阅读说明：本技术 改进的用于治疗肌营养不良的组合物 (Improved composition for treating muscular dystrophy ) 是由 E·M·凯 于 2014-03-14 设计创作，主要内容包括：本发明涉及改进的用于治疗肌营养不良的组合物。描述了通过施用反义分子来治疗肌营养不良的改进的组合物和方法,所述反义分子能够结合人肌养蛋白基因中的选定靶位以诱导外显子跳跃。(The present invention relates to improved compositions for treating muscular dystrophy. Improved compositions and methods for treating muscular dystrophy by administering antisense molecules capable of binding to selected targets in the human dystrophin gene to induce exon skipping are described.)

1. A method of treating duchenne muscular dystrophy in a patient in need thereof, said method comprising administering to said patient a dose of about 30mg/kg of eteplirsen 1 time per week.

2. The method according to claim 1, wherein eteplirsen is administered in a single dose.

3. The method according to claim 1, wherein eteplirsen is administered intravenously.

4. The method of claim 1, wherein the patient has one or more out-of-frame deletions that can be corrected by skipping exon 51 of the dystrophin gene.

5. The method of claim 1, wherein the patient is a pediatric patient.

6. The method according to claim 1, wherein the patient is administered an oral corticosteroid for at least 24 weeks prior to the first dose of eteplirsen.

7. A method for treating duchenne muscular dystrophy in a patient in need thereof, the method comprising: intravenously administering a single dose of eteplirsen of about 30mg/kg 1 time per week to the patient, wherein the patient has one or more out-of-frame deletions that can be corrected by skipping exon 51 of the dystrophin gene.

8.A method of treating duchenne muscular dystrophy in a patient in need thereof, said method comprising administering to said patient a dose of about 50mg/kg of eteplirsen 1 time per week.

9. The method according to claim 8, wherein eteplirsen is administered in a single dose.

10. The method according to claim 8, wherein eteplirsen is administered intravenously.

11. The method of claim 8, wherein the patient has one or more out-of-frame deletions that can be corrected by skipping exon 51 of the dystrophin gene.

12. The method of claim 8, wherein the patient is a pediatric patient.

13. The method according to claim 8, wherein the patient is administered an oral corticosteroid for at least 24 weeks prior to the first dose of eteplirsen.

14. A method of treating duchenne muscular dystrophy in a patient in need thereof, the method comprising: intravenously administering a single dose of eteplirsen of about 50mg/kg 1 time per week to the patient, wherein the patient has one or more out-of-frame deletions that can be corrected by skipping exon 51 of the dystrophin gene.

15.A method of increasing dystrophin production in a patient with duchenne muscular dystrophy, said method comprising administering to said patient a dose of eteplirsen of about 30mg/kg 1 time per week.

16. The method according to claim 15, wherein eteplirsen is administered in a single dose.

17. The method according to claim 15, wherein eteplirsen is administered intravenously.

18. The method of claim 15, wherein the patient has one or more out-of-frame deletions that can be corrected by skipping exon 51 of the dystrophin gene.

19. The method of claim 15, wherein the patient is a pediatric patient.

20. The method according to claim 15, wherein the patient is administered an oral corticosteroid for at least 24 weeks prior to the first dose of eteplirsen.

21. A method of increasing dystrophin production in a patient with duchenne muscular dystrophy, said method comprising administering to said patient a dose of about 50mg/kg of eteplirsen 1 time per week.

22. The method according to claim 21, wherein eteplirsen is administered in a single dose.

23. The method according to claim 21, wherein eteplirsen is administered intravenously.

24. The method of claim 21, wherein the patient has one or more out-of-frame deletions that can be corrected by skipping exon 51 of the dystrophin gene.

25. The method of claim 21, wherein the patient is a pediatric patient.

26. The method according to claim 21, wherein the patient is administered an oral corticosteroid for at least 24 weeks prior to the first dose of eteplirsen.

Technical Field

The present invention relates to improved methods for treating muscular dystrophy in a patient. It also provides compositions suitable for use in promoting exon skipping in the human dystrophin gene.

Background

Antisense technology is being developed using a variety of chemical agents to affect gene expression at a variety of different levels (transcription, splicing, stability, translation). Much of this research has focused on the use of antisense compounds to correct or compensate for aberrant or disease-associated genes within a wide range of indications. Antisense molecules are capable of specifically inhibiting gene expression, and therefore, much research effort on oligonucleotides as modulators of gene expression has 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 typically directed against RNA, whether the sense strand (e.g., mRNA) or the negative strand. To achieve the desired effect of a particular gene down-regulation, oligonucleotides typically promote degradation of the targeted mRNA, block translation of the mRNA, or block the function of cis-acting RNA elements, thereby effectively preventing de novo synthesis of the target protein or replication of the viral RNA.

However, such techniques are not useful if the objective is to up-regulate the production of the native protein or to compensate for mutations that induce premature termination of translation (such as nonsense or frameshift mutations). In these cases, the defective gene transcript does not undergo targeted degradation or steric inhibition, so the antisense oligonucleotide chemical does not promote target mRNA degradation or block translation.

In a variety of genetic diseases, the effect of mutations on the ultimate expression of a gene can be modulated by the process of targeted exon skipping during splicing. The splicing process is guided by a complex multi-component mechanism that brings adjacent exon-intron junctions in the pre-mRNA into close proximity and cleaves the phosphodiester bonds at the ends of the introns, which subsequently reform between the exons that are to be spliced together. This complex and highly precise process is mediated by sequence motifs in pre-mRNA, which are relatively short, semi-conserved RNA fragments to which multiple nuclear splicing factors involved in the splicing reaction will subsequently bind. By altering the splicing machinery in a way that reads out or recognizes motifs involved in pre-mRNA processing, it is possible to generate differentially spliced mRNA molecules. It is now recognized that most human genes are alternatively spliced during normal gene expression, although the mechanisms involved have not been identified. Bennett et al (U.S. Pat. No. 6,210,892) describe antisense modulation of wild type cellular mRNA processing using antisense oligonucleotide analogs that do not induce RNase H mediated cleavage of the target RNA. This has utility in the following respects: alternatively spliced mrnas can be generated that lack specific exons used to make soluble TNF superfamily receptors (e.g., as described (Sazani, Kole, et al. 2007)) that lack exons encoding transmembrane domains.

In the case of premature termination of normal functional proteins by mutations therein, it has been demonstrated that the way of restoring the production of some functional proteins by antisense technology is possible by interference in the splicing process and that if the exon associated with a pathogenic mutation can be specifically deleted from some gene, a shortened protein product can sometimes be produced which has similar biological properties to the native protein or has biological activity sufficient to improve the disease caused by the mutation associated with said exon (see, for example, Sierakowska, Sambade et al 1996; Wilton, Lloyd et al 1999; van Deutekom, Bremmer-Bout et al 2001; Lu, Mann et al 2003; Aarta-Rus, Janson et al 2004). Kole et al (U.S. patent nos. 5,627,274, 5,916,808, 5,976,879 and 5,665,593) disclose methods of combating aberrant splicing using modified antisense oligonucleotide analogs that do not promote degradation of targeted pre-mRNA. Bennett et al (U.S. Pat. No. 6,210,892) describe antisense modulation of wild type cellular mRNA processing also using antisense oligonucleotide analogs that do not induce RNase H mediated cleavage of the target RNA.

The process of targeted exon skipping may be particularly useful in long genes where there are many exons and introns, where there is redundancy in the genetic makeup of the exons, or where the protein is able to function without one or more specific exons.

Efforts to alter gene processing in order to treat genetic diseases associated with truncations caused by mutations in various genes have focused on the use of antisense oligonucleotides that: (1) overlap completely or partially with elements involved in the splicing process; or (2) bind to the pre-mRNA at a location that is sufficiently close to the element to disrupt the binding and function of splicing factors that normally mediate a particular splicing reaction that occurs at the element.

Duchenne Muscular Dystrophy (DMD) is caused by a defect in the expression of the protein dystrophin. The gene encoding this protein contains 79 exons, which are spread over more than 2 million nucleotides of DNA. Any exon mutation that alters the reading frame of an exon, or introduces a stop codon, or is characterized by the removal of one or more full-frame exoexons or one or more copies of an exon, has the potential to disrupt the production of functional dystrophin, resulting in DMD.

Onset of disease may be described as elevated creatine kinase levels at birth, and significant motor deficits may exist in the first year of life. Before the age of 7 or 8, most DMD patients have an increasingly strenuous gait and lose the ability to lift and climb stairs from the ground; most were wheelchair-dependent by the age of 10-14 years. DMD is generally fatal; affected individuals often die of respiratory and/or heart failure in their late teens or early twenties. The continuous progression of DMD allows therapeutic intervention at all disease stages; however, treatment is currently limited to glucocorticoids, which are associated with numerous side effects, including weight gain, behavioral changes, adolescent changes, osteoporosis, cushing's syndrome-like facial appearance, growth inhibition, and cataracts. As a result, it is critical to develop better therapies to treat the underlying cause of the disease.

It has been found that a less severe form of muscular dystrophy, Becker Muscular Dystrophy (BMD), occurs where 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 mRNA into protein is not prematurely terminated. If the joining of upstream and downstream exons would maintain the correct reading frame of the gene in the processing of the mutated dystrophin pre-mRNA, the result would be an mRNA encoding a protein with short internal deletions that retains some activity, resulting in a Becker phenotype.

It has been known for many years that deletion of one or more exons that do not alter the reading frame of dystrophin will give rise to the BMD phenotype, while deletion of exons that cause frame-shifting will give rise to DMD (Monaco, Bertelson et al 1988). Generally, dystrophin mutations (including point mutations and exon deletions that alter the reading frame and thereby block proper protein translation) result in DMD. It should also be noted that some BMD and DMD patients have exon deletions covering multiple exons.

Modulation of mutant dystrophin pre-mRNA splicing with antisense oligoribonucleotides has been reported in vitro and in vivo (see, e.g., Matsuo, Masumura et al 1991; Takeshima, Nishio et al 1995; Pramono, Takeshima et al 1996; Dunckley, Eperon et al 1997; Dunckley, Manohara et al 1998; Errington, Mann et al 2003).

Wilton et al (Wilton, Lloyd et al 1999) reported the first example of specific and reproducible exon skipping in an mdx mouse model. By targeting the antisense molecule to the donor splice site, consistent and efficient exon 23 skipping is induced in dystrophin mRNA within 6 hours of treatment of cultured cells. Wilton et al also describe the use of longer antisense oligonucleotides to target the receptor region of mouse dystrophin pre-mRNA. Although the first antisense oligonucleotide directed to the intron 23 donor splice site induced consistent exon skipping in primary cultured myoblasts, the compound was found to be much less potent in immortalized cell cultures expressing higher levels of dystrophin. However, with refined targeting and antisense oligonucleotide design, the efficiency of specific exon removal is increased by almost 1 order of magnitude (Mann, Honeyman et al 2002).

Recent studies have begun to address the challenge of achieving sustained dystrophin expression with minor adverse effects in tissues affected by dystrophin deficiency. Intramuscular injection of antisense oligonucleotide targeting exon 51(PRO051) into tibialis anterior muscle in 4 patients with DMD resulted in specific skipping of exon 51 without any clinically significant adverse effects (Mann, Honeyman et al 2002; van Deutekom, Janson et al 2007). Studies in mdx mice attempting to deliver antisense phosphorodiamidate morpholino oligomers conjugated with a cell Penetrating Peptide (PPMO) targeting exon 23 systemically produced high and sustained dystrophin production in skeletal and cardiac muscle with no detectable toxicity (Jearawiriyapaiisarn, Moulton et al 2008; Wu, Moulton et al 2008; Yin, Moulton et al 2008).

A recent clinical trial to test the safety and efficacy of splice-switch oligonucleotides (SSOs) for the treatment of DMD is based on SSO technology to induce alternative splicing of pre-mRNA by steric blockade of the spliceosome (Cirak et al, 2011; Goemans et al, 2011; Kinali et al, 2009; van Deutekom et al, 2007). However, despite these successes, the pharmacological options available for treating DMD are limited. Notably, antisense oligonucleotides using negatively charged phosphorothioate backbones (drisapersen) have been associated in clinical trials with proteinuria, increased urinary α 1-microglobulin, thrombocytopenia, and injection site reactions such as erythema and inflammation.

Eteplirsen, which is being developed by the assignee of the present application, has been the subject of clinical studies testing its safety and efficacy, and clinical development is ongoing. Eteplissen is a structurally different oligonucleotide than drisapren. Specifically, the chemical backbone of eteplirsen is Phosphorodiamidate Morpholino (PMO) and the chemical backbone of drisapersen is 2 'O-methyl phosphorothioate (2' -OMe). These structural differences and their potential impact on clinical outcomes have recently been described. See Molecular Therapy Nucleic Acids (2014)3, e 152; doi: 10.1038/mtna.2014.6 (online publication on 3/11/2014).

The sequence of eteplirsen has been described previously. See, for example, U.S. Pat. No. 7,807,816, which is exclusively licensed to applicants. However, U.S. Pat. No. 7,807,816 does not explicitly discuss the optimal dosing schedule and route of administration of eteplirsen.

Thus, there remains a need for improved compositions and methods for treating muscular dystrophy (such as DMD and BMD) in patients.

Disclosure of Invention

The present invention is based, at least in part, on the compelling evidence of therapeutic efficacy of exon skipping antisense oligonucleotide eteplirsen, which represents a significant advance in the treatment of DMD by addressing the underlying cause of the disease. The novel finding of treatment with exon 51 skipping antisense oligonucleotide eteplirsen, as measured by the 6 minute walk test (6MWT), resulted in reliable increases in novel dystrophins and stable walking ability (e.g., stabilization of out-of-bed activity), highlighting the potential to alter the course of the disease. Importantly, no drug-related adverse events were observed in 576 infusions administered over a1 year period. The use of exon skipping antisense oligonucleotides, when applied to other exons, can treat an estimated 70% to 80% of patients with DMD due to a deletion in the dystrophin gene.

Accordingly, in one aspect, the invention relates to a method of treating duchenne muscular dystrophy in a patient in need thereof, said method comprising administering to said patient a dose of about 30mg/kg eteplirsen 1 time per week. In certain embodiments, eteplirsen is administered in a single dose. In certain embodiments, eteplirsen is administered intravenously. In certain embodiments, the patient has an out-of-frame deletion that can be corrected by skipping exon 51 of the dystrophin gene. In certain embodiments, the patient is a pediatric patient.

In another aspect, the invention relates to a method of treating duchenne muscular dystrophy in a patient in need thereof, said method comprising administering to said patient a dose of about 30mg/kg 1 time per week of eteplirsen, wherein said patient is administered an oral corticosteroid for at least 24 weeks prior to the first dose of eteplirsen.

In another aspect, the invention provides a method of treating duchenne muscular dystrophy in a patient in need thereof, the method comprising: intravenously administering a single dose of eteplirsen of about 30mg/kg 1 time per week to the patient, wherein the patient has an out-of-frame deletion that can be corrected by skipping exon 51 of the dystrophin gene.

Other embodiments of the present invention relate to a method of treating duchenne muscular dystrophy in a patient in need thereof, said method comprising administering to said patient a dose of about 50mg/kg eteplirsen 1 time per week. In certain embodiments, eteplirsen is administered in a single dose. In certain embodiments, eteplirsen is administered intravenously. In certain embodiments, the patient has an out-of-frame deletion that can be corrected by skipping exon 51 of the dystrophin gene. In certain embodiments, the patient is a pediatric patient. In other embodiments, the patient is administered an oral corticosteroid for at least 24 weeks prior to the first dose of eteplirsen.

In another aspect, the invention relates to a method of treating duchenne muscular dystrophy in a patient in need thereof, the method comprising: intravenously administering a single dose of eteplirsen of about 50mg/kg 1 time per week to the patient, wherein the patient has an out-of-frame deletion that can be corrected by skipping exon 51 of the dystrophin gene.

In another aspect, the invention provides a method of increasing dystrophin production in a patient with duchenne muscular dystrophy, said method comprising administering to said patient a dose of eteplirsen of about 30mg/kg 1 time per week. In certain embodiments, a dose of about 50mg/kg eteplirsen is administered 1 time per week. In certain embodiments, eteplirsen is administered in a single dose. In certain embodiments, eteplirsen is administered intravenously. In other embodiments, the patient has an out-of-frame deletion that can be corrected by skipping exon 51 of the dystrophin gene. In certain embodiments, the patient is a pediatric patient. In other embodiments, the patient is administered an oral corticosteroid for at least 24 weeks prior to the first dose of eteplirsen.

Accordingly, the present invention relates to a method of treating Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) in a patient by administering an effective amount of a composition comprising an antisense oligonucleotide 20-50 nucleotides in length comprising at least 10 contiguous nucleotides complementary to a target region in an exon of a human dystrophin gene to specifically hybridize to the target region, induce exon skipping and thereby treat the disease. In one embodiment, the effective amount is at least 20mg/kg for a time sufficient to increase the number of dystrophin positive fibers in the subject to at least 20% of normal and stabilize, maintain or improve the distance walked by the patient relative to a 20% shortage of healthy equivalents (e.g., in 6 MWT). In another embodiment, an effective amount is at least 20mg/kg to about 30mg/kg, about 25mg/kg to about 30mg/kg, or about 30mg/kg to about 50 mg/kg. In another embodiment, the effective amount is about 30mg/kg or about 50 mg/kg.

In another aspect, the effective amount is at least 20mg/kg, about 25mg/kg, about 30mg/kg, or about 30mg/kg to about 50mg/kg for at least 24 weeks, at least 36 weeks, or at least 48 weeks, thereby increasing the number of dystrophin positive fibers in the subject to at least 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of normal, and stabilizing or improving the distance walked by the patient relative to a 20% shortage of healthy equivalents (e.g., in 6 MWT). In one embodiment, treatment increases the number of dystrophin positive fibers to 20-60% or 30-50% of normal in said patient. In certain embodiments, the treatment is by systemic administration, such as once weekly infusion. In other embodiments, treatment comprises administering to the subject another therapeutic agent, such as a steroid.

In another aspect, the invention provides a method of treating DMD or BMD in a patient by administering from about 30mg/kg to about 50mg/kg of a composition comprising an antisense oligonucleotide 20-50 nucleotides in length comprising at least 10 contiguous nucleotides complementary to a target region in an exon of a human dystrophin gene, wherein said antisense oligonucleotide specifically hybridizes to a target region that induces exon skipping, thereby treating said subject. In one embodiment, the antisense oligonucleotide is substantially uncharged. In another embodiment, the antisense oligonucleotide comprises morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit. In another embodiment, the antisense oligonucleotide comprises morpholino subunits linked by substantially uncharged phosphorus-containing intersubunit linkages linking a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit. In other aspects, the antisense oligonucleotide comprises a morpholino subunit and a phosphorodiamidate intersubunit linkage.

In certain embodiments, the antisense oligonucleotide is 20-50, 30-50, or 20-30 nucleotides in length comprising at least 10, 12, 15, 17, or 20 contiguous nucleotides complementary to a target region in an exon of a human dystrophin gene, said exon selected from the group consisting of exon 51, exon 50, exon 53, exon 45, exon 46, exon 44, exon 52, exon 55, and exon 8. In one embodiment, the antisense oligonucleotide is 20-50, 30-50, or 20-30 nucleotides in length and includes at least 20 contiguous nucleotides of eteplirsen (SEQ ID NO: 1). In another embodiment, the antisense oligonucleotide is 20-50, 30-50, or 20-30 nucleotides in length and comprises the sequence as set forth in SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 9, at least 10, 12, 15, 17, or 20 contiguous nucleotides of an antisense oligonucleotide set forth in seq id no. In another embodiment, the antisense oligonucleotide is 20-50, 30-50, or 20-30 nucleotides in length and comprises at least 10, 12, 15, 17, or 20 consecutive nucleotides of a nucleotide sequence as set forth in tables 3 and 4, wherein the uracil base in the antisense oligonucleotide is optionally a thymine base.

In one embodiment, the composition comprises eteplirsen (SEQ ID NO: 1) and optionally a pharmaceutically acceptable carrier. In another embodiment, the composition comprises a nucleic acid sequence selected from SEQ ID NOs: 1-9, such as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 and SEQ ID NO: 9. in another embodiment, the antisense oligonucleotide is any one or combination of the nucleotide sequences set forth in tables 3 and 4, wherein the uracil base in the antisense oligonucleotide is optionally a thymine base. In certain aspects, the antisense oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide (such as an arginine-rich peptide).

In another aspect, the invention provides a method of treating DMD or BMD in a patient by administering at least 20mg/kg of a composition comprising eteplirsen (SEQ ID NO: 1) for a time sufficient to increase the number of dystrophin-positive fibers in the subject to at least about 20% of normal, and to stabilize or improve the distance walked by the patient relative to a 20% shortage of healthy equivalents (e.g., in 6 MWT). In another embodiment, an effective amount is at least 20mg/kg to about 30mg/kg, about 25mg/kg to about 30mg/kg, or about 30mg/kg to about 50mg/kg of a composition comprising eteplirsen (SEQ ID NO: 1) and optionally a pharmaceutically acceptable carrier, such as phosphate buffered saline.

In another aspect, an effective amount of a composition comprising eteplirsen (SEQ ID NO: 1) is at least 20mg/kg, about 25mg/kg, about 30mg/kg, or about 30mg/kg to about 50mg/kg for at least 24 weeks, at least 36 weeks, or at least 48 weeks, thereby increasing the number of dystrophin positive fibers in a subject to at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of normal, and stabilizing or improving the distance walked by the patient relative to a 20% shortage of healthy equivalents (e.g., in 6 MWT). In certain embodiments, treatment with the antisense oligonucleotides of the invention will slow or alleviate the lack of bed-out activity that would be expected in the absence of treatment. In certain embodiments, treatment with the antisense oligonucleotides of the invention stabilizes, maintains, or increases the stable walking distance of the patient. For example, treatment may increase the patient's steady walking distance from baseline to greater than 3, 5,6, 7,8, 9, 10, 15, 20, 25, 30, or 50 meters (including all integers in between).

Other aspects of the invention relate to treatment with the antisense oligonucleotides of the invention (such as eteplirsen) that slow or reduce progressive respiratory muscle dysfunction and/or failure in patients with DMD that would be expected in the absence of treatment. In one embodiment, treatment with the antisense oligonucleotides of the invention may reduce or eliminate the need for ventilatory assist devices that would be anticipated in the absence of treatment. In one embodiment, the measurements of respiratory function and the evaluation of potential therapeutic interventions to track disease progression include Maximum Inspiratory Pressure (MIP), Maximum Expiratory Pressure (MEP), and Forced Vital Capacity (FVC).

Drawings

Figure 1A shows an exemplary morpholino oligomer structure with phosphorodiamidate linkages.

Figure 1B shows a conjugate of an arginine-rich peptide and an antisense oligomer, according to one embodiment of the invention.

FIG. 1C shows a conjugate as in FIG. 1B, wherein the backbone linkage contains one or more positively charged groups.

Fig. 1D-G show the repeating subunit segments of exemplary morpholino oligonucleotides, designated D through G.

Figure 2 is a schematic of a study design for treating DMD patients. 12 DMD patients were randomized to one of 3 cohorts of double-blind placebo-controlled study 201: cohort 1, eteplirsen 30 mg/kg/wk; cohort 2, eteplirsen 50 mg/kg/wk; and cohort 3, placebo/delayed eteplirsen. At week 25, placebo-treated patients in cohort 3 switched to treatment with 30 or 50 mg/kg/week eteplirsen open markers. Patients were maintained at their same dose of eteplirsen under an open-label extension study 202. For dystrophin analysis, patients received a biceps biopsy at baseline and a deltoid biopsy at week 48. Additional biceps biopsies were obtained at week 12 (patients from cohort 2 and 2 patients from cohort 3) or week 24 (patients from cohort 1 and 2 patients from cohort 3). Efficacy evaluation 6MWT was used as a functional outcome measure and was performed before and every 12 weeks until week 48 after treatment.

Figure 3 depicts dystrophin positive muscle fibers after eteplirsen at 12, 24 and 48 weeks. Panels a and B show the mean absolute change from baseline in the percentage of dystrophin positive fibers at weeks 12, 24 and 48 of the treatment group. In fig. a:^*p-values are for the comparison between eteplirsen and placebo using pooled results from weeks 12 and 24 and are based on a covariance analysis model on graded data with treatment as the fixed effect and baseline values and time since DMD diagnosis as covariates. The average change shown is based on descriptive statistics.Values are from paired t-tests comparing week 48 values to baseline.Results from placebo-treated patients biopsied at week 12 and 24 were pooled. Placebo/delayed eteplirsen patients received eteplirsen starting at week 25 and a total of 24 doses already at week 48. Abbreviations: BL is baseline; NA is not applicable; ND is not performed; NS is not significant; SE is the standard error.

Figure 4 shows the effect of eteplirsen on dystrophin-related glycoprotein complexes. (A) Representative examples of time-dependent increases in dystrophin positive fibers associated with treatment of all study patients enrolled. (B) nNOS μ staining of muscle from dmd (a) and normal (c) control patients (not under study) and from patient 6 at baseline (b) and week 48 (d) confirmed the recovery of nNOS μ binding to eteplirsen. The β -and γ -sarcoglycan (e) and (f) staining in patient 6 at week 48 confirmed the recovery of the complex of sarcoglycan with eteplirsen. (C) RT-PCR showed the product of jumping in the muscle of patient 12 after treatment (289 bp).

Figure 5 graphically depicts the functional efficacy of eteplirsen. The dark purple line shows the change from baseline in distance walked over 6MWT over time for 6 evaluable patients who received eteplirsen from the beginning of 201 (excluding 2 boys from this analysis that were unacceptable at week 24 or later). The grey line shows the change from baseline in the distance walked on 6MWT for 4 patients receiving placebo in the first 24 weeks and eteplirsen in the last 24 weeks.

Detailed Description

Embodiments of the invention relate to improved methods of treating muscular dystrophy (such as DMD and BMD) by administering antisense compounds specifically designed to induce exon skipping in the human dystrophin gene. Dystrophin plays an extremely important role in muscle function and a variety of muscle-related diseases are characterized by mutated forms of this gene. Thus, in certain embodiments, the improved methods described herein may be used to induce exon skipping in mutant forms of the human dystrophin gene, such as the mutant dystrophin gene found in DMD and BMD.

These mutated human dystrophin genes express defective dystrophins or dystrophins that are not measurable at all due to aberrant mRNA splicing events caused by the mutation, a condition that leads to various forms of muscular dystrophy. To treat this condition, the antisense compounds of the invention will hybridize to selected regions of the pre-processed RNA of the mutated human dystrophin gene, inducing exon skipping and differential splicing in an otherwise aberrantly spliced dystrophin mRNA, and thereby allowing the myocyte to produce an mRNA transcript encoding a functional dystrophin. In certain embodiments, the resulting dystrophin protein is not necessarily the "wild-type" form of dystrophin, but is a truncated, but functional or semi-functional form of dystrophin.

By increasing the level of functional dystrophin in muscle cells, these and related embodiments are useful for preventing and treating muscular dystrophy, particularly those forms of muscular dystrophy such as DMD and BMD, which are characterized by defective dystrophin expression caused by aberrant mRNA splicing. The methods described herein further provide improved treatment options for patients with muscular dystrophy and provide significant and practical advantages over alternative methods of treating related forms of muscular dystrophy. For example, in certain embodiments, the improved methods involve administering antisense compounds for inducing exon skipping in the human dystrophin gene at higher doses and/or for longer durations than previous regimens.

Thus, the present invention relates to improved methods of treating muscular dystrophy (such as DMD and BMD) by inducing exon skipping in a patient. In certain embodiments, exon skipping is induced by administering an effective amount of a composition comprising a charge neutral, Phosphorodiamidate Morpholino Oligomer (PMO), such as eteplirsen, that selectively binds to a target sequence in an exon of a dystrophin pre-mRNA. In certain embodiments, the invention relates to methods of treating DMD or BMD in which an effective amount of a composition, e.g., at least 20mg/kg, about 25mg/kg, about 30mg/kg, or about 30mg/kg to about 50mg/kg, comprising an antisense compound as described herein, such as eteplirsen, is administered for a period of time sufficient to treat the disease.

Some embodiments of the invention relate to the use of eteplirsen as a disease modifying therapy for the treatment of DMD. Without being bound by theory, the clinical efficacy observed so far with eteplirsen can be derived from its safety profile due to its unique chemical composition, characterized by nucleotides bound to a morpholine ring linked by a charge neutral phosphorodiamidate moiety.

In DMD patients treated with eteplirsen for 1 year, the average percentage of dystrophin-positive fibers increased to 47% of normal compared to baseline. The magnitude of the increase depends on the duration of treatment. A significant increase in dystrophin levels was observed in 24-week biopsies of patients taken from cohort 1(30mg/kg) and in 48-week biopsies of patients taken from cohort 3 (which started eteplirsen at week 25).

The clinical benefit of Eteplirsen reflects its ability to induce exon skipping and restore functional dystrophin production. Clinical effects were evaluated with 6MWT, a measure of endurance and muscle capacity that exceeded the intensity evaluation in individual muscle groups. Patients receiving 30 or 50mg/kg eteplirsen from the beginning maintain a steady walking distance in 48 weeks, which is consistent with the increase in eteplirsen-induced expression of novel dystrophins between weeks 12 and 24. In contrast, patients in the placebo/delayed eteplirsen cohort decreased 70 meters before week 36, but appeared to stabilize before week 48 (24 weeks after the start of eteplirsen). This is the same time frame in which clinical effects were observed in patients receiving 30 or 50mg/kg eteplirsen 1 time per week from the start of the study. Both cohorts have maintained stable 6MWT results over 120 weeks, as described below.

At 120 weeks, patients capable of 6MWT in both the 30mg/kg and 50mg/kg eteplirsen cohorts (modified intent-to-treat or mITT population; n ═ 6) experienced general stability with walking ability slightly reduced by 13.9 meters or less than 5% from baseline. A statistically significant therapeutic benefit of 64.9 meters (p ≦ 0.006) was observed for the mITT population compared to the placebo/delayed treatment cohort, which started treatment at week 25 after 24 weeks of placebo (n ═ 4). After experiencing a large decline in early study period (prior to treatment with etiprelsen), the placebo/delayed treatment cohort also showed a steady walking ability for more than 1.5 years, from week 36 to week 120, at which time significant levels of dystrophin may be produced, declining by 9.5 meters over this time frame. These analyses were based on the maximum 6MWT score when the experiments were performed on 2 consecutive days.

Respiratory muscle function from baseline to week 120 in both dosing groups as measured by maximum inspiratory and expiratory pressure (MIP and MEP) showed a 14.6% mean increase in MIP and a 15.0% mean increase in MEP. Analysis of the predicted MIP% (for weight adjusted MIP) and predicted MEP% (for age adjusted MEP) demonstrated an average increase in the predicted MIP% from 90.2% at baseline to 95.2% at week 120, and a slight average increase in the predicted MEP% from 79.3% at baseline to 79.6% at week 120. In addition, there was an average increase in Forced Vital Capacity (FVC) (a measure of lung volume) from baseline to 8.7% at week 120, and the predicted FVC% (FVC for age and height adjustments) remained above the average of 90% at week 120, 101% at baseline and 93% at week 120.

The present invention is based, at least in part, on evidence of the therapeutic efficacy of eteplirsen, which represents a significant advance in the treatment of DMD by addressing the underlying cause of the disease. Accordingly, the present invention relates to methods of treating DMD or BMD in a patient by administering an effective amount of a composition comprising an antisense oligonucleotide, such as eteplirsen, complementary to a target region in an exon of a human dystrophin gene, to specifically hybridize to said target region, induce exon skipping and treat said disease. In one embodiment, treatment is by administering to a subject in need thereof one or more antisense oligonucleotides of the invention (e.g., the nucleotide sequences shown in tables 3 and 4), optionally as part of a pharmaceutical formulation or dosage form. Treatment comprises inducing exon skipping in a subject by administering an effective amount of one or more antisense oligonucleotides, wherein said exon is any one or more of exons 1 to 79 from the dystrophin gene. Preferably, the exon is exon 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 8 from the human dystrophin gene.

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

I.Definition of

"about" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

The terms "complementary" and "complementarity" refer to polynucleotides (i.e., sequences of nucleotides) related by the base-pairing rules. For example, the sequence "T-G-A (5 '-3')" is complementary to the sequence "T-C-A (5 '-3')". Complementarity may be "partial," in which only some of the nucleic acid bases are matched according to the base pairing rules. Alternatively, "complete" or "total" complementarity may exist between nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. Although perfect complementarity is often desired, some embodiments may include one or more, but preferably 6, 5, 4, 3,2, or 1 mismatches with the target RNA. Including variations at any position within the oligomer. In certain embodiments, variations in the sequence near the ends of the oligomer are generally more preferred than variations in the interior, and if present, are generally within about 6, 5, 4, 3,2, or 1 nucleotides of the 5 'and/or 3' ends.

The terms "cell penetrating peptide" and "CPP" are used interchangeably and refer to a cationic cell penetrating peptide, also known as a transit peptide, a carrier peptide, or a peptide transduction domain. The peptides presented herein have the ability to induce cell penetration in 100% of the cells of a given cell culture population and allow translocation of macromolecules after systemic administration within multiple tissues in vivo. One preferred embodiment of a CPP is an arginine-rich peptide as described further below.

The terms "antisense oligomer" and "antisense compound" and "antisense oligonucleotide" are used interchangeably and refer to a sequence of cyclic subunits connected by intersubunit linkages, each cyclic subunit carrying a base-pairing moiety, which permits the base-pairing moiety to hybridize by watson-crick base pairing to a target sequence in a nucleic acid (typically RNA) to form the nucleic acid within the target sequence: oligomeric heteroduplexes. The cyclic subunit is based on ribose or another pentose, or in a preferred embodiment, on a morpholino group (see description of morpholino oligomers below). The oligomer can have exact or approximate sequence complementarity to the target sequence; variations in the sequence near the ends of the oligomer are generally preferred over variations in the interior.

Such antisense oligomers may be designed to block or inhibit translation of mRNA or inhibit the natural pre-mRNA splicing process, and may be said to "target" or "target" the target sequence to which it hybridizes. The target sequence is typically the region: it includes the AUG start codon of the mRNA (oligomers that inhibit translation) or the splice site of the pre-processed mRNA (oligomers that inhibit splicing (SSO)). Target sequences for splice sites may include mRNA sequences: it has 1 to about 25 base pairs at its 5' end located downstream of the normal splice acceptor junction of the pre-processed mRNA. A preferred target sequence is any region of the pre-processed mRNA that includes a splice site or is wholly contained within an exon coding sequence or spans a splice acceptor or donor site. When an oligomer is targeted to a nucleic acid of a target in the manner described above, the oligomer is more generally said to be "targeted" to a biologically relevant target, such as a protein, virus or bacterium.

The term "morpholino oligomer" or "PMO" (phosphoramidate-or phosphorodiamidate morpholino oligomers) refers to an oligonucleotide analog consisting of morpholino subunit structures wherein (i) the structures are linked together by phosphorus-containing bonds having a length of 1-3 atoms, preferably a length of 2 atoms, and preferably are uncharged or cationic, which link the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring bears a purine or pyrimidine base-pairing moiety which effectively binds a base in a polynucleotide by base-specific hydrogen bonding. See, for example, the structure in FIG. 1A, which shows one preferred type of phosphorodiamidite linkage. Changes may be made to the bonds as long as they do not interfere with binding or activity. For example, the oxygen attached to the phosphorus may be replaced by sulfur (thiophosphoryl diamine). The 5' oxygen may be substituted with amino or lower alkyl substituted amino. The pendant nitrogen attached to the phosphorus may be unsubstituted, or mono-or di-substituted with (optionally substituted) lower alkyl. The purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil, thymine or inosine. The synthesis, structure, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337, 8,076,476, 8,299,206, and 7,943,762 (cationic bonds), all of which are incorporated herein by reference. Improved intersubunit linkages and terminal groups are detailed in PCT application US2011/038459 and publication WO/2011/150408, which are incorporated herein by reference in their entirety.

"Eteplirsen", also known as "AVN-4658", is a PMO having base sequence 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: 1). Eteplissen is registered under CAS registry number 1173755-55-9. The chemical names include:

RNA, [ P-deoxy-P- (dimethylamino) ] (2 ', 3 ' -dideoxy-2 ', 3 ' -imino-2 ', 3 ' -ring-opened) (2 ' a → 5 ') (C-m5U-C-C-A-A-C-A-m 5U-C-A-G-A-m 5U-G-C-A-m 5U-m5U-m5U-C-m5U-A-G), 5 ' - [ P- [4- [ [2- [2- (2-hydroxyethoxy) ethoxy ] carbonyl ] -1-piperazinyl ] -N, N-dimethylphosphonic acid amide ester (phosphonamide) ].

And

p, 2 ', 3 ' -trideoxy-P- (dimethylamino) -5 ' -O- { P- [4- (10-hydroxy-2, 5, 8-trioxadecanoyl) piperazin-1-yl ] -N, N-dimethylphosphonic acid amidoyl (phosphonamidoyl) } -2 ', 3 ' -imino-2 ', 3 ' -secocytidinoyl- (2 ' a → 5 ') -P, 3 ' -dideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 ' -secothymidine- (2 ' a → 5 ') -P, 2 ', 3 ' -trideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 '-Cyclocyclocytidinoyl- (2' a → 5 ') -P, 2', 3 '-trideoxy-P- (dimethylamino) -2', 3 '-imino-2', 3 '-cycloadenylyl- (2' a → 5 ') -P, 2', 3 '-trideoxy-P- (dimethylamino) -2', 3 ' -imino-2 ', 3 ' -secocytidinoyl- (2 ' a → 5 ') -P, 2 ', 3 ' -trideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 ' -secoadenylyl- (2 ' a → 5 ') -P, 3 ' -dideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 ' -secothymidine acyl- (2 ' a → 5 ') -P, 2 ', 3 ' -trideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 ' -secocytidinoyl- (2 ' a → 5 ') -P, 2 ', 3 '-trideoxy-P- (dimethylamino) -2', 3 '-imino-2', 3 '-cycloadenylyl- (2' a → 5 ') -P, 2', 3 '-trideoxy-P- (dimethylamino) -2', 3 '-imino-2', 3 '-cycloguanoylyl- (2' a → 5 ') -P, 2', 3 '-trideoxy-P- (dimethylamino) -2', 3 '-imino-2', 3 '-secoguanylyl- (2' a → 5 ') -P, 2', 3 '-trideoxy-P- (dimethylamino) -2', 3 '-imino-2', 3 '-secoadenylyl- (2' a → 5 ') -P, 2', 3 '-trideoxy-P- (dimethylamino) -2', 3 '-imino-2', 3 '-secoguanylyl- (2' a → 5 ') -P, 2', 3 '-trideoxy-P- (dimethylamino) -2', 3 ' -imino-2 ', 3 ' -secoadenylyl- (2 ' a → 5 ') -P, 3 ' -dideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 ' -secothymidine acyl- (2 ' a → 5 ') -P, 2 ', 3 ' -trideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 ' -secoguanylyl- (2 ' a → 5 ') -P, 2 ', 3 '-trideoxy-P- (dimethylamino) -2', 3 '-imino-2', 3 '-secocytidylcytidinoyl- (2' a → 5 ') -P, 2', 3 '-trideoxy-P- (dimethylamino) -2', 3 '-imino-2', 3 '-secoadenylyl- (2' a → 5 ') -P, 3' -dideoxy-P- (dimethylamino) -2 ', 3' -imino-2 ', 3' -secothymidine- (2 'a → 5') -P, 3 ' -dideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 ' -secothymidine acyl- (2 ' a → 5 ') -P, 2 ', 3 ' -trideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 ' -secocytidine acyl- (2 ' a → 5 ') -P, 3 ' -dideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 ' -secothymidine acyl- (2 ' a → 5 ') -P, 2 ', 3 ' -trideoxy-P- (dimethylamino) -2 ', 3 ' -imino-2 ', 3 ' -secoadenylyl- (2 ' a → 5 ') -2 ', 3 ' -dideoxy-2 ', 3 ' -imino-2 ', 3 ' -ring-opened guanosine.

Eteplirsen has the following structure:

structural formula (I)

"amino acid subunit" or "amino acid residue" may mean an alpha-amino acid residue (-CO-CHR-NH-) or a beta-or other amino acid residue (e.g. -CO- (CH)₂)_nCHR-NH-), wherein R is the side chain (which may include hydrogen) and n is 1 to 6, preferably 1 to 4.

The term "naturally occurring amino acid" refers to an amino acid that occurs in a protein found in nature. The term "unnatural amino acid" refers to those amino acids not found in proteins found in nature, examples include: beta-alanine (. beta. -Ala), 6-aminocaproic acid (Ahx) and 6-aminopentanoic acid.

"exon" means a defined portion of a nucleic acid encoding a protein, or a nucleic acid sequence that is present in the mature form of an RNA molecule after any portion of the pre-processed (or precursor) RNA has been removed by splicing. The mature RNA molecule can be messenger RNA (mrna) or a functional form of non-coding RNA, such as rRNA or tRNA. The human dystrophin gene has about 79 exons.

"Intron" means a region of nucleic acid (within a gene) that is not translated into a protein. Introns are non-coding portions that are transcribed into precursor mRNA (pre-mRNA) and subsequently removed by splicing in the formation of mature RNA.

An "effective amount" or "therapeutically effective amount" means an amount of a therapeutic compound (such as an antisense oligonucleotide) administered to a human subject as a single dose or as part of a series of doses effective to produce a desired therapeutic effect. For antisense oligonucleotides, this effect is typically achieved by inhibiting translation or native splicing processing of the selected target sequence. In certain embodiments, an effective amount is at least 20mg/kg of a composition comprising an antisense oligonucleotide for a period of time to treat a subject. In one embodiment, an effective amount is at least 20mg/kg of a composition comprising an antisense oligonucleotide to increase the number of dystrophin positive fibers in a subject to at least 20% of normal. In another embodiment, an effective amount is at least 20mg/kg of a composition comprising an antisense oligonucleotide to stabilize, maintain or improve the distance walked by a patient relative to a 20% shortage (e.g., in 6MWT) of healthy equivalents. In another embodiment, an effective amount is at least 20mg/kg to about 30mg/kg, about 25mg/kg to about 30mg/kg, or about 30mg/kg to about 50 mg/kg. In another embodiment, the effective amount is about 30mg/kg or about 50 mg/kg. In another aspect, the effective amount is at least 20mg/kg, about 25mg/kg, about 30mg/kg, or about 30mg/kg to about 50mg/kg for at least 24 weeks, at least 36 weeks, or at least 48 weeks, thereby increasing the number of dystrophin positive fibers in the subject to at least 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of normal, and stabilizing or improving the distance walked by the patient relative to a 20% shortage of healthy equivalents (e.g., in 6 MWT). In one embodiment, the treatment increases the number of dystrophin positive fibers to 20-60% or 30-50% of normal in the patient.

"exon skipping" generally refers to the process of: by this process, the entire exon or a portion thereof is removed from a given pre-processed RNA and thereby excluded from being present in mature RNA (such as mature mRNA translated into protein). Thus, the portion of the protein otherwise encoded by the skipped exon is not present in the expressed form of the protein, usually resulting in an altered but still functional form of the protein. In certain embodiments, the skipped exon is an aberrant exon from the human dystrophin gene, which may contain mutations or other alterations in its sequence that would otherwise cause aberrant splicing. In certain embodiments, the exon that is skipped is any one or more of exons 1-79, such as 3-8, 10-16, 19-40, 42-47, and 50-55, of the human dystrophin gene, although exons 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, and 8 of the human dystrophin gene are preferred.

"dystrophin" is a rod-like cytoplasmic protein and is an important part of the protein complex that links the cytoskeleton of muscle fibers to the surrounding extracellular matrix through the cell membrane. Dystrophin contains multiple functional domains. For example, dystrophin contains an actin binding domain at about amino acids 14-240 and a central rod domain at about amino acids 253-3040. The large central domain is formed by 24 spectrin-like triple helical elements of about 109 amino acids, which share homology with alpha-actinin and spectrin. These repetitive sequences are usually interrupted by four proline-rich non-repetitive segments (also called hinge regions). The repeats 15 and 16 are separated by a 18 amino acid segment which appears to provide the primary site for proteolytic cleavage of dystrophin. The sequence identity between most of the repeated sequences is in the range of 10-25%. One repeat contains three alpha helices: 1.2 and 3. Alpha helices 1 and 3 are each formed by 7 helical turns, which may interact through hydrophobic interfaces as coiled coils. Alpha helix 2 has a more complex structure and is formed from segments of four and three helical turns separated by glycine or proline residues. Each repeat is encoded by two exons, which are typically interrupted by an intron between amino acids 47 and 48 in the first part of alpha helix 2. Other introns are found elsewhere in the repetitive sequence, usually dispersed within helix 3. Dystrophin also contains a cysteine rich domain at about amino acids 3080-3360, including a cysteine rich segment (i.e., 15 cysteines in 280 amino acids) that shows homology to the C-terminal domain of alpha-actinin from myxobacteria (Dictyostelium discoideum). The carboxy-terminal domain is at about amino acids 3361-3685.

The amino terminus of dystrophin binds to F-actin and the carboxy terminus binds to the dystrophin-related protein complex (DAPC) on the sarcolemma. DAPC includes dystrophic glycans, myoglycans, integrins and caveolin, and mutations within any of these components can cause autosomal inherited muscular dystrophy. DAPC is unstable in the absence of dystrophin, which results in decreased membrane protein levels, and in turn, progressive fiber damage and membrane leakage. In various 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 produce dystrophin at all, primarily due to mutations in the gene sequences that result in incorrect splicing. As mentioned above, the predominant expression of defective dystrophins, or the complete absence of dystrophin or dystrophin-like protein, results in a rapid progression of muscle degeneration. In this regard, a "defective" dystrophin may be characterized by the form of dystrophin produced in certain subjects with DMD or BMD as is known in the art, or by the absence of detectable dystrophin.

The terms "function" and "functional" and the like as used herein mean a biological function, an enzymatic function, or a therapeutic function.

"functional" dystrophin generally means a dystrophin: it typically has sufficient biological activity to reduce the progressive degradation of muscle tissue, which is otherwise characteristic of muscular dystrophy, compared to the altered or "defective" form of dystrophin present in certain subjects 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 vitro or in vivo biological activity of a wild-type dystrophin, as measured according to conventional techniques in the art. As an example, dystrophin-related activity in muscle culture in vitro can be measured in terms of myotube size, myofibrillar organization (or breakdown), 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 the pathogenesis of diseases and provide a means to test the activity associated with dystrophin. Two of the animal models most widely used in the DMD study are mdx mice and Golden Retriever Muscular Dystrophy (GRMD) dogs, both of which are dystrophin negative (see, e.g., Collins & Morgan, Int J Exp Pathol 84: 165-. 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 invention.

The term "recovery" of dystrophin synthesis or production generally refers to the production of dystrophin (including truncated forms of dystrophin) in a patient having muscular dystrophy after treatment with an antisense oligonucleotide as described herein. In certain embodiments, the treatment results in an increase in production of the novel dystrophin protein in the patient of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (including all integers in between). In certain embodiments, the treatment increases the number of dystrophin positive fibers to at least 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% to 100% of normal in the subject. In other embodiments, the treatment increases the number of dystrophin positive fibers to about 20% to about 60%, or about 30% to about 50% of normal in the subject. The percentage of dystrophin positive fibres in a patient after treatment can be determined by muscle biopsy using known techniques. For example, a muscle biopsy may be taken from a suitable muscle, such as the biceps brachii muscle in a patient.

The percentage analysis of positive dystrophin fibres may be performed before treatment and/or at time points after treatment or during treatment. In certain embodiments, the post-treatment biopsy is taken from the contralateral muscle of the pre-treatment biopsy. Both pre-treatment and post-treatment dystrophin expression studies can be performed using any suitable dystrophin assay. In one embodiment, immunohistochemical detection is performed on tissue sections obtained from muscle biopsies using antibodies (which are markers of dystrophin), such as monoclonal or polyclonal antibodies. For example, the MANDYS106 antibody, which is a very sensitive marker of dystrophin, can be used. Any suitable secondary antibody may be used.

In certain embodiments, the percent dystrophin positive fibers is calculated by dividing the number of positive fibers by the total fibers counted. Normal muscle samples have 100% dystrophin positive fibers. Thus, the percent dystrophin positive fiber can be expressed as a percentage of normal. To control the presence of trace levels of dystrophin in the pre-treatment muscles as well as in the revertant (revertant) fibers, a baseline can be set using pre-treatment muscle sections from each patient when counting dystrophin positive fibers in the post-treatment muscles. This can be used as a threshold for counting dystrophin positive fibers in the patient's post-treatment muscle sections. In other embodiments, antibody stained tissue sections may also be used for dystrophin quantification using Bioquant Image Analysis software (Bioquant Image Analysis Corporation, Nashville, TN). Total dystrophin fluorescence signal intensity may be reported as a percentage of normal. Additionally, western blot analysis using monoclonal or polyclonal anti-dystrophin antibodies may be used to determine the percentage of dystrophin positive fibers. For example, an anti-dystrophin antibody, NCL-Dys1, from Novacastra may be used. The percentage of dystrophin positive fibers can also be analyzed by determining the expression of the components of the sarcoglycan complex (β, γ) and/or neuronal NOS.

In certain embodiments, treatment with an antisense oligonucleotide of the invention (such as eteplirsen) slows or reduces progressive respiratory muscle dysfunction and/or failure in patients with DMD that may be expected without treatment. In one embodiment, treatment with the antisense oligonucleotides of the invention can reduce or eliminate the need for ventilatory assist devices that may be foreseen in the absence of treatment. In one embodiment, the measurements of respiratory function and the evaluation of potential therapeutic interventions to track disease progression include Maximum Inspiratory Pressure (MIP), Maximum Expiratory Pressure (MEP), and Forced Vital Capacity (FVC). MIPs and MEPs measure the level of pressure that a person can develop during inspiration and expiration, respectively, and are sensitive measures of respiratory muscle strength. MIP is a measure of diaphragm muscle weakness.

In one embodiment, MEP may decline prior to changes in other lung function tests (including MIP and FVC). In another embodiment, MEP may be an early indication of respiratory dysfunction. In another embodiment, the FVC may be used to measure the total volume of air expelled during forced expiration after maximum inspiration. In patients with DMD, FVC increases with physical growth until early decade. However, as growth slows or is hindered by disease progression and muscle weakness develops, lung capacity enters the decline phase and declines at an average rate of about 8-8.5% per year after the age of 10-12 years. In another embodiment, the predicted MIP% (for weight adjusted MIP), the predicted MEP% (for age adjusted MEP) and the predicted FVC% (for age and height adjusted FVC) are supportive analyses.

By "isolated" is meant a material that: it is substantially or essentially free of components that normally accompany it in its natural state. For example, an "isolated polynucleotide" as used herein may refer to a polynucleotide that: which has been purified or removed from the sequences which flank it in its naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment.

As used herein, "sufficient length" refers to an antisense oligonucleotide that: which is complementary to at least 8, more typically 8-30 consecutive nucleobases in the target dystrophin pre-mRNA. In certain embodiments, a sufficient length of antisense oligonucleotide comprises at least 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleobases in a target dystrophin pre-mRNA. In other embodiments, sufficient length of the antisense oligonucleotide comprises at least 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleobases in the target dystrophin pre-mRNA. An antisense oligonucleotide of sufficient length has at least the minimum number of nucleotides that can specifically hybridize to any one or more of exons 1-79 of the dystrophin gene. Preferably, the antisense oligonucleotides of the invention have a minimum number of nucleotides capable of specifically hybridizing to any one or more of exons 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 8 of a human dystrophin gene. Preferably, a sufficient length of oligonucleotide is about 10 to about 50 nucleotides in length, including 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, and 40 or more nucleotides in length. In one embodiment, a sufficient length of oligonucleotide is 10 to about 30 nucleotides in length. In another embodiment, a sufficient length of the oligonucleotide is 15 to about 25 nucleotides in length. In another embodiment, a sufficient length of oligonucleotides is 20-30 or 20-50 nucleotides in length. In another embodiment, oligonucleotides of sufficient length are 25-28 nucleotides in length.

"enhance" or "increase" or "stimulate" generally refers to the ability of one or more antisense compounds or compositions to produce or cause a greater physiological response (i.e., downstream effect) in a cell or subject as compared to the response caused by the absence of the antisense compound or control compound. In addition to other responses apparent from the understanding in the art and the description herein, a measurable physiological response may include an increase in expression of a functional form of dystrophin, or an increase in a dystrophin-related biological activity in muscle tissue. 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 that express functional dystrophin can also be measured, including increased expression of dystrophin in about 1%, 2%, percent, 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 demonstrated that about 40% improvement in muscle function can occur if 25-30% of the fibers express dystrophin (see, e.g., DelloRusso et al, Proc Natl Acad Sci USA 99: 12979-. An "increased" or "enhanced" amount is typically a "statistically significant" amount, and may include such an increase: it is 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 in between and greater than 1) the amount produced without the antisense compound (lacking the agent) or the control compound, e.g., 1.5, 1.6, 1.7, 1.8, etc.

The term "reduce" or "inhibit" may generally relate to the ability of one or more antisense compounds of the invention to "reduce" an associated physiological or cellular response (such as a symptom of a disease or disorder described herein), as measured according to conventional techniques in the diagnostic art. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction in the symptoms or condition of muscular dystrophy, or a reduction in the expression of defective forms of dystrophin, such as altered forms of dystrophin expressed in individuals with DMD or BMD. A "reduction" in response compared to a response generated without an antisense compound or control composition may be statistically significant and may include a reduction of 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%, including all integers in between.

Also included are vector delivery systems capable of expressing the oligodystrophin targeting sequences of the present invention, such as vectors expressing polynucleotide sequences comprising any one or more of the sequences shown in tables 3 and 4, and variants thereof, as described herein. "vector" or "nucleic acid construct" refers to a polynucleotide molecule, preferably a DNA molecule, derived from, for example, a plasmid, bacteriophage, yeast or virus into which a polynucleotide may be inserted or cloned. The vector preferably contains one or more unique restriction sites, and may be capable of autonomous replication in a defined host cell (including a target cell or tissue or progenitor thereof) or integration with the genome of a defined host such that the cloned sequence is reproducible. Thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

"treatment" of an individual (e.g., a mammal, such as a human) or cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and can be effected prophylactically, or after a pathological event has begun or has been exposed to a pathogen. Treatment includes any desirable effect on the symptoms or pathology of a dystrophin-associated disease or disorder (as in some forms of muscular dystrophy), and may include, for example, minimal change or improvement in one or more measurable markers of the disease or disorder being treated. Also included are "prophylactic" treatments, which can be intended to reduce the rate of progression of the disease or disorder being treated, delay the onset of the disease or disorder, or reduce the severity of its onset. "treating" or "prevention" does not necessarily indicate completely eradicating, curing, or preventing the disease or disorder or symptoms associated therewith.

In one embodiment, treatment with the antisense oligonucleotides of the invention increases novel dystrophin production and slows or reduces the loss of bed exit activity that would be expected in the absence of treatment. For example, treatment may stabilize, maintain, improve, or increase the walking ability of the subject (e.g., stabilization of out-of-bed activity). In certain embodiments, treatment maintains or increases the stable walking distance of the patient, as by, for example, McDonald, et al (Muscle Nerve, 2010; 42: 966-74, incorporated by reference)Incorporated herein) was measured by the 6 minute walk test (6 MWT). The change in 6 minute walk distance (6MWD) can be expressed as an absolute value, a percent change, or a percent change predicted value. In certain embodiments, the treatment maintains or improves a 20% shortage of stable walking distance in 6MWT in subjects compared to healthy equivalents. By calculating the predicted value percentage, the performance of DMD patients in 6MWT compared to the typical performance of healthy equivalents can be determined. For example, for males, the predicted 6MWD percentage may be calculated using the following equation: 196.72+ (39.81x age) - (1.36x age)²) + (132.28x height in meters). For females, the predicted 6MWD percentage can be calculated using the following equation: 188.61+ (51.50x age) - (1.86x age)²) + (86.10x height in meters) (Henricson et al PLoS Curr., 2012, 2 nd edition, incorporated herein by reference). In certain embodiments, treatment with antisense oligonucleotides increases the stable walking distance of the patient from baseline to greater than 3, 5,6, 7,8, 9, 10, 15, 20, 25, 30, or 50 meters (including all integers in between).

Loss of muscle function in patients with DMD may occur in the context of normal child growth and development. Indeed, younger children with DMD may exhibit an increase in distance walked in 6MWT over the course of about 1 year, despite the presence of progressive muscle lesions. In certain embodiments, the 6MWD from patients with DMD is compared to a control subject that is normally developing and to existing standard data from age and gender matched subjects. In certain embodiments, normal growth and development may be explained using an age and height based equation fitted to standard data. Such an equation can be used to convert the 6MWD to a percentage of the predicted value in subjects with DMD. In certain embodiments, analysis of the predicted 6MWD percentage data represents a method to understand normal growth and development, and may indicate that an increase in function at an early stage (e.g., less than or equal to 7 years) represents a stable ability, rather than an improved ability, in patients with DMD (Henricson et al PLoS curr., 2012, 2 nd edition, incorporated herein by reference).

As used herein, "subject" includes any animal exhibiting symptoms or at risk of exhibiting symptoms that can be treated with an antisense compound of the invention, e.g., a subject that: it has or is at risk of having DMD or BMD or any symptom associated with these conditions (e.g., loss of muscle fibers). Suitable subjects (patients) include laboratory animals (such as mice, rats, rabbits or guinea pigs), domestic animals and domestic animals or pets (such as cats or dogs). Including non-human primates, and preferably human patients.

As used herein, a "pediatric patient" is a patient that is 1-21 years old (including endpoints).

Both "alkyl" and "alkylene" represent saturated straight or branched chain hydrocarbon groups containing from 1 to 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" denotes an alkyl group as defined herein containing from 1 to 8 carbons.

"alkenyl" means an unsaturated straight or branched chain hydrocarbon group containing 2 to 18 carbons and containing 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" denotes an alkenyl group as defined herein containing 2 to 8 carbons.

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

"cycloalkyl" means a monocyclic or multicyclic alkyl residue. Examples include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

"aryl" means a cyclic aromatic moiety containing-18 carbons and having one or more closed rings. Examples include, but are not limited to, phenyl, benzyl, naphthyl, anthryl, phenanthryl, and biphenyl.

"aralkyl" denotes a residue of formula RaRb, wherein Ra is an alkylene chain as defined above and Rb is one or more aryl residues as defined above, e.g., benzyl, diphenylmethyl, and the like.

"Thioalkoxy" means a residue of the formula-SRc, wherein Rc is an alkyl residue as defined herein. The term "lower thioalkoxy" denotes an alkoxy group as defined herein containing 1 to 8 carbons.

"alkoxy" means a residue of the formula-ORda, wherein Rd is an alkyl residue as defined herein. The term "lower alkoxy" denotes an alkoxy group as defined herein containing 1 to 8 carbons. Examples of alkoxy groups include, but are not limited to, methoxy and ethoxy.

"alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group.

"carbonyl" represents a C (═ O) -residue.

"guanidino (Guanidynyl)" means H₂N(C＝NH₂) -an NH-residue.

"Amidinyl" means H₂N(C＝NH₂) A CH-residue.

"amino" means NH₂And (c) a residue.

"alkylamino" denotes a residue of the formula-NHRd or-NRdRd, wherein each Rd is independently an alkyl residue as defined herein. The term "lower alkylamino" denotes alkylamino as defined herein containing 1 to 8 carbons.

"heterocycle" means a 5-7 membered monocyclic or 7-10 membered bicyclic heterocycle which is saturated, unsaturated or aromatic, and which contains 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, including bicyclic heterocycles in which any of the above heterocycles are fused to a benzene ring. The heterocyclic ring may be attached through any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Thus, in addition to heteroaryl groups listed below, heterocyclic rings also include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactonyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiopyranyl, and the like.

"heteroaryl" refers to 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, furyl, benzofuryl, thienyl, benzothienyl, quinolyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl and quinazolinyl.

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 oxo substituent (═ O), both hydrogen atoms are replaced. In this regard, substituents include: deuterium, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted cycloalkyl, oxo, halogen, -CN, -ORx, 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 are independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle or optionally substituted cycloalkyl, and said optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, Each of the optionally substituted heterocyclic ring and the optionally substituted cycloalkyl substituents may be further substituted by one or more of oxo, halogen, -CN, -ORx, NRxRy, NRxC (═ O) Ry, NRxSO2Ry, -NRxC (═ O) NRxRy, C (═ O) Rx, C (═ O) ORx, C (═ O) NRxRy, -SOmRx and-SOmNRxRy.

An antisense molecule naming system is proposed and disclosed to distinguish between different antisense molecules (see Mann et al, (2002) J Gen Med 4, 644-654). This nomenclature becomes particularly relevant when several slightly different antisense molecules are tested (all directed to the same target region, as shown below):

H#A/D(x：y)。

the first letter indicates the species (e.g., H: human, M: murine, C: canine). "#" indicates the target dystrophin exon numbering. "A/D" indicates acceptor or donor splice sites at the beginning and end of an exon, respectively. (x y) represents an annealing counterpart, wherein "-" or "+" indicates an intron or exon sequence, respectively. For example, A (-6+18) indicates the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. The closest splice sites are the acceptors that lead to these counterparts having an "a" in front. The annealing counterpart depicted at the donor splice site is D (+2-18), where the last 2 exon bases and the first 18 intron bases correspond to the annealing site of the antisense molecule. The annealing counterpart of a full exon is denoted by A (+65+85), which is the site between the 65 th and 85 th nucleotides from the start of the exon.

II.Construction of antisense oligonucleotides

Exemplary embodiments of the invention relate to morpholino oligonucleotides having a phosphorus-containing backbone linkage, as shown in figures 1A-1C. Preferred are phosphorodiamidate-linked morpholino oligonucleotides, such as shown in figure 1C, modified to contain a positively charged group, preferably 10% to 50% of its backbone linkages, according to one aspect of the invention. Morpholino oligonucleotides (including antisense oligonucleotides) having uncharged backbone linkages are described in detail, for example (Summerton and Weller 1997) and in commonly owned U.S. patent nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185, 444, 5,521,063, 5,506,337, 8,076,476, 8,299,206 and 7,943,762, all of which are expressly incorporated herein by reference.

Important properties of morpholino-based subunits include: 1) the ability to be linked in oligomeric form by stable, uncharged or positively charged backbone linkages; 2) the ability to support nucleotide bases (e.g., adenine, cytosine, guanine, thymidine, uracil, and inosine) such that the formed polymer can hybridize to a complementary base target nucleic acid (including a target RNA), a Tm value above about 45 ℃ in relatively short oligonucleotides (e.g., 10-15 bases); 3) the ability of the oligonucleotide to be actively or passively transported into a mammalian cell; and 4) antisense oligonucleotides: RNA heteroduplexes are resistant to degradation by rnases and rnase H, respectively.

Exemplary backbone structures of antisense oligonucleotides of the claimed subject matter include the morpholino subunit types shown in figures 1D-G, each linked by an uncharged or positively charged, phosphorus-containing subunit linkage. FIG. 1D shows phosphorus-containing linkages forming a five atom repeat unit backbone in which morpholino rings are linked by a 1-atom phosphorus amide linkage. FIG. 1E shows the bonds that result in a 6 atom repeat unit backbone. In this structure, the atom Y connecting the 5' morpholino carbon to the phosphorus group can be sulfur, nitrogen, carbon, or preferably oxygen. The moiety X extending from the phosphorus may be fluorine, alkyl or substituted alkyl, alkoxy or substituted alkoxy, thioalkoxy or substituted thioalkoxy, or unsubstituted, mono-or di-substituted nitrogen, including cyclic structures such as morpholine or piperidine. The alkyl, alkoxy and thioalkoxy groups preferably comprise 1 to 6 carbon atoms. The moiety Z is sulfur or oxygen, preferably oxygen.

The bonds shown in FIGS. 1F and 1G are 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 the morpholino subunit structure of the form shown in figure 1E, wherein X ═ NH₂、N(CH₃)₂Or 1-piperazine or other charged group, Y ═ O, and Z ═ O.

According to one aspect of the invention, substantially uncharged oligonucleotides can be modified to include charged bonds, e.g., up to about 1 charged bond per 2-5 uncharged bonds, such as about 4-5 charged bonds per 10 uncharged bonds. In certain embodiments, the best improvement in antisense activity may be observed when about 25% of the backbone linkages are cationic. In certain embodiments, enhancement may be observed with a small number, e.g., 10-20%, of cationic bonds, or where the number of cationic bonds is in the range of 50-80%, such as about 60%.

Oligomers having any number of cationic linkages, including all cationically linked oligomers, are provided. Preferably, however, the oligomer is partially charged, having for example 10% -80%. In a preferred D embodiment, about 10% to 60% and preferably 20% to 50% of the linkages are cationic.

In one embodiment, the cationic bonds are interspersed along the backbone. The partially charged oligomer preferably contains at least two consecutive uncharged bonds; that is, the oligomer preferably does not have a strictly alternating pattern along its entire length.

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

In certain embodiments, antisense compounds can be prepared by stepwise solid phase synthesis using methods detailed in the references cited above and below for the synthesis of oligonucleotides having mixtures or uncharged and cationic backbone linkages. In certain instances, it may be desirable to add additional chemical moieties to the antisense compound, for example, to enhance pharmacokinetics or to facilitate capture or detection of the compound. These moieties may be covalently linked according to standard synthetic methods. For example, the addition of polyethylene glycol moieties or other hydrophilic polymers, e.g., hydrophilic polymers having 1-100 monomeric subunits, may be used to enhance solubility.

For detection purposes, a reporter moiety, such as fluorescein or a radiolabeled group, may be attached. Alternatively, the reporter label attached to the oligomer may be an antibody or streptavidin-binding ligand, such as an antigen or biotin, capable of binding the label. In selecting a moiety for attaching or modifying an antisense compound, it is, of course, often desirable to select a compound that is biocompatible and that is likely to be tolerated by a subject without undesirable side effects.

Oligomers for antisense applications typically range in length from about 10 to about 50 subunits, more preferably about 10-30 subunits, and typically 15-25 bases. For example, oligomers of the invention having 19-20 subunits (useful lengths for antisense compounds) may desirably have 2-10 (e.g., 4-8) cationic linkages, with the remainder being uncharged linkages. Oligomers having 14-15 subunits may desirably have 2-7 (e.g., 3,4, or 5) cationic linkages, with uncharged linkages remaining. In a preferred embodiment, the oligomer has 25-28 subunits.

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

As noted above, certain embodiments relate to oligomers comprising novel intersubunit linkages, including PMO-X oligomers and those having modified terminal groups. In certain embodiments, these oligomers have higher affinity for DNA and RNA than the corresponding unmodified oligomers and exhibit improved cell delivery, potency, and/or tissue distribution properties compared to oligomers having other intersubunit linkages. The structural features and properties of the various bond types and oligomers are described in more detail in the discussion that follows. The synthesis of these and related oligomers is described in commonly owned U.S. application No. 13/118,298, which is incorporated by reference in its entirety.

In certain embodiments, the invention provides oligonucleotides having a sequence complementary to a target sequence associated with a human disease and comprising a nucleotide sequence having the formula:

wherein Nu is a nucleobase;

R₁having the formula

q is 0, 1 or 2;

R₂selected from hydrogen, C₁-C₅Alkyl radical, C₁-C₅Aralkyl and formamidine groups, and

R₃selected from hydrogen, C₁-C₁₀Acyl radical, C₁-C₁₀Aminoacyl, acyl moiety of a natural or unnatural alpha or beta amino acid, C₁-C₁₀Aralkyl and C₁-C₁₀Alkyl, or

R₂And R₃Are linked to form a 5-7 membered ring, wherein said ring may optionally be selected from C₁-C₁₀Alkyl, phenyl, halogen and C₁-C₁₀Aralkyl substituent;

R₄selected from the group consisting of electron pairs, hydrogen, C₁-C₆Alkyl and C₁-C₆Aralkyl group;

rx is selected from the group consisting of sarcosine, hydroxyl, nucleotide, cell penetrating peptide moiety and piperazinyl;

ry is selected from hydrogen and C₁-C₆Alkyl, nucleotide, cell penetrating peptide moiety, amino acid,Amidino and C₁-C₆An acyl group; and is

Rz is selected from the group consisting of electron pair, hydrogen, C₁-C₆Alkyl and C₁-C₆Acyl groups and pharmaceutically acceptable salts thereof.

Nu may be selected from adenine, guanine, thymine, uracil, cytosine and hypoxanthine. More preferably Nu is thymine or uracil.

In a preferred embodiment, the present invention provides an oligonucleotide comprising a nucleotide sequence having the formula:

wherein Nu is a nucleobase;

R₁is selected from R₁' and R₁", wherein R₁' is dimethyl-amino, and R₁"having the formula

Wherein at least one R₁Is R₁”；

q is 0, 1 or 2; with the proviso that at least one R₁Is a piperidinyl moiety;

R₂selected from hydrogen, C₁-C₅Alkyl radical, C₁-C₅Aralkyl and formamidine groups, and

R₃selected from hydrogen, C₁-C₁₀Acyl radical, C₁-C₁₀Aminoacyl, acyl moiety of a natural or unnatural alpha or beta amino acid, C₁-C₁₀Aralkyl and C₁-C₁₀Alkyl, or

R₂And R₃Are linked to form a 5-7 membered ring, wherein said ring may optionally be selected from C₁-C₁₀Alkyl, phenyl, halogen and C₁-C₁₀Aralkyl substituent;

R₄selected from the group consisting of electron pairs, hydrogen, C₁-C₆Alkyl and aralkyl groups;

rx is selected from the group consisting of sarcosine, hydroxyl, nucleotide, cell penetrating peptide moiety and piperazinyl;

ry is selected from hydrogen and C₁-C₆Alkyl, nucleotide, cell penetrating peptide moiety, amino acid, carbamimidoyl and C₁-C₆An acyl group; and,

Rz is selected from the group consisting of electron pair, hydrogen, C₁-C₆Alkyl and C₁-C₆Acyl groups and pharmaceutically acceptable salts thereof.

Nu may be selected from adenine, guanine, thymine, uracil, cytosine and hypoxanthine. More preferably Nu is thymine or uracil.

About 90-50% R₁The radical being dimethylamino (i.e. R)₁'). More preferably, 90-50% of R₁The radical is dimethylamino. Most preferably, about 66% R₁The radical is dimethylamino.

R₁"may be selected from

Preferably, at least one nucleotide of the oligonucleotide has the formula:

wherein Rx, Ry, Rz and Nu are as described above. Most preferably, Nu is thymine or uracil.

Although thymine (T) is the preferred base-pairing moiety (Nu or Pi) containing the above-described chemical modification, any base subunit known to those skilled in the art may be used as the base-pairing moiety.

The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond to each other. Thus, "specifically hybridizable" and "complementary" are terms such as: which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It will be understood in the art that the sequence of an antisense molecule need not be 100% complementary to the sequence of its target sequence to be specifically hybridizable. Antisense molecules are specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired (i.e., under physiological conditions for in vivo assays or therapeutic treatments, and under conditions in which assays are performed for in vitro assays).

Although the above method can be used to select antisense molecules that can delete any exon from within a protein (which can be shortened without affecting its biological function), the exon deletion should not result in a frame shift in the shortened transcribed mRNA. Thus, if in a linear sequence of three exons the end of the first exon encodes two of the three nucleotides in a codon and the next exon is deleted, then the third exon in the linear sequence must start with a single nucleotide that is capable of completing a nucleotide triplet of a codon. If the third exon does not start with a single nucleotide, there will be a frame shift that results in the production of a truncated or non-functional protein.

It will be appreciated that the codon arrangement at the end of an exon of a structural protein may not always be broken at the end of a codon, so more than one exon may need to be deleted from a pre-mRNA to ensure in-frame reading of the mRNA. In such cases, it may be desirable to select, by the methods of the invention, a plurality of antisense oligonucleotides, each directed against a different region responsible for inducing splicing in the exon to be deleted.

The length of the antisense molecule can vary so long as it is capable of selectively binding to the desired location within the pre-mRNA molecule. The length of such sequences may be determined according to the selection procedure described herein. Typically, the antisense molecule will be about 10 nucleotides in length up to about 50 nucleotides in length. However, it will be appreciated that any length of nucleotides within this range may be used in the method. Preferably, the antisense molecule is between 10-30 nucleotides in length.

The most common method of generating antisense molecules is methylation of the 2' hydroxyl ribose position, and incorporation of a phosphorothioate backbone will result in such a molecule: its surface is similar to RNA, but its resistance to nuclease degradation is much higher.

To avoid degradation of the pre-mRNA during duplex formation with the antisense molecule, the antisense molecule used in the method can be engineered to minimize or prevent cleavage by endogenous RNase H. This property is highly preferred because treatment of RNA with unmethylated oligonucleotides (either in cells or in crude extracts containing RNase H) results in pre-mRNA: degradation of the antisense oligonucleotide duplex. Any form of modified antisense molecule that is capable of bypassing or not inducing such degradation may be used in the methods of the invention. An example of an antisense molecule that is not cleaved by cellular rnase H when forming a duplex with RNA is the 2' -O-methyl derivative. 2' -O-methyl-oligoribonucleotides are very stable in the cellular environment and in animal tissues and their duplexes with RNA have a higher Tm than their ribose-or deoxyribose-counterparts.

Antisense molecules that do not activate rnase H can be prepared according to known techniques (see, e.g., U.S. patent No. 5,149,797). Such antisense molecules, which may be deoxyribonucleotide or ribonucleotide sequences, contain only any structural modification that sterically hinders or prevents rnase H binding to a duplex molecule containing an oligonucleotide as one member thereof, which structural modification does not substantially hinder or disrupt duplex formation. Since the portions of the oligonucleotides involved in duplex formation are substantially different from those involved in binding of rnase H thereto, numerous antisense molecules are available that do not activate rnase H. For example, such antisense molecules can be oligonucleotides, at least one of which isSome internucleotide bridging phosphate residues are modified phosphates such as methylphosphonate, methylphosphonothioate, phosphomorpholinolate, phosphopiperazinate and phosphoramidate. For example, every other internucleotide bridging phosphate residue can be modified as described. In another non-limiting embodiment, such antisense molecules are those in which at least one or all of the nucleotides contain a 2' lower alkyl moiety (e.g., C)₁-C₄Linear or branched, saturated or unsaturated alkyl groups such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl and isopropyl). For example, every other nucleotide may be modified as described.

Although antisense oligonucleotides are a preferred form of antisense molecule, the invention includes other oligomeric antisense molecules, including, but not limited to, oligonucleotide mimetics, such as those described below.

Specific examples of preferred antisense compounds useful in the invention include oligonucleotides containing modified backbones or unnatural internucleoside linkages. As defined in the specification, oligonucleotides with modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone may also be considered oligonucleosides.

In other preferred oligonucleotide mimetics, the sugar and internucleoside linkages (i.e., the backbone) of the nucleotide units are replaced by novel groups. The base units are maintained for hybridization with the appropriate nucleic acid target compounds. One such oligomeric compound, an oligonucleotide mimetic that has been demonstrated to have excellent hybridization properties, is known as Peptide Nucleic Acid (PNA). In PNA compounds, the sugar-backbone of the oligonucleotide is replaced by an amide-containing backbone (in particular an aminoethylglycine backbone). The core-base is retained and directly or indirectly bonded to the aza nitrogen atom of the amide portion of the backbone.

The modified oligonucleotide may also contain one or more substituted sugar moieties. Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. Certain nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methyl cytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ℃ and are the presently preferred base substitutions, even more particularly when combined with 2' -O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the invention involves chemically linking the oligonucleotide with one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to: lipid moieties such as cholesterol moieties, cholic acids, thioethers, e.g. hexyl-5-tritylthiol, thiocholesterol, aliphatic chains, e.g. dodecanediol or undecyl residues, phospholipids, e.g. bis-hexadecyl-rac-glycerol or triethylammonium 1, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate, polyamine or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-hydroxycholesterol moieties.

All positions in a given compound are not necessarily uniformly modified, and indeed more than one of the foregoing modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The invention also includes antisense compounds that are chimeric compounds. In the context of the present invention, a "chimeric" antisense compound or "chimera" is an antisense molecule, in particular an oligonucleotide, which contains 2 or more chemically distinct regions, each region being composed of at least one monomeric unit (i.e. nucleotide in the case of oligonucleotide compounds). These oligonucleotides typically contain at least one region, wherein the oligonucleotide is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and another region for increased binding affinity to the target nucleic acid.

III.Peptide transporter

The antisense compounds of the invention may include an oligonucleotide moiety conjugated to a CPP, preferably an arginine-rich peptide trafficking moiety effective to enhance transport of the compound into a cell. The transport moiety is preferably attached to the end of the oligomer as shown, for example, in fig. 1B and 1C. The peptides have the ability to induce cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (including all integers in between) of the cells of a given cell culture population and allow for macromolecular translocation in a variety of in vivo tissues following systemic administration. In one embodiment, the cell penetrating peptide may be an arginine-rich peptide transporter. In another embodiment, the cell penetrating peptide may be a Penetratin (pennetratin) or a Tat peptide. These peptides are well known in the art and are disclosed, for example, in U.S. publication No. 2010-0016215a1, which is incorporated by reference in its entirety. One particularly preferred approach to conjugating peptides to antisense oligonucleotides can be found in PCT publication WO2012/150960, which is incorporated by reference in its entirety. A preferred embodiment of the peptide-conjugated oligonucleotide of the present invention utilizes glycine as a linker between the CPP and the antisense oligonucleotide. For example, one preferred peptide-conjugated PMO is represented by R₆-G-PMO.

It has been demonstrated that a trafficking moiety as described above greatly enhances cellular entry of the ligated oligomer compared to the uptake of the oligomer in the absence of the ligated trafficking moiety. Uptake is preferably enhanced at least 10-fold, and more preferably 20-fold, compared to the unconjugated compound.

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 into primary cells, including muscle cells (Marshall, Oda et al 2007; Jearawiriyapaiisam, Moulton et al 2008; Wu, Moulton et al 2008). Furthermore, the peptide transporters described herein exhibit an enhanced ability to alter the splicing of several gene transcripts when conjugated to antisense PMOs, compared to other known peptide transporters such as the penetratin and Tat peptides (Marshall, Oda et al 2007).

Exemplary peptide transporters, excluding linkers, are given in table 1 below.

TABLE 1 exemplary peptide transporters

Name (nomenclature)	Sequence of	SEQ ID NOA
			rTAT	RRRQRRKKR	876
Tat	RKKRRQRRR	877
			R9F2	RRRRRRRRRFF	878
R5F2R4	RRRRRFFRRRR	879
			R4	RRRR	880
R5	RRRRR	881
			R6	RRRRRR	882
R7	RRRRRRR	883
			R8	RRRRRRRR	884
R9	RRRRRRRRR	885
			(RX)8	RXRXRXRXRXRXRXRX	886
(RAhxR)4；(P007)	RAhxRRAhxRRAhxRRAhxR	887
			(RAhxR)5；(CP04057)	RAhxRRAhxRRAhxRRAhxRRAhxR	888
(RAhxRRBR)2；(CP06062)	RAhxRRBRRAhxRRBR	889
			(RAR)4F2	RARRARRARRARFF	890
(RGR)4F2	RGRRGRRGRRGRFF	891

^AThe sequence assigned to SEQ ID NO does not include a linking moiety (e.g., C, G, P, Ahx, B, AhxB, where Ahx and B represent 6-aminocaproic acid and β -alanine, respectively).

IV.Formulations and treatments

In certain embodiments, the present invention provides formulations and compositions suitable for therapeutic delivery of antisense oligomers as described herein. Thus, in certain embodiments, the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more oligomers described herein formulated with one or more pharmaceutically acceptable carriers (additives) and/or diluents. Although it is possible to administer the oligomers of the invention alone, it is preferred to administer the compounds as pharmaceutical formulations (compositions).

The compositions of the present invention may be administered alone or in combination with another therapeutic agent. The other therapeutic agent may be administered prior to, concurrently with, or subsequent to administration of the composition of the invention. For example, the composition may be administered in combination with a steroid and/or an antibiotic. The steroid may be a glucocorticoid or prednisone. Glucocorticoids such as cortisol control carbohydrate, fat and protein metabolism and are anti-inflammatory by preventing phospholipid release, reducing eosinophil action and many other mechanisms. Mineralocorticoids such as aldosterone control electrolyte and water levels, primarily by promoting sodium retention in the kidney. Corticosteroids are a class of chemicals that include steroid hormones that are naturally produced in the adrenal cortex of vertebrates and analogs of these hormones that are synthesized in the laboratory. Corticosteroids are involved in a wide range of physiological processes including stress response, regulation of immune response and inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels and behaviour. Corticosteroids include betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone.

Other agents that may be administered include antagonists of the ryanodine receptor, such as dantrolene, which has been shown to enhance antisense-mediated exon skipping in patient cells and DMD mouse models (g.kendall et al. Sci Tranl Med 4164 ra160(2012), incorporated herein by reference).

Methods for delivering nucleic acid molecules are described, for example, in Akhtar et al, 1992, Trends Cell bio, 2: 139; and the design of Delivery Strategies for Antisense Oligonucleotide Therapeutics, Akhtar; sullivan et al, PCT WO 94/02595. These and other protocols can be used to deliver essentially any nucleic acid molecule, including the isolated oligomers of the invention.

As detailed below, the pharmaceutical compositions of the present invention may be specifically formulated for administration in solid or liquid form, including those suitable for use in: (1) oral administration, e.g., drench (aqueous or non-aqueous solution or suspension), tablets, e.g., those for buccal, sublingual, and systemic absorption, pills, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous or epidural injection, e.g., as a sterile solution or suspension, or a sustained release formulation; (3) topical application, e.g., as a cream, ointment, or controlled release patch or spray applied to the skin; (4) intravaginally or intrarectally, e.g., as pessaries, creams or foams; (5) lingually; (6) through the eye; (7) transdermally; or (8) nasally.

The phrase "pharmaceutically acceptable" as used herein means a compound, substance, composition, and/or dosage form that: it is suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment.

The phrase "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium or zinc stearate, or stearic acid), or such a solvent encapsulating material: which are involved in transporting or transporting the subject compounds from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

Some examples of materials 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (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) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline water; (18) ringer's solution; (19) ethanol; (20) a pH buffer solution; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible materials employed in pharmaceutical formulations.

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, lipophilic moiety-containing nucleic acids, phosphorothioates, P-glycoprotein inhibitors that can enhance drug entry into various tissues (such as Pluronic P85); biodegradable polymers such as DL-lactide-glycolide copolymer microspheres for sustained release delivery after implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) Alkermes, inc. cambridge, mass; and loaded nanoparticles, such as those made from polybutylcyanoacrylates, that can deliver drugs across the vascular brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-.

The invention also features the use of a composition comprising surface-modified liposomes (PEG-modified, branched and linear or combinations thereof, or long-circulating liposomes or stealth liposomes) containing poly (ethylene glycol) lipids. The oligomers of the invention may also comprise covalently linked PEG molecules of various molecular weights. These formulations would provide a means of increasing drug accumulation in the target tissue. Such drug carriers are resistant to opsonization and clearance by the monocyte macrophage system (MPS or RES), thereby achieving longer blood circulation times and enhanced tissue exposure of the encapsulated drug (Lasic et al chem. Rev.1995, 95, 2601-. Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the target tissues of neovascularization (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 the tissues of MPS (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 liver and spleen), long circulating liposomes may also protect drugs from nuclease degradation to a greater extent than cationic liposomes.

In another embodiment, the invention includes preparing oligomer compositions 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 invention provides an oligomer of the invention in a composition comprising a copolymer of lysine and Histidine (HK), as described in U.S. Pat. nos. 7,163,695, 7,070,807, and 6,692,911, either alone or in combination with PEG (e.g., branched or linear PEG or a mixture of both), in combination with PEG and a targeting moiety, or in combination with any of the foregoing crosslinking agents. In certain embodiments, the present invention provides antisense oligomers in compositions comprising a gluconic acid modified polyhistidine or a gluconylated polyhistidine/transferrin-polylysine. One skilled in the art will also recognize that amino acids having properties similar to His and Lys may be substituted within the composition.

Certain embodiments of the oligomers described herein may contain basic functional groups, such as amino or alkylamino, and thus are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. In this regard, the term "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds of the present invention. These salts may be prepared in situ during the administration vehicle or dosage form preparation, or as follows: separately, the purified compound of the invention, in its free base form, is reacted with a suitable organic or inorganic acid and the salt thus formed is isolated in a subsequent purification process. Representative salts include: hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate, and the like (see, e.g., Berge et al (1977) "Pharmaceutical Salts", J.drug. Sci.66: 1-19).

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

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

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

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

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

In certain embodiments, the formulations of the present invention comprise: an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle-forming agents (e.g., bile acids), and polymeric carriers (e.g., polyesters and polyanhydrides); and oligomers of the invention. In certain embodiments, the aforementioned formulations render the oligomers of the present invention orally bioavailable.

The method of making these formulations or compositions includes the step of combining the oligomer of the present invention with a carrier and optionally one or more adjuvants. In general, the formulations are prepared as follows: the compound of the present invention is uniformly and intimately admixed with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, the product is shaped.

Formulations of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia 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 pastilles (using an inert base such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as the active ingredient. The oligomers of the invention may also be administered as a bolus, electuary or paste.

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

Tablets may be prepared by compression or moulding, optionally together with one or more accessory ingredients. Compressed tablets may be prepared using binders (for example, gelatin or hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrating agents (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agents. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may also be formulated using, for example, hydroxypropylmethyl cellulose in various proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres to provide slow or controlled release of the active ingredient therein. They may be formulated for rapid release, e.g., freeze-drying. They can be sterilized as follows: for example, by filtration through a bacteria-retaining filter, or by inclusion of a sterilizing agent in the form of a sterile solid composition soluble in sterile water, or by inclusion of some other sterile injectable medium immediately prior to use. These compositions may also optionally contain opacifying agents and may be of such a composition to release the active ingredient only, or preferably in certain parts of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be present in microencapsulated form, if appropriate together with one or more of the abovementioned excipients.

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 forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

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

Formulations for rectal or vaginal administration may be presented as suppositories, which may be prepared as follows: one or more compounds of the invention are mixed with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and 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.

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

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

Transdermal patches have the additional advantage of providing controlled delivery of the oligomers of the present invention to the body. Such dosage forms may be prepared by dissolving or dispersing the oligomer in a suitable medium. Absorption enhancers may also be used to increase the flux of the agent through the skin. Such flux rates may be controlled as follows: in addition to other methods known in the art, rate controlling membranes are provided, or the pharmaceutical agent is dispersed in a polymer matrix or gel.

Pharmaceutical compositions suitable for parenteral administration may comprise one or more oligomers of the present invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the effects of microorganisms on the subject oligomers may be ensured by inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved as follows: liquid suspensions of crystalline or amorphous materials with poor water solubility are used, among other methods known in the art. The rate of absorption of the drug then depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are prepared by forming a microcapsule matrix of the subject oligomers in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of oligomer to polymer and the nature of the particular polymer used, the rate of oligomer release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable depot formulations can also be prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

When the oligomers of the invention are administered as medicaments to humans and animals, they may be administered as such or as a pharmaceutical composition containing, for example, 0.1-99% (more preferably, 10-30%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

As indicated above, the formulations or preparations of the invention may be administered orally, parenterally, systemically, topically, rectally or intramuscularly. They are usually administered in a form suitable for each route of administration. For example, they are administered in the form of tablets or capsules by injection, inhalation, eye lotion, ointment, suppository, etc., by injection, infusion, or inhalation; topically applied by lotion or ointment; and rectal administration by suppository.

The phrases "parenteral administration" and "parenterally administered" as used herein refer to modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

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

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

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

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start a dose of a compound of the invention used in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, a suitable daily dose of a compound of the invention is the amount of such compound: which is the lowest dose effective to produce a therapeutic effect. Such effective dosages will generally depend upon the factors described above. Typically, when used to specify an effect, the oral, intravenous, intracerebroventricular, intramuscular, and subcutaneous dosages of the compounds of the invention range from about 0.0001 to about 100mg per kilogram body weight per day for a patient.

The preferred dose of the oligomer of the invention (e.g., phosphorodiamidate morpholino oligomer; eteplirsen) administered is generally about 20-100 mg/kg. In some cases, a dose of greater than 100mg/kg may be necessary. For intravenous administration, the preferred dose is about 0.5mg to 100 mg/kg. In certain embodiments, at about 20mg/kg, 21mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg, 32mg/kg, 33mg/kg, 34mg/kg, 35mg/kg, 36mg/kg, 37mg/kg, 38mg/kg, 39mg/kg, 40mg/kg, 41mg/kg, 42mg/kg, 43mg/kg, 44mg/kg, 45mg/kg, 46mg/kg, 47mg/kg, 48mg/kg, 49mg/kg 50mg/kg, 51mg/kg, 52mg/kg, 53mg/kg, 54mg/kg, 55mg/kg, 56mg/kg, 57mg/kg, 58mg/kg, 26mg/kg, 23 mg/kg, 50mg/kg, 51mg/kg, 52mg/kg, 53mg/kg, 54mg/kg, 55mg/kg, 56mg/kg, 57mg/kg, or, 59mg/kg, 60mg/kg, 65mg/kg, 70mg/kg, 75mg/kg, 80mg/kg, 85mg/kg, 90mg/kg, 95mg/kg, 100mg/kg (including all integers in between) of the oligomer. In a preferred embodiment, the oligomer is administered at 30 mg/kg. In another preferred embodiment, the oligomer is administered at 50 mg/kg.

If desired, an effective daily dose of the active compound may optionally be administered in unit dosage form as 2, 3,4, 5,6 or more sub-doses administered separately at appropriate intervals throughout the day. In some cases, the administration is once daily. In certain embodiments, the administration is one or more times 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 functional dystrophin expression.

In certain embodiments, the oligomers of the invention (e.g., phosphorodiamidate morpholino oligomers; eteplirsen) are typically administered at regular intervals (e.g., daily, weekly, every 2 weeks, monthly, every 2 months). The oligomer may be administered at regular intervals, e.g., 1 time per day; 1 time every 2 days; 1 time every 3 days; 1 time every 3-7 days; 1 time every 3-10 days; 1 time every 7-10 days; 1 time per week; every 2 weeks for 1 time; 1 time per month. For example, the oligomer may be administered once a week by intravenous infusion. The oligomer may be administered intermittently over a longer period of time, e.g., for weeks, months, or years. For example, the oligomer may be administered 1 time every 1, 2, 3,4, 5,6, 7,8, 9, 10, 11, or 12 months. In addition, the oligomer may be administered 1 time every 1, 2, 3,4, or 5 years. Administration may be prior to or concurrent with administration of antibiotics, steroids or other therapeutic agents. Based on the results of the immunoassay of the subject under treatment, other biochemical tests and physiological examinations, the treatment regimen (dose, frequency, route, etc.) can be adjusted as indicated.

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 encapsulation in liposomes, iontophoresis, or incorporation in other vehicles (such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres) as described herein and known in the art. In certain embodiments, microemulsion techniques can be used to improve the bioavailability of lipophilic (water-insoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo, S.K., et al, Drug Development and Industrial Pharmacy, 17(12), 1685-.

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

While all suitable amphiphilic carriers are contemplated, the presently preferred carriers are generally those having a Generally Recognized As Safe (GRAS) status, and which can solubilize the compounds of the present invention and microemulsify them at a later stage when the solution is contacted with a complex aqueous phase, such as found in the human gastrointestinal tract. Typically, amphiphilic components meeting these requirements have an HLB (hydrophilic-lipophilic ratio balance) value of 2-20, and their structure contains straight chain aliphatic residues in the range of C-6 to C-20. Examples are polyethylene-glycolated fatty acid glycerides and polyethylene glycols.

Examples of amphiphilic carriers include saturated and monounsaturated polyglycolized fatty acid glycerides such as those obtained from various vegetable oils that are fully or partially hydrogenated. Such oils may advantageously be composed of di-and mono-polyethylene glycol esters of tri-, di-, and mono-fatty acid glycerides and corresponding fatty acids, with particularly preferred fatty acid compositions including 4-10% capric acid, 3-9% capric acid, 40-50% lauric acid, 14-24% myristic acid, 4-14% palmitic acid and 5-15% stearic acid. Another useful class of amphiphilic carriers includes partially esterified sorbitan and/or sorbitol having saturated or monounsaturated fatty acids (SPAN-series) or corresponding ethoxylated analogues (TWEEN-series).

Commercially available amphiphilic carriers may be particularly useful, including the Gelucire-series, Labrafil, Labrasol or Lauroglicol (both manufactured and sold by Gattefose Corporation, Saint Prest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurate and di-laurate, lecithin, polysorbate 80, and the like (manufactured and sold by a number of companies in the United states and throughout the world).

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

Hydrophilic polymers suitable for use in the present invention are hydrophilic polymers: it is readily soluble in water, can be covalently linked to vesicle-forming lipids, and it is tolerated in vivo with no toxic effects (i.e., is biocompatible). 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 polymer has a molecular weight of about 100 or 120 daltons up to about 5,000 or 10,000 daltons, or about 300 daltons to about 5,000 daltons. In other embodiments, the polymer is polyethylene glycol having a molecular weight of about 100 to about 5,000 daltons or having a molecular weight of about 300 to about 5,000 daltons. In certain embodiments, the polymer is polyethylene glycol of 750 daltons (PEG (750)). The polymer may also be defined by the number of monomers therein; a preferred embodiment of the invention utilizes a polymer of at least about 3 monomers, such a PEG polymer consisting of 3 monomers (about 150 daltons).

Other hydrophilic polymers that may be suitable for use in the present invention include polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide and derivatized cellulose such as hydroxymethyl cellulose or hydroxyethyl cellulose.

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

Cyclodextrins are cyclic oligosaccharides consisting of 6, 7 or 8 glucose units, designated by the greek letters α, β or γ, respectively. The glucose units are linked by alpha-1, 4-glycosidic linkages. As a result of the chair-type conformation of the saccharide units, all secondary hydroxyl groups (at C-2, C-3) are located on one side of the ring, while all primary hydroxyl groups at C-6 are located on the other side. The outer face is therefore hydrophilic, thereby making the cyclodextrin water-soluble. In contrast, the cavities of cyclodextrins are hydrophobic in that they are linked by the hydrogen of the atoms C-3 and C-5 and by ether-like oxygen. These matrices allow for complexation with a variety of relatively hydrophobic compounds, including, for example, steroid compounds such as 17 α -estradiol (see, e.g., van Uden et al Plant Cell tissue. org. cut. 38: 1-3-113 (1994)). Complexation occurs through van der waals interactions and through hydrogen bond formation. For a general review of the chemical nature of cyclodextrins, see Wenz, agnew. 803-822(1994).

The physicochemical properties of cyclodextrin derivatives strongly depend on the kind and degree of substitution. For example, their solubility in water ranges from insoluble (e.g., 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 enable the solubility of various formulation components to be controlled by increasing or decreasing their solubility.

Numerous cyclodextrins and methods for their preparation have been described. For example, parmeter (i), et al (U.S. patent No. 3,453,259) and Gramera, et al (U.S. patent No. 3,459,731) describe charge neutral cyclodextrins. Other derivatives include cyclodextrins with cationic character [ Parmeter (II), U.S. Pat. No. 3,453,257], insoluble cross-linked cyclodextrins (Solms, U.S. Pat. No. 3,420,788) and cyclodextrins with anionic character [ Parmeter (III), U.S. Pat. No. 3,426,011 ]. Among cyclodextrin derivatives having anionic character, carboxylic acids, phosphorous acids, phosphinic acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfinic acids and sulfonic acids have been appended to the parent cyclodextrin [ see, parmeter (iii), supra ]. In addition, Stella, et al (U.S. Pat. No. 5,134,127) have described thioalkyl ether cyclodextrin derivatives.

Liposomes consist of at least one lipid bilayer membrane encapsulating an aqueous internal compartment. Liposomes can be characterized by membrane type and size. Small Unilamellar Vesicles (SUVs) have a unilamellar membrane, typically ranging between 0.02-0.05 μm in diameter; large Unilamellar Vesicles (LUVs) are typically greater than 0.05 μm. Multilamellar macrovesicles and multilamellar vesicles have multiple, generally concentric membrane layers and are typically greater than 0.1 μm. Liposomes with several non-concentric membranes (i.e., several smaller vesicles contained within larger vesicles) are called multivesicular vesicles.

One aspect of the invention relates to a formulation comprising liposomes containing the oligomers of the invention, wherein the liposomal membrane is formulated to provide liposomes with increased carrying capacity. 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 present invention can be aggregated with lipid surfactants and carried within the interior space of liposomes; in these cases, the liposome membrane is formulated to resist the destructive effects of the active agent-surfactant aggregates.

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

The active agent contained within the liposomes of the invention is in dissolved form. Aggregates of surfactants and active agents (such as emulsions or micelles containing the active agent of interest) can be entrapped in the interior space of liposomes according to the invention. The surfactant functions to disperse and solubilize the active agent and may be selected from any suitable aliphatic, cycloaliphatic or aromatic surfactant, including, but not limited to, biocompatible Lysophosphatidylcholine (LPG) of varying chain lengths (e.g., about C14 to about C20). Polymer-derivatized lipids (such as PEG-lipids) can also be used for micelle formation because they will act to inhibit micelle/membrane fusion, and because the addition of a polymer to the surfactant molecule will reduce the CMC of the surfactant and aid in micelle formation. Preferred are surfactants having CMO in the micromolar range; higher CMC surfactants can be used to prepare micelles that are entrapped within the liposomes of the invention.

Liposomes according to the invention can be prepared by any of a variety of techniques known in the art. See, for example, U.S. Pat. nos. 4,235,871; published PCT applications WO 96/14057; new RRC, liposomees: 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, liposomes of the invention can be prepared as follows: the lipids derivatized with the hydrophilic polymer are diffused into the preformed liposomes, such as exposing the preformed liposomes to micelles composed of the lipid-grafted polymer at a lipid concentration corresponding to the final mole percent of derivatized lipid desired in the liposomes. Liposomes containing hydrophilic polymers may also be formed by homogenization, lipid field hydration or extrusion techniques known in the art.

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

In one aspect of the invention, the liposomes are prepared to have a substantially uniform size within a selected size range. An efficient sizing method comprises: extruding an aqueous suspension of liposomes through a series of polycarbonate membranes having a selected uniform pore size; the pore size of the membrane will correspond approximately to the maximum size of the liposomes produced by extrusion through the membrane. See, for example, U.S. patent No. 4,737,323 (12/4/1988). In certain embodiments, agents such asAndcan be used to introduce polynucleotides or proteins into cells.

The release profile of the formulations of the present invention depends on the encapsulating material, the concentration of the encapsulated drug, and the presence of the release modifier. For example, the release may be manipulated to be pH dependent, e.g., using a pH sensitive coating that releases only at low pH (as in the stomach) or higher pH (as in the intestine). Enteric coatings may be used to prevent release until after passage through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach followed by a later release in the intestine. Release may also be manipulated by inclusion of salts or pore formers which may increase water uptake of the drug by diffusion from the capsuleOr released. Excipients that modify the solubility of the drug may also be used to control the release rate. Agents that enhance matrix degradation or release from the matrix may also be incorporated. They may be added to the drug, added as a separate phase (i.e., as microparticles), or may be co-dissolved in the polymer phase, depending on the compound. 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 asAndpore formers (i.e., water-soluble compounds such as inorganic salts and sugars) are added as microparticles, which add microstructure to the matrix. The range is generally between 1-30% (w/w polymer).

Uptake can also be manipulated by varying the residence time of the particles in the intestine. This can be achieved as follows: for example, the particles are coated with a mucoadhesive polymer, or a mucoadhesive polymer is selected as the encapsulating material. Examples include polymers having mostly free carboxyl groups such as chitosan, cellulose and in particular polyacrylates (polyacrylate as used herein means polymers comprising acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates).

The oligomer may be formulated for inclusion in, or modified for release by, a surgical or medical device or implant. In certain aspects, the implant may be coated or otherwise treated with the oligomer. For example, hydrogels or other polymers (such as biocompatible and/or biodegradable polymers) can be used to coat implants with the compositions of the present invention (i.e., the compositions can be adapted for use with medical devices through the use of hydrogels or other polymers). Polymers and copolymers useful for coating medical devices with agents are well known in the art. Examples of implants include, but are not limited to, stents, drug eluting stents, sutures, prostheses, vascular catheters, dialysis catheters, vascular grafts, prosthetic heart valves, cardiac pacemakers, implantable cardioverter defibrillators, intravenous needles, devices for bone fixation and formation (such as pins, screws, plates, and other devices), and prosthetic tissue matrices for wound healing.

By analogy with other drugs, in addition to the methods provided herein, the oligomers used according to the invention may be formulated for administration in any convenient manner for use in human or veterinary medicine. In the treatment of muscular dystrophy, antisense oligomers and their corresponding formulations can be administered alone or in combination with other therapeutic strategies such as myoblast transplantation, stem cell therapy, administration of aminoglycoside antibiotics, proteasome inhibitors, and upregulation therapies (e.g., upregulation of utrophin, an autosomal paralogue of dystrophin).

The route of administration described is intended merely as a guide, as the skilled practitioner will be able to readily determine the route of administration and any dosage which is optimal for any particular animal and condition. Various protocols have been tried to introduce new genetic material with functionality into cells in vitro and in vivo (Friedmann (1989) Science, 244: 1275-. These protocols involve integration of the gene to be expressed into the modified retrovirus (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), supplement: 5074S-5079S); integration into non-retroviral vectors (e.g., adeno-associated viral vectors) (Rosenfeld, et al (1992) Cell, 68: 143-; or delivery of a transgene linked to a heterologous promoter-enhancer element via liposomes (Friedmann (1989), supra; Brigham, et al (1989) am. J. Med. Sci., 298: 278;. 281; Nabel, et al (1990) Science, 249: 1285-; coupled to ligand-specific, cation-based transport systems (Wu and Wu (1988) J.biol.chem., 263: 14621-14624) or using naked DNA expression vectors (Nabel et al (1990), supra); wolff et al (1990) Science, 247: 1465-1468). Direct injection of the transgene into the tissue only results in localized expression (Rosenfeld (1992) supra); rosenfeld et al (1991) supra; brigham et al (1989) supra; nabel (1990) supra; as in Hazinski et al (1991). The Brigham et al group (am.J.Med.Sci. (1989) 298: 278-. An example of a review article of human gene therapy programs is: anderson, Science (1992) 256: 808-813.

V.Reagent kit

The invention also provides a kit for treating a patient having a genetic disease, the kit comprising at least an antisense molecule (e.g., one or more antisense oligonucleotides capable of specifically hybridizing to any one or more of exons 1-79 of a dystrophin gene (e.g., exon 51 set forth in tables 3 and 4 herein) packaged in a suitable container with instructions for its use. The kit may also contain secondary reagents such as buffers, stabilizers, and the like. It will be appreciated by those of ordinary skill in the art that the application of the above methods has broad utility for identifying antisense molecules suitable for use in the treatment of many other diseases.

VII.Examples

Materials and methods

Patient's health

Eligible patients were between 7-13 years of age (inclusive), with an out-of-frame deletion of the DMD gene that could be corrected by skipping exon 51. At least 24 weeks prior to enrollment, patients were confirmed to have stable cardiac and pulmonary function and a stable dose of glucocorticoid. Patients who could walk between 200 and 400 meters (+ -10%) in the 6-minute walk trial (6MWT) at baseline were only enrolled.

Design of research

The 1 year test was performed in 2 stages: (1) treatment was double-blind until week 24, and (2) open-label thereafter. The primary endpoint was the percent change in dystrophin fiber and out-of-bed activity measured by the 6-minute walk test (6 MWT).

Study 201 is a single-site, randomized, double-blind, placebo-controlled, multiple-dose efficacy, safety and tolerability trial of eteplirsen. 12 patients with DMD were randomized to one of 3 groups: eteplirsen 30 mg/kg/week (cohort 1); eteplirsen 50 mg/kg/week (cohort 2); or placebo/delayed eteplirsen (cohort 3). All patients received weekly intravenous eteplirsen or placebo/delayed eteplirsen dosing. Placebo-treated patients switched to eteplirsen 30 (n-2) or 50mg/kg (n-2) weekly at week 25. Efficacy and Safety were assessed at the planned visit and the independent Data Safety Monitoring committee (Data Safety Monitoring Board) ensured the welfare of all patients. All patients had a biceps biopsy at baseline. Follow-up biopsies were performed in the opposite arm (biceps) at week 12 for the 50mg/kg group and 2 placebo-treated patients and at week 24 for the 30mg/kg group and 2 placebo-treated patients.

Patients continued to be dosed with 30 or 50mg/kg eteplirsen weekly under study 202, a long-term open-label extension study. During study 202, all efficacy assessments were continued, including a third biopsy (in the left deltoid) at week 48 in all patients. Monitoring of adverse events continued throughout the study. A schematic of the study design is shown in figure 2.

Research medicine

Eteplirsen [ sequence 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' ] (SEQ ID NO: 1) (100mg/ml) was provided in phosphate buffered saline in a disposable vial by Sarepta Therapeutics, Inc. Eteplirsen was reconstituted with 150ml of saline and infused over 60 minutes. Placebo administered in the first 24 weeks of study 201 was supplied as phosphate buffered saline from the same vial and administered in the same manner as eteplirsen.

Safety and tolerability monitoring

Safety was assessed by evaluating adverse events, vital signs, physical examination, electrocardiogram, echocardiogram, and clinical laboratory tests. In addition, renal function was monitored by periodic assessment of serum cystatin C as well as urinary cystatin C and KIM-1.

Pharmacokinetic and immunological evaluation

Validated and sensitive anion exchange high performance liquid chromatography was used with fluorescence detection bioanalytical methods to establish the pharmacokinetic parameters of eteplirsen from plasma and urine taken after the twelfth dose. Single samples for analysis of plasma concentrations were taken at weeks 24, 25 and 36. The immune response to the novel dystrophin protein was measured every 6 weeks with ELISPOT up to 24 weeks according to previously disclosed methods.

Biochemical efficacy assessment

Pre-and post-treatment dystrophin expression studies were based on MANDYS106[ Glen Morris, MDA Monoclonal Antibody Library ], a very sensitive marker of dystrophin used in previous studies of eteplirsen and other exon skipping candidates. 3 10 μm cryosections (separated by at least 200 μm) were stained with MANDYS106 followed by secondary antibody (Alexa Fluor 594 goat anti-mouse antibody). The percent dystrophin positive fibers was calculated by dividing the number of positive fibers by the total fibers counted. Since normal muscle samples had 100% dystrophin positive fibers, the percentage of dystrophin positive fibers was expressed as a percentage of normal values. Sections stained with the same antibodies were used for dystrophin quantification using Bioquant image analysis software. Total dystrophin fluorescence signal intensity is reported as a percentage of normal.

Supportive measures include expression of components of the myoglycan complex (β, γ), neuronal NOS, and western blotting (using anti-dystrophin antibody NCL-Dys1 from Novacastra). RT-PCR analysis to confirm exon skipping was performed on 400ng total RNA using the dystrophin-specific reverse primer as described previously.

Clinical efficacy assessment

6MWT is administered using a protocol established by McDonald, et al (Muscle Nerve, 2010; 42: 966-74, incorporated herein by reference) for patients with DMD. Exploratory functional results include arctic star ambulatory assessment, quantitative muscle testing, 9-hole stake testing, Pulmonary Function Testing (PFT), timed function testing, and quality of life assessment.

Statistical analysis

SAS version 9.3 (Cary, NC) was used for all statistical analyses. A hybrid model was used with treatment as the fixed effect, subjects staying within treatment as the random effect, baseline values, and time since DMD diagnosis as covariates for analysis of muscle biopsy data. Mixed Model Repeat Measurements (MMRM) were used with treatment, time and treatment x time (treatment-by-time) interaction terms as fixed effects, subjects staying within treatment as random effects, and baseline values and time since DMD diagnosis as covariates for analysis of 6MWT data. Performing safety and muscle biopsy analysis on the intended treatment population; analysis of results related to bed-escape activity, including 6MWT, used a modified intent-to-treat (mITT) population that did not include 2 patients in cohort 1 who exhibited signs of disease progression and a significant decline in 6MWT within weeks of enrollment, and were unable to make measurements of bed-escape activity at or after week 24.

Example 1: subject characteristics

The baseline characteristics of 12 patients in this study are summarized in table 2. 5 different genotypes susceptible to exon 51 skipping were represented in the study population. The average distance at baseline on the 6-minute walk trial (6MWT) was similar to those in other studies of children with DMD and, as expected, was well below 600 more meters typically observed in age-matched healthy children. Due to the random nature of the sampling, the 30mg/kg cohort was slightly older, heavier and higher than the other cohorts, and had a lower baseline average 6MWT distance. All patients received all study drug infusions as all assessments planned and completed.

TABLE 2

Example 2: loss of security and adverse events

Eteplirsen is well tolerated with no treatment-related adverse events, severe adverse events, discontinuation of medication, or missed dosing in 48 weeks of treatment. Furthermore, no clinically significant changes in physical examination or vital signs were observed. Electrocardiogram, echocardiogram and PFT remain stable and no clinically significant changes in blood function, renal function, coagulation function or liver function are indicated by chemical tests. Mild and transient proteinuria was observed in single placebo-treated subjects.

Example 3: pharmacokinetic profile

Analysis of PK parameters at week 12 revealed rapid absorption. The mean values for plasma clearance were 339. + -. 75.8mL/hr/kg (for 30mg/kg) and 319. + -. 125mL/hr/kg (for 50 mg/kg). Half-life averages 3.30 ± 0.341 hours (for 30mg/kg) and 3.17 ± 0.249 hours (for 50mg/kg), with renal clearance accounting for approximately 65-70% of total systemic clearance.

Example 4: efficacy

At week 48, eteplirsen caused a robust increase in the number and intensity of dystrophin-positive fibers. As shown in FIG. 3, patients receiving 30 or 50mg/kg eteplirsen without interruption for 48 weeks showed an average increase in the percentage of dystrophin-positive fibers of 47% (p.ltoreq.0.001) to reach normal compared to baseline. The increase was similar when the 30 (52%; p.ltoreq.0.001) and 50 (43%; p.ltoreq.0.008) mg/kg groups were analyzed, respectively, suggesting that the effect of eteplirsen on the production of novel dystrophins is independent of the dose within this range.

Biopsies were taken at staggered time points (see figure 2) to assess the effect of treatment duration on the production of novel dystrophins. At week 12, the 50mg/kg cohort had undetectable novel dystrophin levels. At week 24, the 30mg/kg cohort showed an increase in the percentage of dystrophin positive fibers reaching 23% (p ≦ 0.002) of normal, and at week 48, after 24 weeks of treatment with 30 or 50mg/kg eteplirsen, 4 patients in the placebo/delayed eteplirsen cohort showed an increase of 38% (p ≦ 0.009) of normal compared to baseline. Taken together, these data suggest that the duration of treatment plays an important role in eteplirsen's ability to uniformly restore novel dystrophin production. Consistent with these findings, eteplirsen also significantly increased the mean fluorescence signal intensity at week 48 in all 3 treatment groups (all p-values ≦ 0.023).

Figure 4 explains the time-dependent effect of eteplirsen on the percentage of dystrophin-positive fibres (panel a), with recovery of β -and γ -myoglycans and nNOS μ at the sarcolemma (panel B). Dystrophin expression and exon skipping was confirmed in all patients by western blot and RT-PCR. RT-PCR results from representative patients are shown in FIG. C. These data confirm the increase in functional dystrophin in patients.

Example 5: functional results

Progressive loss of ambulation is a common hallmark of DMD, with most patients exhibiting impaired function before the ages of 7 or 8 and becoming wheelchair-dependent before the ages of 10-14. Consistent with this, boys assigned to the placebo/delayed eteplirsen cohort in this study showed a decline in walking ability after week 12 at a rate predicted by previous studies, reaching the peak of approximately 60 meters of loss before week 48 (fig. 5). In a significant comparison, eteplirsen treated patients maintained a steady walking distance during the study, increasing by an average of about 7 meters from baseline before week 48. The differences between eteplirsen treated patients and those in the placebo/delayed eteplirsen cohort became statistically significant for the first time at week 32 (39-meter difference; p ≦ 0.05). Interestingly, patients in the placebo/delayed eteplirsen cohort appeared stable after week 36 (i.e., between 12-24 weeks after starting treatment with eteplirsen at week 25). As previously noted, 2 boys were excluded from the analysis, who showed signs of rapid disease progression and a significant drop in 6MWT within weeks of enrollment, and were unable to measure bed-exit activity at 24 weeks or later. However, both remained on eteplirsen treatment until week 48, with no treatment-related adverse events and maintained stable lung and upper limb function as measured by PFT and 9-hole stake tests, respectively.

Notably, patients receiving eteplirsen for 48 weeks, which can be evaluated at 6MWT (n ═ 6), improved significantly (p ≦ 0.001) at 6MWT compared to the placebo/delayed cohort (67.3 m).

Both cohorts maintained stable 6MWT results over 120 weeks as described below. At 120 weeks, patients in the 30mg/kg and 50mg/kg eteplirsen cohort (improved intent-to-treat or mITT population; n ═ 6) capable of 6MWT experienced overall stability with walking ability slightly decreased from baseline by 13.9 meters or less than 5%. A statistically significant therapeutic benefit of 64.9 meters (p ≦ 0.006) was observed for the mITT population compared to the placebo/delayed treatment cohort (n ≦ 4) with treatment initiated at week 25 after the placebo at week 24. After experiencing a large decline in early study (prior to treatment with etiprelsen), the placebo/delayed treatment cohort also exhibited a stabilization of walking ability for more than 1.5 years (from week 36 to week 120), at which time significant levels of dystrophin may be produced, declining by 9.5 meters over this time frame. These analyses were based on the maximum 6MWT score when the experiments were performed on 2 consecutive days.

Example 6: immune response

At any of the time points evaluated (including week 24), there was no difference in the number of interferon- γ induced spot-forming colonies relative to the collection of dystrophin peptides (extending throughout the protein) between eteplirsen-and placebo-treated patients, indicating that no T-cell response was elicited by newly expressed dystrophin in eteplirsen-treated patients.

Example 7: pulmonary function

Respiratory muscle function from baseline to week 120 in both dosing groups showed a mean increase in MIP of 14.6% and a mean increase in MEP of 15.0% as measured by maximum inspiratory and expiratory pressures (MIP and MEP). Analysis of the predicted MIP% (for weight adjusted MIP) and predicted MEP% (for age adjusted MEP) confirmed an average increase in the predicted MIP% from 90.2% at baseline to 95.2% at week 120 and a slight average increase in the predicted MEP% from 79.3% at baseline to 79.6% at week 120. In addition, there was an average increase in Forced Vital Capacity (FVC) (a measure of lung volume) from baseline to 8.7% at week 120, and the predicted FVC% (FVC for age and height adjustments) maintained an average of greater than 90% prior to week 120, 101% at baseline, and 93% at week 120.

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

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