Abstracts that will be presented in World Muscle Society Meeting 2014

Exon Skipping

Oral small molecule compounds that promote the skipping of exon 51 in the DMD gene
N.A. Naryshkin,  A. Dakka, V. Gabbeta, J. Pichardo,  M.G. Woll, N. Zhang, G.M. Karp,  E.M. Welch

Duchenne muscular dystrophy (DMD) is a severe progressive X-linked genetic disorder that affects 1 in 3500 males. It is a monogenic disease caused by mutations in the gene encoding the dystrophin protein. Approximately two thirds of the mutations are deletions in the open reading frame that result in the production of C-terminus-truncated non-functional dystrophin protein. One potential therapeutic approach to address a subset of the reading frame-altering deletions, located in the central rod domain, is to cause skipping of an exon downstream of the deletion thereby restoring the reading frame. The resulting protein, containing an internal deletion, would be partially functional but expected to lead to a milder, Becker muscular dystrophy-like phenotype. The largest fraction (∼13%) of all DMD patients have the above described mutations that can be addressed by skipping exon 51. We are pursuing an innovative approach to discover and develop orally bioavailable tissue-penetrable small molecules that induce skipping of DMD exon 51. Using PTC’s proprietary drug discovery platform technology referred to as GEMSAS (Gene Expression Modulation by Small-molecules – Alternative Splicing™) we have designed and conducted a high throughput screen of our compound library. We identified several structurally distinct classes of small molecules that promote the skipping of DMD exon 51. These scaffolds are undergoing structural optimization to improve biological, pharmacological and pharmaceutical properties with the ultimate goal of identifying a Drug Candidate for preclinical and clinical development.

Prospects for single antisense oligonucleotide-induced multiple exon skipping for rare non-hotspot mutations in Duchenne muscular dystrophy
J.A.M. Janson, I.G.M. Kolfschoten, R.E.Y. van den Eijnde, R. Weij, R.C. Verheul, A. Baghat, M.M. Plug, P.C. de visser, J.C.T. van Deutekom
 Antisense oligonucleotides (AONs) for Duchenne muscular dystrophy (DMD) are designed to induce skipping of a single exon during pre-mRNA splicing, thus restoring the transcript’s open reading frame and consequently synthesis of the deficient dystrophin protein. Although this approach is mutation-dependent, the majority of mutations cluster in a hotspot region from exon 45 to 53, allowing application of one AON to a subpopulation of patients with grouped mutations. Some mutations however are ultra-rare with only few patients worldwide, and require the skipping of an exon outside the hotspot region. Combining these ultra-rare mutations by applying multiple exon skipping is therefore attractive. The feasibility of multiple exon skipping using a cocktail of AONs has been demonstrated, but a drawback is the increased complexity of the development pathway and manufacturing costs. We therefore studied the possibility of designing a single AON capable of inducing simultaneous skipping of multiple exons. The repetitive nature of the central rod domain in dystrophin implies the occurrence of homologous stretches within the transcript. Indeed, in the exon 10 to 40 region several exon pairs were identified sharing such a homologous stretch, which we explored as a potential dual target for a single AON. In-frame exon combinations that would mimic a Becker-like mutation associated with a relatively mild phenotype and that would apply to a considerable patient subpopulation (up to ∼15%) were selected. In vitro proof-of-concept was obtained for healthy donor as well as for DMD patient-derived muscle cells and resulted in novel dystrophin expression. Results demonstrating the applicability in the mdx mouse model in vitro and in vivo will be presented. The data support further (pre) clinical development of such multiple exon skipping AONs, not only for more rare mutations that are not targeted by current single skip programs but also for mutations in the hotspot region.
Induction of the N-truncated dystrophin by out-of-frame exon 2 skipping restores muscle function in the Dup2 mouse, providing further support for a therapeutic pathway for 5′ DMD mutations

N. Wein, A. Vulin,T. Simmons, K.N. Heller, A. Rutherford, L.R. Rodino-Kaplac, D. Johnson, R.B. Weiss, F. Muntoni, K.M. Flanigan

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon. We demonstrated that this IRES is active in patients expressing the N-truncated dystrophin, raising the possibility of the therapeutic use of this isoform. To explore this we developed a novel out-of-frame exon-skipping approach that uses AAV-mediated U7snRNA to efficiently skip exon 2. By injecting this AAV vector into a DMD mouse model carrying a duplication of exon 2 (Dup2), this generates a truncated reading frame, leading to activation of the IRES and synthesis of the N-truncated isoform. We now demonstrate that despite lacking the first half of the canonical actin binding domain 1, this N-truncated protein is highly functional. Intramuscular injection of the AAV1. U7snRNA vector into Dup2 mice results in high levels of expression of the N-truncated isoform by 4 to 6 weeks post-injection, along with complete correction of the physiologic and pathologic features as measured by Evans blue dye uptake, hindlimb grip strength, tibialis anterior specific force, and force correction after eccentric contraction. Notably, utrophin levels remain unchanged. This level of correction to that of control mice supports the idea that this novel therapeutic approach should be beneficial for the 6% of patients with mutations within the first five exons of DMD. The efficiency of this treatment in inducing expression of the N-truncated dystrophin in 6 patient cell lines with different 5′ mutations is underway, and will be presented as well.

Transient proteinuria with oligonucleotide therapy due to interference with tubular protein reabsorption

C. den Besten, S. Jones, M. Wilmer, R. Masereeuw, G. Campion,

Prosensa Therapeutics is developing 2′O-modified phosphorothioate antisense oligonucleotides (AONs) to provide a mutation-specific therapy in the treatment of Duchenne muscular dystrophy (DMD). Drisapersen is the lead compound in development for treatment of a subset of DMD patients amenable to skip exon 51. Chronic treatment with 6 mg/kg subcutaneous has been associated with subclinical elevations in urinary protein. These elevations are mild (generally less than 1 g/L) and concern mainly low molecular weight proteins (LMWP). Safety data show that there is no progression with prolonged treatment and that values return to baseline either spontaneously or following treatment interruption. These mild and transient elevations in urinary LMWP in patients receiving drisapersen are considered to reflect interference with the tubular re-absorption of proteins from the glomerular filtrate rather than indications of overt renal toxicity. AONs demonstrate class predictive kinetic and distribution profiles, with the kidney being the main organ of accumulation, primarily in proximal tubular cells. A dose related appearance of basophilic granules in proximal tubular cells is commonly observed in animals treated with AONs. These granules represent endosomal accumulation of AONs and are not regarded as toxicologically relevant in the absence of associated degenerative pathology. The exact mechanism (s) and receptor (s) involved in renal uptake of AONs have not yet been established, but are likely to be similar to those associated with reabsorption of LMWP, i.e. endocytosis via the megalin–cubulin receptors. In vitro experiments using a human tubular cell line, ciPTEC, could be a suitable tool to investigate the mechanism of tubular oligonucleotide absorption and the potential interference with tubular protein reabsorption.

Evaluation of exon skipping activity of 2′- deoxy-2′-fluoro antisense oligonucleotides for Duchenne muscular dystrophy

S.M.G. Jirka, J.W. van der Meulen,  C.L. Tanganyika-de Winter, M. van Putten,
M. Hiller, R. Vermue, P.C. de Visser, A. Aartsma-Rus

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by out of frame mutations in the dystrophin gene. Restoration of the reading frame would in theory allow the production of a shorter but partly functional dystrophin protein as seen in patients with Becker muscular dystrophy (BMD). This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons in the pre-mRNA. Over the years chemical modifications have been developed to increase AON binding affinity to the target RNA, increase resistance against nuclease degradation and improve cellular uptake. The 2′-deoxy-2′-fluoro AON (2FPS) is an attractive candidate chemistry for exon skipping. It has been shown that ILF2/3 proteins are recruited to the 2FPS/pre-mRNA duplex, resulting in (undesired) enhanced exon skipping in a model ofspinal muscular atrophy (SMA). In this study we compared 2′-F phosphorothioate (2FPS) and 2′-O-methyl phosphorothioate AONs (2OMePS) targeting different human and mouse dystrophin exons in vitro and in vivo. In vitro, the 2FPS AONs induced higher exon skipping levels than their 2OMePS AON counterparts, and were effective at concentrations where their 2OMePS AON counterparts were not. Surprisingly, a 2FPS AON targeting mouse exon 23 appeared to be ineffective after local intra muscular administration (2 times 2.9 nmol) and after 8 weeks of systemic treatment in mdx mice (4 times 50 mg/kg/week). To assess whether this is a generalized effect of 2FPS AON or specific for mouse exon 23 skip, we are currently evaluating a 2FPS AON targeting a human exon in our hDMD mouse model, which contains the complete human DMDgene integrated into the mouse genome.

Safety and pharmacokinetic profile of eteplirsen, SRP-4045, and SRP-4053, three phosphorodiamidate morpholino oligomers (PMOs) for the treatment of patients with Duchenne muscular dystrophy (DMD)

Eteplirsen, SRP-4045 and SRP-4053 are three PMOs developed as treatments for DMD. DMD is mostly caused by mutations in the dystrophin gene that lead to a reading frame shift and premature translation termination. PMOs promote exon skipping during splicing of dystrophin pre-mRNA, restoring the reading frame and translation of internally truncated, but functional dystrophin protein. Eteplirsen, SRP-4045, and SRP-4053 are intended to treat patients with mutations amenable to skipping exon 51, 45 and 53, respectively. IND enabling preclinical studies have been completed for eteplirsen, SRP-4045, and SRP-4053. In vitro metabolism data and PK profiles in animals were similar for all three PMOs. No adverse responses were detected in safety pharmacology studies in non-human primates (NHPs). Genotoxicity studies were negative. NHPs given 12 weekly IV doses up to 320 mg/kg showed no significant sequence-specific toxicities. A clinical study of eteplirsen with doses of 30 and 50 mg/kg/wk reported no clinically significant treatment-related adverse events through 120 weeks of treatment. A few cases of transient, mild urine protein elevation resolved without intervention or drug interruption and one femur fracture was determined to be treatment-unrelated. There were no hospitalizations, discontinuations, or clinically significant treatment-related changes for lab parameters, including liver enzymes, kidney function, coagulation, or platelet counts. Plasma Cmax and AUC increased proportionally with dose, and half-life was approximately 3 h with no plasma accumulation observed. The consistent preclinical profiles and lack of significant, sequence-specific toxicities for eteplirsen, SRP-4045, and SRP-4053 demonstrate PMOs to be a well-tolerated therapeutic class, with potential applications in a wide variety of disease indications.

Pulmonary function is stable through week 120 in patients with Duchenne muscular dystrophy (DMD) treated with exon-skipping drug eteplirsen in phase 2b study

J.R. Mendell, L.P. Lowes, L. Alfano, J. Saoud, P. Duda, E. Kaye

DMD is a rare, degenerative, genetic disease that results in progressive muscle loss and premature death and affects 1:5000 male births. Clinically manifest pulmonary dysfunction often occurs when DMD patients become non-ambulant and is preceded by subclinical deterioration of pulmonary function tests (PFTs). Specifically, MIP and MEP % predicted deteriorate by approximately 4 % per year between the ages of 8–19. Eteplirsen is an investigational drug designed to enable functional dystrophin production in boys who are amenable to skipping exon 51. When dosed for up to 120 weeks, stabilization of 6 min walk distance was demonstrated in a Phase 2b study. 12 boys were randomized 1:1:1 to 30/50 mg/kg or placebo. Upon completion of a 24-week double-blind, placebo-controlled phase, all patients were enrolled in an open-label extension and the placebo-treated patients initiated eteplirsen treatment. FVC, FVC % predicted, MIP, MEP and MIP and MEP % predicted were assessed every 12 to 24 weeks. For all 12 patients changes in function at Week 120 were examined from Week 1, and from last assessment pre-eteplirsen administration (Week 1 for 8 patients and Week 24 for 4 patients). One-sample t-test was used for statistical analysis. Reported here are the results for all 12 patients, including two patients who became non ambulant by Week 24. Median age at Week 120 was 12 years. The 120 week data for 5 of the 6 PFT parameters were not statistically different from baseline, with the exception of a statistically significant increase in MEP. Furthermore, individual patient values for all 12 patients continue to be in the age-adjusted normal ranges indicative of continued normal pulmonary function without the need for any ventilation assistance or respiratory failure. A PFT update after 3 years of treatment (Week 144) will be provided. Eteplirsen dosed for up to 120 weeks demonstrated stability on PFT measures contrary to a steady decline expected in DMD patients of this age.

Drisapersen (DRIS) treatment for Duchenne muscular dystrophy (DMD): Results of up to 188 weeks’ follow-up of an open-label extension study

N. Goemans, M. Tulinius, R. Wilson, C. Wardell, P. Bedwell, G. Campion

Drisapersen (DRIS) is a 2’-O-methyl-phosphorothioate antisense oligonucleotide that induces skipping of exon 51 to correct Duchenne muscular dystrophy (DMD) mutations. This open-label extension (OLE) to a 5-week dose-escalation study assessed the long-term safety and efficacy of 6 mg/kg DRIS in 12 boys (5–16 years) with DRIS-correctable mutations. Dosing was once weekly for 72 weeks, with a planned interruption (weeks 73–80), followed by intermittent dosing (Weeks 81–188). Median (mean [SD]) 6-min walking distance (6MWD) declined between original baseline and OLE baseline (6–15 months) from 394 (402 [73]) to 362 (384 [121]) m at OLE baseline (n = 10). At Week 177, the mean (range) age of boys who completed the 6MWD at OLE baseline (n = 10) was 12.9 (9.3–15.4) years. The median (mean [SD]) change from OLE baseline was +7.5 (−24.5 [161]) m for these 10 boys; 2 boys (baseline 6MWD < 300 m) lost ambulation and scored 0. Five boys were able to walk further (range 37–163 m), with 2 walking >140 m further than at OLE baseline. The mean (SD) change from OLE baseline to Week 177 in rise from floor was 1.0 (0.9) s for boys (n = 6) who attempted the test, and 6.9 (10.7) s when missing data for boys unable to attempt the test were replaced by 30s (n = 11). There was a mean (SD) change from OLE baseline to Week 177 in absolute forced vital capacity of 0.06 (0.29) L (n = 12). There were no clear trends in other timed tests. Over 188 weeks, all 12 boys had ⩾1 adverse event (study drug-related injection-site reactions, n = 12;gastrointestinal disorders, n = 6; decreased platelet counts, n = 5). All had increases in urine α-1-microglobulin levels and transient mild proteinuria; these were non-progressive and reversible in drug-free periods. No boys permanently withdrew from treatment. Data from this OLE are encouraging, as the natural history of DMD in steroid-treated boys receiving standard care at one of the study sites shows a median (mean [SD]) 6MWD decline of −106 (−125 [139])m over 104 weeks.

Evaluation of efficacy and safety baseline parameters in patients with Duchenne muscular dystrophy (DMD) from three placebo-controlled studies of drisapersen (DRIS)

E. Mercuri, T. Voit, N. Goemans, C.M. McDonald, R. Wilson, C. Wardell,G. Campion

In three placebo (PBO)-controlled studies (24–48 wks), each with a primary endpoint of 6-min walking distance (6MWD), DRIS (6 mg/kg/wk) showed clinically meaningful and statistically significant effects in one Phase II study (DMD114117 [117]), clinically meaningful but not statistically significant effects in another Phase II study (DMD114876 [876]) and neither clinically meaningful nor statistically significant effects in a Phase III study (DMD114044 [044]), although there was a benefit for 6MWD of 21 m in a subset of boys ⩽7y. As DMD is rare, these study populations were small (N = 53, 51 and 186, respectively). Although mean ages for each study were similar, in 044, boys were older with a longer time since first diagnosis and treatment, enrolled boys were aged ⩽16y, whereas in 117 and 876 the oldest were 11 and 13y, respectively. Study populations differed with respect to disease severity at baseline (BL). Notably, patients from 044 had lower BL 6MWD than patients from 876 or 117: 337 and 348 m for DMD and PBO arms, respectively, vs 396 and 415 m (876) and 428 and 403 m (117). Similarly, rise from floor time was slower at BL for patients from 044 than the other two studies: 12.3 and 13.4 s for DMD and PBO arms, respectively, vs 5.0 and 4.5 s (876) and 4.8 and 4.7 s (117). Similar trends between groups were also seen for 10 m walk/run time, 4 stairs climb-ascent/descent. For some BL safety parameters, e.g. urinary protein, albumin and α-1 microglobulin, a small proportion of boys had values above the high range of normal at screening. Other parameters e.g. CK, LFTs were elevated as expected in this population. The trajectory of DMD is progressive, escalating more rapidly in severity after 7y. BL parameters suggest that boys in 044 may have had a poorer prognosis and be more likely to experience substantial deterioration in gross motor skills and ambulatory capacity. These findings are important to understand the different outcomes in the three DRIS PBO-controlled studies.

Pooled analyses of efficacy parameters in patients with Duchenne muscular dystrophy (DMD): Results from the drisapersen (DRIS) clinical trial programme

N. Goemans, T. Voit, C.M. McDonald,E. Mercuri, R. Wilson, C. Wardell,G. Campion

DRIS is a 2′-O-methyl-phosphorothioate antisense oligonucleotide designed to skip exon 51 in dystrophin pre-mRNA of boys with DMD. In a Ph 3 study (DMD114044), a 10 m treatment difference (NS) on 6-min walking distance (6MWD) in favour of DRIS was seen in the overall study population but there was a treatment benefit of 21 m in a pre-specified subset of boys aged ⩽7y (n = 79), suggesting that younger, less progressed boys could benefit more from DRIS in terms of ambulatory endpoints vs older boys in whom DMD is more advanced. In an open-label extension (OLE; DMD114349) of two 48-wk placebo (PBO)-controlled trials (DMD114117, DMD114044), 96 wks’ DRIS resulted in a clinically meaningful difference from 48 wks’ PBO (feeder study) followed by DRIS (OLE) in 6MWD of 46 m (n = 113; completed 48 wks’, DMD114349). By feeder study, the treatment effect was 52 m (n = 30) for DMD114117 and 49 m for DMD114044 (n = 83), for those on DRIS for 96 wks vs PBO/delayed treatment. Data suggest that treatment benefits that manifest early in less severe DMD are maintained, while in more advanced DMD, benefits may emerge only after prolonged exposure. In a pooled analysis of two comparable Ph 2 trials (DMD114117, DMD114876), a subset of boys with similar BL demographic and functional characteristics who received DRIS 6 mg/kg/wk (n = 36) was compared with PBO-treated boys (n = 34; Mixed-Effect Model Repeated Measure model including Treatment, Visit, Treatment by Visit, Country/Study, BL 6MWD, BL 6MWD by Visit). At Wk 24, there was a clinically meaningful and statistically significant difference in 6MWD between DRIS- and PBO-treated boys of 31 m (n = 70; 95% confidence interval: 11, 51; p = 0.003), based on an adjusted mean (standard error) difference of −11(7) m for placebo and  + 20(7) m for DRIS. DMD114044 enrolled boys aged ⩽16y, whereas in the pooled dataset, the oldest boys were only 13y, supporting the premise that treatment at a younger age is more immediately beneficial for preserving ambulatory capacity.

Eteplirsen in Duchenne Muscular Dystrophy (DMD): 3 year update on Six-Minute Walk Test (6MWT) and Safety
J.R. Mendell, L. Rodino-Klapac,Z. Sahenk, K. Roush, L. Bird, L.P. Lowes, L. Alfano, A.M. Gomez, S. Lewis,V. Malik, K. Shontz, K.M. Flanigan, C. Shilling, P. Sazani, J. Saoud, P. Duda, E. Kaye

DMD, a rare, degenerative, genetic disease that results in progressive muscle loss and premature death affects 1:5000 male births. It is caused by deletions in the dystrophin gene, which prevents production of the dystrophin protein. There are no approved treatments available, although corticosteroids have shown some benefit. Eteplirsen is an investigational drug designed to enable functional dystrophin production in boys who are amenable to skipping exon 51. Twelve boys aged 7–13 years with eligible genotypes were randomized 1:1:1 to eteplirsen 30 mg/kg/wk, 50 mg/kg/wk, or placebo IV for 24-weeks. All patients transitioned into an ongoing open-label extension with 30 or 50 mg/kg eteplirsen. Clinical efficacy endpoints included the 6-min walk test (6MWT) and pulmonary function testing. Safety assessments included adverse event recording, EKG, ECHO, hematology, blood chemistry and urinalysis. After 120 weeks of treatment, a significant clinical benefit of 65 m was observed (p = 0.006) on the 6MWT for ambulatory-evaluable patients in the combined eteplirsen-treated cohorts (n = 6) versus the placebo/delayed-treatment cohort (n = 4). The eteplirsen-treated cohorts showed a decline of less than 14 m in walking ability from baseline, which was not statistically significant. After a substantial decline in the first 36 weeks of the study, the placebo/delayed-treatment cohort demonstrated stabilization in walking ability at week 48, i.e., 12 weeks after initiation of treatment with eteplirsen when meaningful levels of dystrophin were likely produced, with <10 m decline through 120 weeks. No deaths, discontinuations due to AEs, treatment-related SAEs, or clinically significant abnormal laboratory, ECG, or ECHO findings were reported. Eteplirsen demonstrated a significant clinical benefit on the 6MWT over 120 weeks, was well tolerated, and exhibited an unremarkable safety profile. Week 144 data will be presented.

Drisapersen: An overview of the clinical programme to date in Duchenne Muscular Dystrophy (DMD)

C. McDonald, E. Mercuri, N. Goemans , T. Voit , R. Wilson , C. Wardell ,  G. Campion

A clinical trial programme to assess safety and efficacy of drisapersen (DRIS) in DMD comprises two Phase 2, one Phase 3 placebo (PBO)-controlled, (total N = 290) and two open-label extension (OLE) trials (N = 233). The Phase 2 studies enrolled comparable populations with relatively mild disease (mean baseline [BL] 6-min walking distance [6MWD] ∼400 m). At Wk 25, DMD114117 (117; N = 53) showed a statistically significant and clinically meaningful treatment difference of +35 m (p = 0.014) in 6MWD for 6 mg/kg/wk continuous DRIS treatment vs PBO (maintained at Wk 49 [+36 m; p = 0.051]); %-predicted 6MWD improved by 6% (p < 0.05) at both time points. After 24 wks’ continuous 6 mg/kg/wk DRIS treatment in DMD114876 (N = 51), a clinically meaningful treatment difference was seen in 6MWD (+27 m; p = 0.069); %-predicted 6MWD improved by 5% (p = 0.051). The Phase 3 study DMD114044 (044; N = 186) enrolled a more severe population (mean BL 6MWD < 350 m). Boys received 6 mg/kg/wk continuous DRIS or PBO for 48 wks resulting in a non-statistically significant +10 m difference in favour of DRIS. At Wk 48, for the combined analysis of 117 and 044 (⩽7 y at study BL), the treatment difference in 6MWD was +24 m (p = 0.048) for DRIS 6 mg/kg/wk vs. PBO. At 96 wks in the OLE fed by 117 and 044, the treatment difference (n = 113) was +46 m from BL for continuous 6 mg/kg DRIS vs starting DRIS treatment after 48 wks. In the Phase 1/2 OLE DMD114673 (n = 10; average age: 12.9 y), the mean change in 6MWD at Wk 177 (3.4 y) was −24.5 m (median +7.5 m) from OLE BL. Most common AEs with DRIS treatment were injection-site reactions, mild reversible proteinuria. Infrequent moderate to severe thrombocytopenia was also seen. The results of the trial programme are promising, as deterioration of muscle function is associated with the natural history of DMD in young boys. Overall, observed improvements or reductions in decline in 6MWD over 24–96 wks at this stage of the disease is indicative of an encouraging effect in reducing disease progression.