-451) were prepared, each with a length of 25 bases, as Figure 4A The exon skipping potency of PPMOs was evaluated by treating RD cells and primary human skeletal muscle cells at various concentrations as described in the Materials and Methods above. Three PPMOs (SEQ ID NOS:428, 429, and 431) that were effective in inducing exon skipping were identified and selected for additional evaluation. Dose-ranging experiments in RD cells were used to confirm the relative potency of these three PPMO sequences. SEQ ID NOS:429 was shown to be the most effective in inducing exon 53 skipping, as shown in Figure 5A. Figure 4B -F. However, when compared to other exon 53 antisense sequences, SEQ ID NOS:429 was shown to be identical to H53A(+23+47), which is listed as SEQ ID NO:195 in WO2006 / 000057 and as SEQ ID NO:609 in this application. Other sequences were compared to SEQ ID NO:429, including H53A(+39+69) and H53A(-12+10) (listed as SEQ ID NOs:193 and 199, respectively, in WO2006 / 000057) and h53AON1 (listed as SEQ ID NO:39 in U.S. Application No. 11 / 233,507), which are listed as SEQ ID NOs:608, 611 and 610, respectively, in this application. All evaluated sequences were prepared as peptide-coupled PMOs using the CP06062 peptide (SEQ ID NO:578). This allows direct comparison of the relative potency of antisense sequences without regard to antisense chemistry or cellular delivery. Fig. 4I and4G -H, indicating that SEQ ID NO: 429 is superior to each of these four sequences.

[0332] Example 4

[0333] Exon 44 scanning

[0334] A series of overlapping antisense PPMOs targeting human dystrophin exon 44 were designed and synthesized. For exon 44, a series of PPMOs (SEQ ID NOS: 1-20) were prepared, each with a length of 25 bases, such as Figure 5A The exon skipping potency of PPMOs was evaluated by treating RD cells at different concentrations as described in Materials and Methods above. Five PPMOs (SEQ ID NOS: 4, 8, 11, 12, and 13) that were effective in inducing exon skipping were identified and selected for additional evaluation. Dose-ranging experiments in RD cells were used to confirm the relative potency of these five PPMO sequences, as shown in FIG. Figure 5C-5H It is shown that SEQ ID NOS: 8, 11 and 12 most effectively induce exon 44 skipping. Figure 5H As shown, SEQ ID NOS: 12 was shown to be the most effective.

[0335] Comparison of SEQ ID NOS: 12 with other exon 44 antisense sequences was performed in both RD cells and human primary skeletal muscle cells. All evaluated sequences were prepared as peptide-coupled PMOs using the CP06062 peptide (SEQ ID NO: 578). This allowed for direct comparison of the relative potency of the antisense sequences without regard to antisense chemistry or cellular delivery.

[0336] The sequences (SEQ ID NOS: 600, 601, 602 and 603) are aligned with SEQ ID NOS: 4, 8, 11 and 12 as shown in Figure 5B In WO2006 / 000057, SEQ ID NOS:601 and 603 are listed as SEQ ID NOS:165 and 167. SEQ ID NO:602 is listed in WO2004 / 083446, which is listed as SEQ ID NO:21 in U.S. Application No. 11 / 233,507. SEQ ID NO:600 was published in 2007 (Wilton, Fall et al. 2007). Comparison in RD cells showed that both SEQ ID NOS:602 and 603 were superior to SEQ ID NO:12 ( Fig.5I ). However, if Figure 5JAs shown, in human primary skeletal muscle cells, SEQ ID NO: 12 (8.86% exon skipping) outperformed SEQ ID NO: 602 (6.42%). Similar experiments were performed with SEQ ID NO: 603.

[0337] Example 5

[0338] Exon 45 scanning

[0339] A series of overlapping antisense PPMOs targeting human dystrophin exon 45 were designed and synthesized. For exon 45, a series of 22 PPMOs (SEQ ID NOS: 21, 23, 25, 27, 29, 31, 32, 34, 35, 37, 39, 41, 43, and 45-53) were prepared, each with a length of 25 bases, such as Fig. 6A The exon skipping potency of PPMOs was evaluated by treating RD cells and human primary skeletal muscle cells at various concentrations as described in the Materials and Methods above. Four PPMOs (SEQ ID NOS: 27, 29, 34, and 39) that were effective in inducing exon skipping were identified and selected for additional evaluation. Dose-ranging experiments in RD cells were used to confirm the relative potency of these four PMO sequences, as shown in FIG. Figure 6C -G, and summarized in Figure 6H SEQ ID NOs: 49 was used as a negative control in these experiments. It was shown that SEQ ID NOs: 29 and 34 were the most effective in inducing exon 45 skipping. Figure 6H shown.

[0340] Comparison of SEQ ID NOS:34 with other exon 45 antisense sequences was performed in both RD cells and human primary skeletal muscle cells. All evaluated sequences were prepared as peptide-coupled PMOs using the CP06062 peptide (SEQ ID NO:578). This allowed for direct comparison of the relative potency of antisense sequences without regard to 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 1. Figure 6B In WO2006 / 000057, SEQ ID NOS:604 and 607 are listed as SEQ ID NOS:211 and 207, respectively. In U.S. Application No. 11 / 233,507, SEQ ID NOS:605 and 606 are listed as SEQ ID NOS:23 and 1, respectively. Comparison in RD cells showed that SEQ ID NOS:34 outperformed all four sequences evaluated, such as Fig.6I As described above, these compounds were tested in different populations of primary human skeletal muscle cells.

[0341] Sequence ID List

[0342] The sequences are represented using the common DNA nucleotide base symbols A, G, C, and T. Other reaction chemistries, such as 2'-O-methyl, replace T with U. Any base may be replaced by inosine (I), particularly in stretches of 3 or more G residues.

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363] *Ahx is aminocaproic acid and B is beta-alanine.

Claims

1. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide has 21 morpholino subunits connected by phosphorus-containing intersubunit linkages, wherein the intersubunit linkages connect the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, wherein the antisense oligonucleotide comprises a variant base sequence of SEQ ID NO.431, wherein the variant base sequence differs from SEQ ID NO.431 by no more than 8 nucleotides, wherein the antisense oligonucleotide is 100% complementary to exon 53 of human dystrophin pre-mRNA, thereby causing exon 53 skipping.

2. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as claimed in claim 1, wherein the inter-subunit link connecting two adjacent morpholino subunits has the following structure: in: X is selected from NH2, N(CH3)2 and optionally substituted 1-piperazinyl; Y1 is O; Z is O; Pi and Pj are purine or pyrimidine base pairing moieties that effectively bind to bases in a polynucleotide by base-specific hydrogen bonding, wherein the purine or pyrimidine base pairing moieties are selected from adenine, cytosine, guanine, uracil, thymine or inosine, wherein the base pairing moieties form a base sequence.

3. A pharmaceutical composition comprising the antisense oligonucleotide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or diluent.

4. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1 or 2 or the pharmaceutical composition according to claim 3, for use in treating muscular dystrophy.

5. The antisense oligonucleotide or pharmaceutically acceptable salt thereof or pharmaceutical composition according to claim 4, wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD).

6. The antisense oligonucleotide or pharmaceutically acceptable salt thereof or pharmaceutical composition according to claim 4, wherein the muscular dystrophy is Becker muscular dystrophy (BMD).

7. A composition for producing exon 44 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit, the antisense compound comprising a sequence selected from SEQ ID NOS: 1-20 and capable of forming a heteroduplex with a complementary mRNA sequence in exon 44 of a dystrophin gene with a Tm of at least 45°C between the compound and the mRNA.

8. A composition for producing exon 45 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, the antisense compound comprising a sequence selected from the group consisting of SEQ ID NOS: 21-76 and 612-624 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 45 of a dystrophin gene between the compound and the mRNA having a Tm of at least 45°C.

9. A composition for producing exon 46 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit, the antisense compound comprising a sequence selected from SEQ ID NOS:77-125 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 46 of a dystrophin gene with a Tm of at least 45°C between the compound and the mRNA.

10. A composition for producing exon 47 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit, the antisense compound comprising a sequence selected from SEQ ID NOS: 126-169 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 47 of a dystrophin gene between the compound and the mRNA having a Tm of at least 45°C.

11. A composition for producing exon 48 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, the antisense compound comprising a sequence selected from the group consisting of SEQ ID NOS: 170-224 and 634 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 48 of a dystrophin gene between the compound and the mRNA having a Tm of at least 45°C.

12. A composition for producing exon 49 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit, the antisense compound comprising a sequence selected from SEQ ID NOS: 225-266 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 49 of a dystrophin gene with a Tm of at least 45°C between the compound and the mRNA.

13. A composition for producing exon 50 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit, the antisense compound comprising a sequence selected from SEQ ID NOS: 267-308 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 50 of a dystrophin gene with a Tm of at least 45°C between the compound and the mRNA.

14. A composition for producing exon 51 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit, the antisense compound comprising a sequence selected from SEQ ID NOS: 309-371 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 51 of a dystrophin gene with a Tm of at least 45°C between the compound and the mRNA.

15. A composition for producing exon 52 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit, the antisense compound comprising a sequence selected from SEQ ID NOS:372-415 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 52 of a dystrophin gene with a Tm of at least 45°C between the compound and the mRNA.

16. A composition for producing exon 53 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, the antisense compound comprising a sequence selected from the group consisting of SEQ ID NOS: 416-475 and 625-633 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 53 of a dystrophin gene between the compound and the mRNA having a Tm of at least 45°C.

17. A composition for producing exon 54 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit, the antisense compound comprising a sequence selected from SEQ ID NOS:476-519 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 54 of a dystrophin gene with a Tm of at least 45°C between the compound and the mRNA.

18. A composition for producing exon 55 skipping in the processing of human dystrophin pre-processed mRNA, comprising A substantially uncharged antisense compound containing 20-35 morpholino subunits connected by phosphorus-containing intersubunit linkages linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, the antisense compound comprising a sequence selected from the group consisting of SEQ ID NOS: 520-569 and 635 and capable of forming a heteroduplex structure with a complementary mRNA sequence in exon 55 of a dystrophin gene with a Tm of at least 45°C between the compound and the mRNA.

Images